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SupercriticalFluid Extraction
of Nutraceuticalsand BioactiveCompounds
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SupercriticalFluid Extraction
of Nutraceuticalsand BioactiveCompounds
Edited by
Jose L. Martínez
CRC Press is an imprint of theTaylor & Francis Group, an informa business
Boca Raton London New York
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CRC PressTaylor & Francis Group6000 Broken Sound Parkway NW, Suite 300Boca Raton, FL 33487-2742
© 2008 by Taylor & Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group, an Informa business
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International Standard Book Number-13: 978-0-8493-7089-2 (Hardcover)
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Library of Congress Cataloging-in-Publication Data
Supercritical fluid extraction of nutraceuticals and bioactive compounds / [edited by] Jose L. Martinez.
p. cm.Includes bibliographical references and index.ISBN 978-0-8493-7089-2 (alk. paper)1. Supercritical fluid extraction. 2. Functional foods. 3. Bioactive compounds. I.
Martinez, José L. (José Luis), 1966-
TP156.E8S835 2007660.6’3--dc22 2007025441
Visit the Taylor & Francis Web site athttp://www.taylorandfrancis.com
and the CRC Press Web site athttp://www.crcpress.com
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Dedication
To Marlene and Alejandro
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vii
ContentsPreface.......................................................................................................................ixAcknowledgments......................................................................................................xiContributors............................................................................................................ xiiiEditor........................................................................................................................xv
Chapter 1 Fundamentals.of.Supercritical.Fluid.Technology.................................1
Selva Pereda, Susana B. Bottini, and Esteban A. Brignole
Chapter 2 Supercritical.Extraction.Plants:.Equipment,.Process,.and.Costs........25
Jose L. Martínez and Samuel W. Vance
Chapter 3 Supercritical.Fluid.Extraction.of.Specialty.Oils................................. 51
Feral Temelli, Marleny D. A. Saldaña, Paul H. L. Moquin, and Mei Sun
Chapter 4 Extraction.and.Purification.of.Natural.Tocopherols.by.Supercritical.CO2............................................................................... 103
Tao Fang, Motonobu Goto, Mitsuru Sasaki, and Dalang Yang
Chapter 5 Processing.of.Fish.Oils.by.Supercritical.Fluids................................ 141
Wayne Eltringham and Owen Catchpole
Chapter 6 Supercritical.Fluid.Extraction.of.Active.Compounds.from.Algae..... 189
Rui L. Mendes
Chapter 7 Application.of.Supercritical.Fluids.in.Traditional.Chinese.Medicines.and.Natural.Products....................................................... 215
Shufen Li
Chapter 8 Extraction.of.Bioactive.Compounds.from.Latin.American.Plants.... 243
M. Angela A. Meireles
Chapter 9 Antioxidant.Extraction.by.Supercritical.Fluids................................ 275
Beatriz Díaz-Reinoso, Andrés Moure, Herminia Domínguez, and Juan Carlos Parajó
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viii Contents
Chapter 10 Essential.Oils.Extraction.and.Fractionation.Using.Supercritical.Fluids...........................................................................305
Ernesto Reverchon and Iolanda De Marco
Chapter 11 Processing.of.Spices.Using.Supercritical.Fluids............................... 337
Mamata Mukhopadhyay
Chapter 12 Preparation.and.Processing.of.Micro-.and.Nano-Scale.Materials.by.Supercritical.Fluid.Technology.................................................... 367
Eckhard Weidner and Marcus Petermann
Index....................................................................................................................... 391
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ix
PrefaceIn.the.last.decade.new.trends.in.the.food.industry.have.emerged,.enhanced.concern.over. the. quality. and. safety. of. food. products,. increased. preference. for. natural..products,. and. stricter. regulations. related. to. the. residual. levels.of. solvents..Addi-tionally,.the.nutraceutical.and.functional.food.sector.represents.one.of.the.fastest.growing.areas.in.a.consumer-driven.trend.market..These.trends.have.driven.super-critical.fluid.(SCF).technology.to.be.a.primary.alternative.to.traditional.solvents.for.extraction,.fractionation,.and.isolation.of.active.ingredients..The.aim.of.this.book.is. to.present. the.current. state.of. the.art. in.extracting.and. fractionating.bioactive.ingredients.by.SCFs.
This.book.contains.twelve.chapters.that.primarily.focus.on.implemented.indus-trial.processes.and.trends.of.the.technology..The.content.of.the.chapters.includes.a.review.of.the.major.active.components.in.the.target.material,.including.chemical,.physical,. nutritional,. and. pharmaceutical. properties;. an. analysis. of. the. specific.SCF. process. used;. a. comparison. of. traditional. processing. methods. versus. SCF.technology;.and.a.set.of.conclusions.with.supporting.data.and.insight..A.review.of.the.fundamentals.of.the.technology.and.an.examination.of.SCF.extraction.systems.and.process.economics.are.also.included.
The. contributing. authors. are. international. experts. on. the. topics. covered,. and.I.would.like.to.thank.them.for.their.thoughtful.and.well-written.contributions..This.book.is.addressed.to.food.scientists,. technologists,.and.engineers.as.well.as.other.professionals.interested.in.the.nutraceutical.and.functional.food.sector..Additionally,.I.hope.that.this.book.will.serve.to.stimulate.academia.and.industry.to.search.for.new.process.and.product.developments.as.well.as.their.industrial.implementation.
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xi
AcknowledgmentsThe.authors.of.the.chapters.of Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds wish.to.acknowledge.the.following.funding.agencies.for.their.support.and.assistance.
Dr.. Feral. Temelli. would. like. to. acknowledge. the. financial. support. from. the.Natural.Sciences.and.Engineering.Research.Council.of.Canada.(NSERC).
Dr..Fang.et.al..gratefully.acknowledge.the.21st.COE.program.“Pulsed.Power.Sicence”.and.Wuhan.Kaidi.Fine.Chemical.Industries.Co.,.Ltd.,.for.their.support.
Dr..Shufen.Li.would.like.to.thank.Dr..Can.Quan,.Dr..Shaokun.Tang,.Dr..Wenqiang.Guan,.Dr..Yongyue.Sun,.Ms..Luan.Xiao,.and.Ms..Ying.Zhang.for.their.contributions.to.the.research.work.as.well.as.their.assistance.in.the.preparation.of.Chapter.7.
Dr..Maria.Angela.Meireles.thanks.CNPq,.CAPES,.and.FAPESP.for.supporting.the.research.done.at.LASEFI.–.DEA/.FEA.–.UNICAMP.
Dr.. Eckhard. Weidner. and. Dr.. Marcus. Petermann. would. like. to. thank. their.coworkers.and.students.from.the.University.Bochum.as.well.as.Prof..Knez.and.his.coworkers.from.the.University.of.Maribor.and.Adalbert-Raps.Research.Center..They.would.also. like. to. thank.Adalbert-Raps.Stiftung,. the.European.Union,. the.Ewald.Doerken.AG,.and.Yara.Industrial.GmbH.for.their.support.
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xiii
Contributors
Susana B. Bottini, Ph.D.Planta.Piloto.de.Ingeniería.QuímicaUniversidad.Nacional.del.SurBahía.Blanca,.Argentina
Esteban A. Brignole, Ph.D.Planta.Piloto.de.Ingeniería.QuímicaUniversidad.Nacional.del.SurBahía.Blanca,.Argentina
Owen J. Catchpole, Ph.D.Industrial.Research.LimitedLower.Hutt,.New.Zealand
Iolanda De Marco, Ph.D.Dipartimento.di.Ingegneria.
Chimica.ed.AlimentareUniversita.di.SalernoSalerno,.Italy
Beatriz Díaz-Reinoso, M.Sc.Department.of.Chemical.EngineeringFacultade.de.Ciencias.de.OurenseUniversidade.de.VigoOurense,.Spain
Herminia Domínguez, Ph.D.Department.of.Chemical.EngineeringFacultade.de.Ciencias.de.OurenseUniversidade.de.VigoOurense,.Spain
Wayne Eltringham, Ph.D.Industrial.Research.LimitedLower.Hutt,.New.Zealand
Tao Fang, Ph.D.Department.of.Applied.Chemistry.and.
BiochemistryKumamoto.UniversityKumamoto,.Japan
Motonobu Goto, Ph.D.Department.of.Applied.Chemistry.and.
BiochemistryKumamoto.UniversityKumamoto,.Japan
Shufen Li, Ph.D.School.of.Chemical.Engineering.&.
TechnologyTianjin.UniversityTianjin,.China
Jose L. Martínez, Ph.D.Thar.Technologies,.Inc.Pittsburgh,.Pennsylvania
M. Angela A. Meireles, Ph.D.LASEFI-DEAFEA.–.UNICAMPSao.Paulo,.Brazil
Rui L. Mendes, Ph.D.Departamento.de.Energias.RenovaveisINETILisboa,.Portugal
Paul H.L. Moquin, B.Sc.Department.of.Agricultural,.Food,.and.
Nutritional.ScienceUniversity.of.AlbertaEdmonton,.Canada
Andrés Moure, Ph.D.Department.of.Chemical.EngineeringFacultade.de.Ciencias.de.OurenseUniversidade.de.VigoOurense,.Spain
Mamata Mukhopadhyay, Ph.D.Chemical.Engineering.DepartmentIndian.Institute.of.TechnologyBombay,.India
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xiv Contributors
Juan Carlos Parajó, Ph.D.Department.of.Chemical.EngineeringFacultade.de.Ciencias.de.OurenseUniversidade.de.VigoOurense,.Spain
Selva Pereda, Ph.D.Planta.Piloto.de.Ingeniería.QuímicaUniversidad.Nacional.del.SurBahía.Blanca,.Argentina
Marcus Petermann, Ph.D.University.BochumParticle.TechnologyBochum,.Germany
Ernesto Reverchon, Ph.D.Dipartimento.di.Ingegneria.
Chimica.ed.AlimentareUniversita.di.SalernoSalerno,.Italy
Marleny D.A. Saldana, Ph.D.Department.of.Agricultural,.Food,.and.
Nutritional.ScienceUniversity.of.AlbertaEdmonton,.Canada
Mitsuru Sasaki, Ph.D.Department.of.Applied.Chemistry.and.
BiochemistryKumamoto.UniversityKumamoto,.Japan
Mei Sun, M.Sc.Department.of.Agricultural,.Food,.and.
Nutritional.ScienceUniversity.of.AlbertaEdmonton,.Canada
Feral Temelli, Ph.D.Department.of.Agricultural,.Food,.and.
Nutritional.ScienceUniversity.of.AlbertaEdmonton,.Canada
Samuel W. Vance, P.E.Thar.Technologies,.Inc.Pittsburgh,.Pennsylvania
Eckhard Weidner, Ph.D.University.BochumProcess.TechnologyBochum,.Germany
Dalang Yang, M.Sc.Wuhan.Kaidi.Fine.Chemical.Industries.
Co..Ltd.Wuhan,.Hubei,.China
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xv
EditorDr. Jose L. Martínez,.a.native.of.León,.Spain,.received.his.B.S..and.Ph.D..degrees.from.the.University.of.Oviedo.(Spain)..He.is.currently.General.Manager.of.Thar.Technologies,. Inc.,. Process. Division. (Pittsburgh,. USA),. a. company. dedicated.exclusively.to.supercritical.fluid.technology..He.has.nearly.two.decades.of.experi-ence. in.conducting.R&D.and. implementing. industrial.processes. in.supercritical.fluid.technology,.including.applications.in.extraction,.fractionation,.chromatogra-phy,.particle.formation,.coating,.and.impregnation.for.the.food,.nutraceutical,.and.pharmaceutical.industries.
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�
1 Fundamentals of Supercritical Fluid Technology
Selva Pereda, Susana B. Bottini, and Esteban A. Brignole
Contents
1.1 Introduction.....................................................................................................11.2 SupercriticalFluids.........................................................................................2
1.2.1 PhysicalPropertiesofSupercriticalFluids..........................................41.3 PhaseEquilibriumwithSupercriticalFluids..................................................4
1.3.1 SolidSolubilities..................................................................................41.3.2 MultipleFluidPhaseEquilibrium.......................................................6
1.4 PhaseEquilibriumEngineeringofSupercriticalProcesses...........................81.4.1 UnderstandingPhaseBehavior............................................................9
1.5 ConceptualSupercriticalProcessDesign..................................................... 111.5.1 OxychemicalExtractionandDehydration......................................... 111.5.2 ParticleMicronizationwithSupercriticalFluids............................... 151.5.3 Extraction,Purification,orFractionationofNaturalProductswith
SupercriticalFluids............................................................................ 171.5.3.1 FractionationofOils............................................................. 171.5.3.2 ExtractionfromVegetableMatrices.................................... 18
1.5.4 SupercriticalReactions...................................................................... 19References................................................................................................................ 21
�.� IntroduCtIon
Solventsareusedinlargeamountsinthechemical,pharmaceutical,food,andnatural-product industries. In thesearch forenvironmentally friendlysolvents, increasingattentionisbeingpaidtosupercriticalfluids(SCFs)forawidevarietyofapplica-tions.Forinstance,supercriticalsolventsareusedinextractions,materialprocessing,micronization,chemicalreactions,cleaning,anddrying,amongotherapplications.SCFsandnear-criticalfluidsaddanewdimensiontoconventional(liquid)solvents: their density-dependent solvent power.ThedensityofSCFscanbeeasilytunedtotheprocessneeds,withchangesintemperature,pressure,and/orcomposition.OtherimportantpropertiesofSCFsaretheirverylowsurfacetensions,lowviscosities,andmoderatelyhighdiffusioncoefficients.
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� Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
Thedesignofprocessesusingsupercriticalsolventsisstronglydependentonthephaseequilibriumscenario,whichishighlysensitivetochangesinoperatingcondi-tions.Therefore,phaseequilibriumengineeringplaysakeyroleinthesynthesisanddesignoftheseprocesses.
�.� superCrItICal FluIds
The different physical states of a pure substance can be visualized in a three-dimensionalpressure–volume–temperature(PVT)diagram,asshowninFigure1.1.Thesurfacesrepresentthedifferentstates—solid,liquid,orvapor—thatcorrespondtoparticularvaluesofpressureandtemperature.Accordingtothephaserule, thetwo-phase(solid–liquid,solid–vapor,andliquid–vapor)regionsofapuresubstancehaveonlyonedegreeoffreedom.Therefore,theequilibriumpressureineachcaseisafunctionoftemperature.ThePTprojectionsofthesolid–liquid,solid–vapor,andliquid–vaporequilibriumlinesareshownontheleftofFigure1.1.Inparticular,thevapor–liquidlinerepresentsthevaporpressurecurvethatstartsatthetriplepoint(TP)ofsolid–liquid–vaporcoexistenceandendsatthecriticalpoint(CP).ThenatureoftheCPcanbeunderstoodfollowingthechangesofthefluidpropertiesalongthevaporpressurecurve.Withincreasingvaluesoftemperature,thedensityoftheliquidphasediminishesandthevapordensityincreasesduetothehighervaporpressure.Eventually,bothdensitiesconvergeat theCPanddifferentiating the liquidor thevaporstateisnolongerpossibleabovethecriticaltemperature.Whenbothtempera-tureandpressureareabovethecriticalvalues(Figure1.1),thesystemisconsideredtobeinthesupercriticalregion.
Withinaregionclosetothecriticalconditions,thesystempropertiesarehighlysensitivetopressureandtemperature;thisregionisconsiderednear-critical.Usually,theSCFsolventisappliedatatemperatureclosetoitscriticalvalueandatapres-surehighenoughforitsdensitytobecomegreaterthanthefluidcriticaldensity.A
Volume
Pc
Tc
Vc
Vapor
Solid
Liquid
sv
lv sl
slv TP
sl
lv
sv
Pres
sure
Tempera
ture
Supercritical Region
CP CP
FIgure �.� PVTdiagramofapuresubstanceanditsprojectiononthePTplane.
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Fundamentals of Supercritical Fluid Technology �
listoffluidsthathavebeenproposedasSCFsolventsisshowninTable1.1.Thesefluidscanbeclassifiedasa) low-critical temperature(low-Tc)andb)high-criticaltemperature(high-Tc)solvents.Somecondensablegases,likecarbondioxide(CO2),ethane, andpropane,areconsidered low-Tcsolvents,whereas thehigheralkanes,methanol, andwater can be considered high-Tc solvents. Strong differences insolventpowerandselectivitycharacterizethelow-Tcandhigh-Tcsolvents.
Francis[1]madeasignificantcontributiononthesubjectofCO2solventproper-tiesbystudyingitsbehaviorwithalargenumberofsolutes.LiquidCO2ismisciblewithalkanesuptoapproximatelycarbonnumber10,while therangeofmiscibil-ity increasesforethaneup to20,andpropaneup to35.Therefore, thesesolventsshowselectivityforrelativelylow-molecular-weightmaterial.StahlandQuirin[2]havereportedtheextractabilityofawiderangeofnaturalproductsusingCO2;theyshowedthat:“1)hydrocarbonsandotherlipophilicorganiccompoundsofrelativelylowmolecularmassandpolarityareeasilyextractable;2)theintroductionofpolarfunctionalgroups,hydroxylorcarboxylgroupsrendertheextractionmoredifficultorimpossible;3)sugarsandaminoacidscannotbeextracted;4)fractionationeffectsarepossibleiftherearemarkeddifferencesinmass,vaporpressureorpolarityoftheconstituentsofamixture.”
Regardingtheuseofhigh-Tcsolvents,suchastolueneorwater,theextractioniscarriedoutat temperaturesfrom500to700K,whereevenamildpyrolysisofhigh-molecular-weightmaterialtakesplace.Thesolventpowerofhigh-Tcfluidsismuchhigherthanthatoflow-Tcsolvents,andhigh-Tcsolventsarepropersolventsfor high molecular weight materials. However, they have low selectivity and thesevereoperatingconditions,ontheotherhand,degradethermallylabilematerials.Agoodfeatureoflow-Tcsolvents,ascomparedwithconventionalliquidsolvents,is that theyoperate atmoderate temperature andhave low solvent power.There-fore, by carefully choosing the pressure and temperature of operation, selectivefractionscanbeextractedfromvegetablematrices,suchasessentialoils,alkaloids,lipids, or oleoresins. These are the preferred solvents for the pharmaceutical andnatural-productindustries.Akeyadvantageoflow-Tcsolventsisthattheyareeasilyseparatedfromtheextract.
table �.�Critical properties of Fluids of Interest in supercritical processes
FluidCritical temperature
tc/KCritical pressure
pc/barCritical Volume Vc/cm�·mol–�
CO2 304.12 73.7 94.07
Ethane 305.3 48.7 145.5
Propane 369.8 42.5 200.0
Water 647.1 220.6 55.95
Ammonia 405.4 113.5 72.47
n-Hexane 507.5 30.2 368.0
Methanol 512.6 80.9 118.0
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� Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
SCF-soluteinteractionsintheliquidphasemayoriginateasecondliquidphase(gas salting out effect), improving process selectivity and making it possible, forinstance, to separate chemical reaction products in situ [3]. A better understand-ingofsupercriticalsolventpropertieswillbeobtainedafterconsideringthephaseequilibriumbehaviorofbinarysystemsthatshowadifferentdegreeofasymmetryinsizeorintermolecularinteractions.
1.2.1 Physical ProPerties of suPercritical fluids
ThephysicalpropertiesofSCFsarein-betweenthoseofagaseousandliquidstates.Typical values of different physical properties for each fluid state are listed inTable1.2.
DensityandviscosityofSCFsarelowerthanthoseofliquids;however,diffusivi-tiesarehigher.ThermalconductivitiesarerelativelyhighinthesupercriticalstateandhaveverylargevaluesneartheCPbecause,inprinciple,theheatcapacityofafluidtendstoinfinityattheCP.Interfacialtensionisclosetozerointhecriticalregion.Ingeneral,thephysicalpropertiesinthecriticalregionenhancemassandheattransferprocesses.
�.� phase equIlIbrIum wIth superCrItICal FluIds
1.3.1 solid solubilities
TheconditionsofphaseequilibriumbetweenaSCF(1)andasolidcomponent(2)areformulatedonthebasisoftheisofugacitycriterion.Ifthesolidphaseisassumedtobeapurecomponent(2),thesolubilityinthegasphasecanbedirectlyobtainedas:
y E
p
P
s
22= (1.1)
table �.�Comparison of the physical properties of gas, liquid, and supercritical Fluidsphysical property gas (tambient) sCF (tc, pc) liquid (tambient)
Densityr(kgm–3) 0.6–2 200–500 600–1600
Dynamicviscositym(mPa.s) 0.01–0.3 0.01–0.03 0.2–3
Kinematicviscosityha(106m2s–1) 5–500 0.2–0.1 0.1–5
Thermalconductivityλ(W/mK) 0.01–0.025 Maximumb 0.1–0.2
DiffusioncoefficientD(106m2s–1) 10–40 0.07 0.0002–0.002
Surfacetensionσ(dyn/cm2) — — 20–40
a Kinematicviscositydefinedasη = µ/ρb Thermalconductivitypresentsmaximumvaluesinthenear-criticalregion,highlydependent
ontemperature
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Fundamentals of Supercritical Fluid Technology �
whereEistheenhancementfactorovertheidealsolubilityand ps2 isthesublima-
tionpressureofthesolute(2).Foralow-volatility,incompressiblesolidsolute,theenhancementfactorcanbecalculatedasfollows:
E
P p vRT
S sol
=
−
exp
( )2 2
2Φ (1.2)
whereΦ2isthefugacitycoefficientofthesolidsoluteinthegasphaseandv2
solisthesolidmolarvolume.Φ2isstronglydependentontheSCFdensity.Figure1.2showstheregionofSCFextraction.Thisregionischaracterizedbyastrongvariationoffluiddensitywithpressure,attemperaturesclosetotheSCFcriticaltemperature.Foragivenisotherm,theincreaseinsolubilitycloselyfollowstheincreaseindensity,as indicated inFigure1.2.Thedrastic increase insolubility in thevicinityof thecriticalregioncanbeofseveralordersofmagnitudeandismainlyduetoasharpdecreaseofthesolutefugacitycoefficientΦ2inthefluidphase.Thisistheclassicalenhancementeffectatthenear-criticalregion.
Theinfluenceoftemperatureonthesolidsolubilityistheresultoftwocompet-ingeffects:theincreaseofsolidvolatilityandthedecreaseofsolventdensitywithtemperaturerise.Nearthecriticalpressure,theeffectoffluiddensityispredominant.Therefore, a moderate increase in temperature leads to a large decrease in fluiddensityandaconsequentreductioninsolutesolubility.However,athigherpressures,the increase of solid sublimation pressure with temperature exceeds the densityreductioneffect,andthesolubilityincreaseswithtemperature.Thisbehaviorleadstoaregionofretrogradebehaviorofthesolidsolubility,asillustratedinFigure1.3.AtpressureswellabovetheSCFcriticalpressure,theisothermsexhibitamaximumin solubility.Thismaximum is usuallyobserved in the rangeof30 to100MPa.
Pressure
Den
sity
(δ)
Near Critical Region
Solu
bilit
y (y 2
)
T1
T1 < Tc1 < T2
T2
Pc1
FIgure �.� Density(δ)andsolidsolubilityinfluidphase(y2)asafunctionofpressure.
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� Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
KurnikandReid [4]have shown that themaximum is achievedwhen thepartialmolarvolumeofthesoluteinthefluidphaseisequaltoitssolidmolarvolume.
Aquantitativecorrelationandpredictionof thesolubilityofapuresolid inaSCFispossibleifthefugacitycoefficientofthesolidinthefluidphaseiscomputedusinganequationofstate.Cubicequationsofstate,withconventionalmixingrulesandadjustablebinaryinteractionparameters,havebeenwidelyusedsincetheearlyworks of Deiters and Swaid [5] and Kurnik and Reid [4]. However, equations ofstate thatuseclassicalmixingrules,evenwithenergyandsizebinaryinteractionparameters, may fail to predict or correlate the solubility of solids with polar orhydrogen-bonding interactions.For instance,Kurnik andReid [4] found that thisapproachisnotabletomodelthesolubilityofstearicacidorn-octanolinCO2.Thelimitationsofcubicequationsofstatetomodelthesolubilityofpolarsolidscanbetackledbyusingcubicequationsofstatewithlocalcompositionmixingrules[6].
Whenanonpolarsupercriticalsolvent isused, theseparationprocessdoesnotpresent specific selectivities; in this case, the addition of a proper cosolvent canenhancesolubilityandselectivity.Nonpolarcosolventsincreasethesolubilityofsolidaromaticsseveraltimes,whereaspolarcosolventsenhancethesolubilityofsolutesthat present specific interactions with the cosolvent. For example, Brenecke andEckert[7]showedadramaticeffectofthecosolventtributilphosphateonthesolubil-ityofhydroquinoneinCO2.Thecosolventselectionfollowsthegeneralrulesappliedforclassicsolventselectioninsolidorliquid-liquidextraction.Brunner[8]studiedtheeffectsofcosolventsontheextractionoflow-volatilityliquidsandshowedthattheuseofacetoneormethanol,forinstance,improvesselectivityandsolventpowerintheextractionofhexadecanolfromoctadecane.
1.3.2 MultiPle fluid Phase equilibriuM
Equilibriumpredictionsinsystemshavingtwoormorefluidphasesaremorecom-plexthanthoseincasesofsolidsolubilitiesduetotheneedtocomputefugacities
T2
T1
Pressure
T1 > T2 > Tc1
y2
Pc1
Supercritical Region
FIgure �.� TypicalisothermsofsolidsolubilityinSCF.
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Fundamentals of Supercritical Fluid Technology �
inseveralphasesofdifferentcompositions.Theuseofthesameequationofstatetocomputefugacitycoefficientsinallphasesgivestherequiredcontinuityinthepre-dictionofphaseequilibriumatthecriticalregion.CubicequationsofstateofthevanderWaalsfamilyhavebeensuccessfullyappliedinthecorrelationandpredictionofphaseequilibria inmixturesofsubcriticalandsupercriticalnonpolarcomponentsinthenaturalgasandpetrochemicalindustries.However,theirapplicationtosizeand energy asymmetric systems, typical of the supercritical extraction of naturalsubtracts, has found little success. De la Fuente et al. [9] tried to correlate bothvapor-liquidequilibrium(VLE)andliquid-liquidequilibrium(LLE)ofthesystemsunfloweroil+propaneusingtheSoave[10]equationofstatewithquadraticmixingrulesandbinaryinteractionparametersforboth,theattractiveenergyparameterandthecovolume.ItwasnotpossibletoquantitativelydescribebothVLEorLLEusingonlyonesetofparametersfortheattractiveenergyparameterandthecovolume.ThisindicatesthelimitationsofthevanderWaalsrepulsivetermtodescribetheseasym-metricmixtures.Thefailureofcubicequationsofstatetomodelphaseequilibriainsizeasymmetricmixturescanbeattributedtothelargedifferencesinthepure-componentcovolumes[11].
Espinosaetal.[12]andFerreiraetal.[13]extensivelydiscussedtheapplicationofequationsofstatetomodelthesupercriticalprocessingofnaturalproducts.Agroupcontribution approach is particularly useful when dealing with natural productsbecausealargenumberofcompounds,suchastriglycerides,fattyacids,esters,andalcohols,canberepresentedwithasmallnumberoffunctionalgroups.Groupcon-tributionequationsofstate,suchasModifiedHuron-Vidal2(MHV2)[14,15]andgroupcontributionequationofstate (GC-EOS)[16,17],areparticularlyuseful tomodelthecomplexphasebehaviorobservedinasymmetricmixturesatnear-criticalconditions.Bottinietal.[18]extendedtheGC-EOSmodeltodescribebothVLEandLLEinmixturesofsupercriticalgases+vegetableoilmixturesusingthesamesetofparameters.Grosetal.[19]andFerreiraetal.[20]extendedthismodeltorepresentassociatingmixtures(GCA-EOS),usingagroupcontributionapproachfordealingwith self- andcross-associations.TheGCA-EOSequationcanbederived fromathree-term(repulsive,attractive,andassociating)Helmholtzresidualenergy:
A=Arep+Aatt+Aassoc (1.3)
The repulsive (rep) term is givenby theCarnahan-Starling equation for hardspheres, the attractive (att) term is a group contribution version of a density-dependent local composition Non-Random Two Liquids (NRTL) model, and theassociation (assoc) term is a group contribution expression based on Wertheim’sstatisticalassociationfluidtheory[21].ThehardspheretermperformsbetterthanthevanderWaalsrepulsivetermwhendealingwithhighlysize-asymmetricsystemsandtheothertwotermsareabletohandlestrongnonidealspecificinteractions.TheGC-EOSmodelwascompared toMHV2andPSRK[22] byEspinosaetal. [23].Allthreemodelsperformsimilarlyformoderatelypolarsystemsoflowmolecularweightcompounds.However,theMHV2andPSRKmodelspresentsomelimitationswhentheyareappliedtoveryasymmetricsystems.
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� Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
�.� phase equIlIbrIum engIneerIng oF superCrItICal proCesses
Phase equilibrium engineering is the systematic application of phase equilibriumknowledgetoprocessdevelopment.Thisknowledgecomprisesdatabanks,experi-mentaldata,phenomenologicalphasebehavior,thermodynamicanalysis,andmath-ematical modeling procedures for phase equilibrium process calculations. EachSCFapplicationhasasetofspecificationsandphysicalrestrictions.Insupercriticalreactions,forinstance,homogeneousphaseconditionsmayberequiredatthereactiontemperature.Thesolutiontothisproblemisgivenbytheselectionofthepropersolventandthedeterminationofafeasibleoperatingpressurerangeandfeedcompositiontoachieve homogeneity in the reaction mixture. On the other hand, aheterogeneoustwo-phasesystemmayberequiredtodevelopsupercriticalextractionorfractionationprocesses.Additionalphaseequilibriumrestrictionsmayincludenosolidphasepre-cipitation,azeotropeformation,specificsolventsolubilities,orsaturationconditions.
Amulticomponentfluidcanbeasupercriticalmixture,asubcooledliquid,asuper-heated vapor, or a heterogeneous liquid-liquid, liquid-vapor, or liquid-liquid-vapormixture.Ausefulplottoidentifyeachregionisapressurevs.temperaturediagramshowingthebubbleanddewpointphasetransitionscurves,aswellastheCPofagivenglobalcomposition.Theselinesdeterminethemixturephaseenvelope.Differentphasescenarioscanbeselectedfromthisphaseenvelope(Figure1.4):a)homogeneouscon-ditionsforasupercriticalreaction,b)homogenousandheterogeneousconditionsforatunablephasesplitreactor,orc)phasesplitforaseparationprocess.Certainly,differentphaseenvelopesareobtainedduringthecourseofthereactionorseparationprocess.However,theprocesstrajectoryshouldalwaysremainattherequiredphasescenario.Generalconditionscanalsobesetfromthisplot;forinstance,abovethemaximumpressureofthephaseenvelopetherewillbeasinglephaseatanytemperature.
Rigoroussimulationsofequilibriumstageseparationsatnear-criticalconditionsare needed for the design and optimization of supercritical processes. However,equilibriumcalculationsinthenear-criticalregioncanpresentseriousconvergence
Sometimes We Look for Bothb) Tunable Reactors
Sometimes We Look for Phase Splitc) Separation Processes
Temperature
Pres
sure
Sometimes We Look for Homogeneitya) Supercritical Reactions
Heterogenous RegionBubble P
oint C
urve
Liquid
Vapor
CriticalPoint
Dew Point Curve
FIgure �.� Possibleprocessphasescenarios.
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Fundamentals of Supercritical Fluid Technology �
difficulties.Inthatrespect,Michelsen’s[24]phasestabilitycriterion,multiple-phaseflashalgorithms,andglobalphasecomputationsareofparticularinterestforsuper-criticalextractionapplications.
Solventrecycleisamajorissueintheeconomicoptimizationoftheseprocesses,becauseitisthemainfactorindeterminingcapitalandoperatingcosts.Designandsynthesis problems have been increasingly solved by formulating mathematicalmodels,whichinvolvecontinuousandintegervariablestorepresentoperatingcon-ditionsandalternativeprocesstopologies[25].Withregardtosupercriticalextrac-tions,Grosetal. [26]haveaddressed thesynthesisofoptimumprocesses for theextractionanddehydrationofoxychemicalsasamixedintegernonlinearprogram-mingproblem.Espinosaetal.[23]andDiazetal.[27]haveappliedtheseproceduresforthesynthesisandoptimizationofcitrusoildeterpenationprocesses.
1.4.1 understanding Phase behavior
VanKonynenburgandScott[28]haveshownthatthefluidphasebehaviorobservedinbinarymixturescanbeclassifiedintofivemaintypes.IntypeIphasebehavior,com-pleteliquidmiscibilityisobservedatalltemperatures.Whenpartialliquidmiscibilityoccursatlowtemperatures,thesystemisoftypeII.TypeIphasebehaviorisusuallyfoundinsystemswithcomponentsofsimilarchemicalnatureandmolecularsize,likemixturesofhydrocarbons,noblegases,orsystemsthatdonotdeviategreatlyfromidealbehavior.TypeII is typicalofnonidealmixturesofsimilarsizecompounds,inwhichnonidealityleadstoliquidphasesplitatsubcriticalconditions.Whentheliquidimmiscibilitypersistsevenathighpressuresandtemperatures, thesystemsareoftypeIII.Thisbehaviorischaracteristic,forexample,ofmixturesofCO2withhigh-molecular-weight alkanes or vegetable oils.When the difference in molecu-larsizebecomessignificant,inalmostidealsystems,liquid-liquidimmiscibilityisobservednearthelight-componentcriticaltemperature(solventTcinsupercriticalprocesses).However,completemiscibilityisrecoveredatlowertemperatures; thiscorrespondstotypeVphasebehavior.TypeIV,ontheotherhand,showsdiscon-tinuedliquid-liquidimmiscibility(i.e.,liquidimmiscibilityoccursatlowandhightemperaturesbutnotatintermediatetemperatures).Figure1.5isamasterchartofthedifferent typesofbinaryfluidphasediagrams [29].Thearrows inFigure1.5qualitativelyindicatethetypeoffluidphasebehaviorthatcanbeexpectedwhenthesystemcomponentsexhibitgreatermolecularinteractions,sizedifferences,orboth.
Figure1.6illustrates,inmoredetail,aTypeVphasediagram.Thelinesinthisdiagram indicate the boundaries of phase transitions and the critical locus. Thethree-phaseequilibriumline(l1l2g)startsatthelowercriticalendpoint(LCEP)andfinishesattheuppercriticalendpoint(UCEP).Thisbehavioristypicalofmixturesofpropanewith triglycerides,suchassunfloweroilor tripalmitin [30].When theprocess operating temperatures are above the critical temperature of the solvent,pressuresshouldbehigherthanthecriticalpressureofthemixtureinordertoensurecompletemiscibility(i.e.,thepressureshouldbeabovethel1=l2line).
In the search for an adequate supercritical solvent to achievehomogenous orheterogeneousconditions,twodifferentapproachescanbefollowed:1)tocomparethephasebehaviorofagivensubstratewithdifferentsolventsor2) to followthe
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�0 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
change in thephasebehaviorofagivensolventwithdifferent familiesofchemi-calcompounds.Inthemoregeneralcase,whenthecomponentsofthemixtureareofdifferentchemicalnature, thesecondapproachshouldbefollowedtotakeintoaccountanypossiblechangeinthephasebehaviorduringprocessevolution.
Theliquid-liquidimmiscibilityoftypeVphasebehaviorappearsinmanybinarymixtures between supercritical solvents and organic substrates beyond a certaincarbon number. Figure1.7 shows the regions of liquid-liquid immiscibility forbinarymixturesofsupercriticalsolvents(ethaneandpropane)withhydrocarbonsofdifferentchainlength[31].Peters[31]alsopresentedsimilardataontheliquid-liquid
T
P
lg(2)
lg(1)
l1 = l2
l1 = g
l1l2g
l2 = g
UCEP
LCEP
FIgure �.� TypeVphasebehavioraccordingtoVanKonynenburgandScottclassification.
CL
CL
CL
CL
CL
CH
CH
CH
CH
CH
T
T T
P
P P
P Type II
Type IV Type I
T
Type V
Type III
Molecular Interaction
Molecular Interaction
Molecular Interaction
+ Size
Size
Size
Pure Component Vapor Pressure Critical Locus �ree Phase Region (LLV)
P T
Molecular Interaction
+ Size
FIgure �.� Modificationsofbinaryphasebehaviorwith sizeandenergyasymmetries.CLandCHarethecriticalpointsofthelightandheavycompounds,respectively.
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Fundamentals of Supercritical Fluid Technology ��
immiscibilitydomainsofthesystemsethane+alcohols,ethane+aromatichydro-carbons,andethane+alkanes.Itbecomesclearfromthesedatathatethaneisnotanadequatesupercriticalsolventfornormalalcoholsbecauseitpresentsliquid-liquidimmiscibilityevenwithmethanol.However,ethaneseemstobeabettersolventforaromatichydrocarbonsorparaffinsbecausetheliquid-liquidimmiscibilityappearsatcarbonnumbersgreaterthan15or18,respectively.
CO2 has been the most studied solvent for supercritical processes. However, itexhibitsstrongliquid-liquidandgas-liquidimmiscibilityforhydrocarbonswithcarbonnumbersgreaterthan13.Inaddition,CO2presentsaratherlowcriticaltemperaturetobeusedas a solvent for reactionscarriedout atmoderateorhigh temperatures.Figure1.8showsdataonthetypeofphasetransitionforthefamiliesofCO2+alkanescompiledbyPeters[32],whoalsoshowedthebehaviorofCO2+alkanolsystems.
Unfortunately,thetypeofdatashowninFigures1.7and1.8isonlyknownforalimitednumberoffamiliesoforganiccompoundswithsomesupercriticalsolvents.Therefore,reliablethermodynamicmodelsareneededtoexplorethepossiblephasescenariosfoundinmixturesbetweenprocesscomponentsandsupercriticalsolvents.
The phase equilibrium engineering approach will be illustrated with severalexamples,where thermodynamicandmodeling toolsareapplied for supercriticalprocessdevelopment.Theexamplestobecoveredarealcoholextractionanddehy-dration,gasantisolventcrystallization,purificationofvegetableoils, supercriticalfractionation,extractionwithnearcriticalfluids,andsupercriticalreactions.
�.� ConCeptual superCrItICal proCess desIgn
1.5.1 oxycheMical extraction and dehydration
Thesupercriticalextractionoforganicoxygenatedcompoundsfromaqueoussolu-tionsisofgreatinterestinbiotechnologicalprocesses.Oxygenatedcompoundsand
280
300
320
340
360
380
400
10 15 20 25 30 35 40 45 50 55 60 65 70
UCEP
UCEP
LCEP
LCEP
Ethane
Propane
SL1L2V
SL1L2V
Number of Carbon Atoms
Tem
pera
ture
, (K)
FIgure �.� Phasetransitionsforthebinariesofethaneandpropanewithparaffinsofdif-ferentchainlength.UCEPandLCEPpointsareupperandlowercriticalendpoints,respec-tively.SL1L2Vstandsforsolid–liquid–liquid–vaporequilibria.
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�� Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
water have strong hydrogen bonding interactions that complicate their separationwithconventionalsolvents.Moreover,anoxygenatedcompounddissolvedinanon-
polarnear-criticalsolventwillhavearatherhighactivitycoefficient( γ oxySCF),leading
toalowvalueofthedistributioncoefficient:
moxyoxyH O
oxySCF
=γγ
2
(1.4)
This is even more pronounced in the case of alcohols or acids that exhibit self-association.AstrategytoovercomethisproblemmaybebasedontheKoenenandGaube [33] diagram that classifies binary mixtures in an excess Gibbs function(GE)versusexcessenthalpy(HE)diagram(Figure1.9).Wecanderivetheeffectoftemperature on the activity coefficients directly from this diagram. The aqueoussolutionsoforganicoxygenatedcompoundsarelocatedonthesecondquadrantofthediagramwithnegativeHEvalues,whereasthesupercriticalsolutionsthatcorrespondtopositiveHEvaluesarelocatedonthefirstquadrant.Inbothcases,therearepositivedeviationstononideality(positiveGE).Fromthisdiagram,wecanseethattheactiv-itycoefficientsintheaqueousphaseincreasewithtemperature;however,thereverseoccurswiththeactivitycoefficientsintheSCFphase.Therefore,extractingathightemperatures leads to more attractive values of the distribution coefficients. ThisbehaviorisfoundintheextractionofisopropanolorethanolfromaqueoussolutionsusingCO2,ethane,orpropaneasnear-criticalsolvents.However,weshouldconsideranotherfacttomakeapropersolventselection:Atoptimumextractiontemperatures(around380 to400K), the solvent powerofCO2or ethane is drastically reducedduetofluiddensitydecreaseattemperatureswellabovethecriticaltemperatureofbothfluids(around304K).Torecoverthesolventpower,relativelyhighpressuresshouldbeusedfortheextractionprocess.Thismakespropaneabettercandidateas
200
220
240
260
280
300
320
340
5 7 9 11 13 15 17 19 21 23 25
Type II Type VType IV
UCEP
LCEP
Number of Carbon Atoms
Tem
pera
ture
, (K)
FIgure �.� PhasetransitionsforthebinariesofCO2withparaffinsofdifferentchainlength.UCEPandLCEPpointsareupperandlowercriticalendpoints,respectively.Dashedline(opensquares):SL1L2Vequilibria.
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Fundamentals of Supercritical Fluid Technology ��
anextractionsolventbecauseitscriticaltemperatureiscloseto370KandithasalowercriticalpressurethanCO2orethane.Horizoeetal.[34]andBrignoleetal.[35]
verifiedthepotentialofpropaneasanextractingsupercriticalsolvent.Dehydrationbynear-critical solventsfinds importantapplications, forexample,
intheextractionofsolutesfromaqueoussolutionsandinthedryingofsolidparticlesaftermicronization.Wewillconsiderfirstthedehydrationofextractedsolutes.Inlow-pressure separations, entrainment agents like cyclohexane or solvents like ethyleneglycolhavebeenusedtoseparatewaterbyazeotropicorextractivedistillations.Incon-nectionwithsupercriticalprocesses,itisofinteresttostudytheequilibriumbetweenwaterandanear-criticalfluidasafunctionoftemperatureandpressure.InthecaseofCO2,thedataofWiebbe[36]andCoanetal.[37]showthesolubilityofwaterinCO2asafunctionofpressureatsubcriticalandsupercriticaltemperatures.Thesedataindicatethatwaterfollowstheclassicalsupercriticaleffect:theconcentrationofwaterintheCO2phaseincreasesoncethesupercriticalpressureisexceeded(Figure1.10).
AttheCO2saturationpressure,atsubcriticalconditions,wewouldhaveathree-phase VLL equilibrium condition, where the concentration of water in the con-densedCO2phaseexceedstheconcentrationofwaterinthevaporphase.Hence,inaCO2–waterseparationprocess,therelativevolatilityofwaterwithrespecttoCO2islowerthanone.ThisbehaviorhasimportantconsequencesfortheseparationofwaterfromCO2extracts.Water,asexpected,islessvolatilethanCO2;therefore,theextractcannotbeobtainedfreefromwaterinthesolventrecoveryoperation.
Whenthesamephaseequilibriaanalysisismadeforwaterandlightalkanes,suchasethaneandpropane,adifferentpictureisobtained.ThedataofKobayashiandKatz[38]forthesolubilityofwaterinpropaneareplottedagainstpressureatdifferenttemperatures(Figure1.11).Fornear-criticalpropane,thesolubilityofwaterdecreaseswhenthecriticalpressureisexceeded(seeTable1.1forthecriticalproper-tiesofpropane).Thisphenomenoncanbecalledanonclassical supercritical effect.
< 0
GE
HE
γ > 1
Regular SolutionSE= 0
> 0δγδT
γ < 1
> 0δγδT
γ < 1
< 0δγδT
γ > 1
< 0δγδT
FIgure �.� Value and temperature derivative of activity coefficients, according to therelativevaluesofGEandHE.
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�� Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
Averyattractivepropertycanbederivedfromthiseffect.Whenworkingatsubcriti-caltemperatures,atthepropanesaturationpressure,weagainhaveVLLequilibria.Inthiscase,thecompositionofwaterinthevaporphaseisgreaterthanthatinthecondensedpropanephase,leadingtoawater-propanerelativevolatilitygreaterthanone.Thismakesitpossibletoobtaindehydratedorganicoxygenatedproductsduringtheprocessofsolventrecoveryfromtheextract[39].
0
0.002
0.004
0.006
0.008
0.01
0.012
0 200 400 600 800Pressure, (bar)
Gra
ms o
f Wat
er P
er L
iter o
f Exp
ande
d G
as at
s.t.p
.
298 K323 K348 K
FIgure �.�0 CompositionoftheCO2-richphaseasafunctionofpressureandtempera-ture.ExperimentaldatafromWiebbe[34].
0.1
1
10
100
0 20 40 60 80 100P(bar)
Mol
e Fra
ctio
n %
of W
ater
327.6 K
377.6 K
369.6 K
FIgure �.�� Experimental water composition in liquid and vapor propane. Data fromKobayashiandKatz[38].
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Fundamentals of Supercritical Fluid Technology ��
On thebasisof thephase equilibriumengineering concepts presented above,a process for the production of bioethanol or for the dehydration of isopropanolwithanear-criticalsolvent(propane)canbedeveloped.Thekeyfeaturesoftheseprocessesare:
a) Hightemperaturesandpressuresofextractionfavorthesolubilityofalcoholinpropane.
b) Liquid-liquidequilibriumatlowtemperaturesisbeneficialforreducingthewatercontentintheextract.
c) Thealcoholproductisobtaineddehydratedbecausetherelativevolatilityofwaterwithrespecttopropaneisgreaterthanoneoveracertainconcen-trationrangeofethanolintheextractmixture.
All these properties were first predicted by group-contribution thermodynamicmodelingandthereafterverifiedbyexperimentalandpilotplantinformation.
1.5.2 Particle Micronization with suPercritical fluids
Supercriticalmicronizationprocessesarebasedoncreatingahighdegreeofsolutionsupersaturationthatleadstotheformationofagreatnumberofnucleationsitesandverysmallcrystals.Theseprocesseshavefoundmanyapplicationsinthelastdecade[40,41],mainlyinthemicronizationofpharmaceuticalsolidcompounds.Usually,several components may participate in the process: the solute to be crystallized,thesolvent,a supercriticalfluid,andacosolvent.Thephaseequilibriumbetweenthesecomponentsplaysakeyroleintheselectionofthepropertechnologyforthemicronizationprocesses.Abetterunderstandingofprocessselectioncanbemadeon thebasis of thebinaries behavior.First,we shall consider the solute+ super-criticalfluidbinary. If the solute solubilityunder supercriticalconditions ishigh,thenonlythesecomponentsparticipateintheprocessandmicronizationisobtaineddirectlybyadrasticreductioninthesolutesolubilitybytherapidexpansionofthesupercriticalsolution(RESSprocess)throughanozzleorotherconvenientdevice.ThemainlimitationofthisRESSprocessisthatitcanonlybeappliedtosoluteswithhighsolubilitiesinthesupercriticalfluid.ThelowsolventpowerofsupercriticalCO2forpolarormedium-tohigh-molecular-weightmaterialmakesthisapproachuneconomicalforthesemixtures.
Whenthesolutecannotbedissolvedinsignificantamountsinthesupercriticalfluid,wecanlookforagoodliquidsolventforboththesoluteandthesupercriticalgas.Inthiscase,aconcentratedsolutionofthesoluteinthesolventispreparedandahighdegreeof supersaturation isobtainedbydissolving the supercriticalfluidintheliquidphaseathighpressure.Thistechnologyiscalledthegasantisolvent(GAS)processanditcanbecarriedoutinabatchorsemicontinuousprocess.Theseprocessescanbeappliedtoavarietyofsolutes,butinthiscase,theternaryphaseequilibria shouldalsobeevaluated toassureahighdegreeof supersaturationattheoperatingpressureand temperature. In the semicontinuousprocess,both thesolutionandthesupercriticalfluidentertogetherintheprecipitationvesselthrough
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�� Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
amixingdevice.Verygoodprecipitationconditionsareachievediftheoperatingconditions are above the CP of the solvent + supercritical fluid mixture. Undertheseconditions,bothfeedsarecompletelymiscibleandnointerfacialresistanceisofferedtomasstransfer[42].
AnotherpossiblephasescenarioappearswhenthesolidsoluteisnotsolubleintheSCF,butthesolubilityoftheSCFinthemeltedsolidishighatelevatedpres-sures.Therefore,ifthesolutionisexpandedtoatmosphericpressure,alargecoolingeffectoccursthatgivesrisetotheprecipitationofmicronizedsoluteparticles.
Adifferentsituationariseswithsolutesthatareonlysolubleinwater,suchassomeorganicsaltsandproteins[41].TypicalnonpolarsupercriticalfluidslikeCO2andethanearenotsolubleinaqueoussolutions,evenathighpressures.Therefore,noantisolventeffectcanbeobtainedinatypicalGASprocess[43].Inthiscase,acosolventthatshowscompletemiscibilitywithboththeSCFandwatercanbeintro-duced.Forexample,ethanolwasusedasapropercosolventfortheprecipitationofanorganicsaltfromaqueoussolution[43].Inthisapplication,theaqueoussolutionisfedasasprayormistintoaprecipitationvesselalreadyfilledupwithamixtureofethanol+CO2attherequiredcomposition.Toobtainafeasibleprocess,theoperat-ingconditionsoftheprecipitationchambershouldlieinsidethehomogeneousregionofthetriangularphasediagramforwater+CO2+ethanolatagivenpressureandtemperature,asshowninFigure1.12.Inthisway,thefinewaterdropletsbecomequicklysupersaturatedbytheethanol+CO2dissolutioninthedropsandthesimulta-neousfastevaporationofwater.Asaresultofthisprocess,highlymicronizeddriedsaltparticlesareobtained[43].Alltheseexamplesillustratethataphaseequilibriumengineeringanalysisisaprerequisiteforpropertechnologyselectionandsuccessfuladequatechoiceofmicronizationoperatingconditions.
CO2
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0
Ethanol
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Water + Lobenzarit
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
Feasible Region
A
B
FIgure �.�� Feasible operating region for Lobenzarit precipitation using supercriticalCO2andethanolascosolvents.
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Fundamentals of Supercritical Fluid Technology ��
1.5.3 extraction, Purification, or fractionation of natural Products with suPercritical fluids
�.�.�.� Fractionation of oils
Intheprocessingofvegetableoils,itispossibletotakeadvantageofthelowsolu-bilityof triglycerides inCO2.For instance,bothpalmoilandsunfloweroilgiveliquid-liquidorliquid-SCFimmiscibilitywithCO2,evenathighpressures,typicaloftypeIIIsystems.Inthesecases,eithersupercriticalorliquidCO2canbeusedasasolventtoremoveundesirablecomponentsfromtheoil—forinstance,removalofoleicacidfromoliveoil[44].Likewise,liquidornear-criticalCO2canbeappliedtorecovervaluablecomponentsliketocopherolsorsqualenefromfishoil[45].Whendealingwiththeseseparationprocesses,itispossibletofindoptimumextractionoperatingconditions thatminimize the solvent-feed ratio and, at the same time,keep the coextraction of oil at a low value. Other solvents that have regions ofliquid-liquidimmiscibilitywithfattyoils,suchasethaneandpropane,maybeusedasalternativesolvents.
SCFsolventscanalsobeusedasfractionatingagents.Thisisofinterestintheseparationof low-volatilesubstancesofcloserelativevolatility.Forinstance,CO2andethanehavebeenproposedasdensegasextractantstoremovetheterpenefrac-tionfromcitrusessentialoils[27]andalsoforthefractionationofhighlyunsaturatedfishoilmethylesterstoobtainricheicosapentaenoicacidanddocosahexaenoicacidfractions[46].ThebinarysystemsbetweenCO2andthesefamiliesofcompoundsare generally of type II, so complete miscibility for all compositions is obtainedabovethemaximumpressureofthevapor-liquidcriticallocus.
Asingledense-gasfractionationcolumnschemeisshowninFigure1.13.Themixturetobefractionatedisfedatanintermediatepointinthecolumn.Adensegas
N=40 N=40 N=40
Feed
Separator
CO2
Heat Exchanger
Extractor
Fresh CO2
Raffinate
N=40
Extract
FIgure �.�� Dense-gasfractionationschemeprocess.
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�� Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
(CO2,forexample)isintroducedatthebottomofthecolumnanditflowscountercur-rentlytotheliquidmixturetobeseparatedorenriched.Atthetopofthecolumn,theextractphaseisheatedandexpandedtoalowerpressuretorecoverthelightfractionandCO2isrecycledtothebottomofthecolumn.Acompressororcondenser-pumpcyclecanbeselectedforthispurpose.Thesetypesofseparationprocessesfollowtheprinciplesofastrippingoperation.Oneofthemaindifferenceswithordinarygasstrippingisthatthedensegasisverysolubleinthefeed.Therefore,theliquidphaseflowrateinthecolumnismuchlargerthanthefeedflowrate.Ontheotherhand,thelowvolatilityofthesubstratesbeingfractionatedleadstoarelativelyhighgas-feedstrippingratio.Botheffectscontributetogiveafairlyconstantmolaroverflowforbothphasesinasimplecounter-currentcolumn.Thedesignoftheseseparationprocessesishighlydependentontherelativevolatilitybetweenthekeycomponentsof theoils ineachseparationstage. Itcanbeshown thatasimplecountercurrentseparationislimitedbytherecoveryofeachkeycomponentinthebottomandtopproducts.Inthiscase,thelimitingrecoveriesofthekeycomponents(φ1, φ2)inthetopandbottomproductsaredeterminedbytherelativevolatility(α12)betweenbothcomponentsunderprocessconditions:
α φ φ12 1 21= −/ ( ) (1.5)
In most simple countercurrent extraction columns, this constraint limits therecoveryandpurityoftheproductsintheseparationofcomponentsofcloserelativevolatility.Therefore,theuseofrecycle(reflux)ofthetopproductisrequired:1)toincreaserecoveryandpurityand2) toassure that the trajectoryof theseparationprocessliesinsidethetwo-phaseregion.Thus,thecolumnandseparatoroperatingconditions(pressure,temperature,andcompositions)shouldalwaysbecheckedinordertoverifyaheterogeneousoperation.
�.�.�.� extraction from Vegetable matrices
The extraction of lipids and oils from vegetable matrices has been extensivelycoveredinthemonographeditedbyKingandList[47].Intheextractionoffattyoilsfromgroundedseeds,itisadvantageoustoselectasolventthatpresentscompletemiscibilitywiththeoil.CO2isacheap,nontoxicsolvent;however,theoilsolubilityinthisSCFisverylowevenatpressuresoftheorderof30MPa(typeIIIbinary).Ontheotherhand,liquidpropaneiscompletelymisciblewithvegetableoilsbelowtheLCEPofthisbinary.PropanehastypeIIortypeIVglobalphasediagramswithvegetableoils.Themaindrawbackofusingpropaneasasolventfortheextractionofoilsfromgroundedseedsisthatitisflammable.Recently,Hegeletal.[48]studiedtheuseofpropane+CO2solventmixturesforoilextraction, lookingforefficientandsafesolventmixtures.Peter[45]hasstudiedthesetypesofmixturestoimproveselectivitiesintheseparationoflecithinfromvegetableoils.IntheworkofHegeletal.[48],theselectedphasescenariowastooperateinaregionofcompleteliquidmiscibilityoftheoil+solventmixture,withanonflammablevaporphase.Theselec-tionofoperatingconditionswasbasedonexperimentaldataontheLLVregionatconstant temperature, for the systemsunfloweroil+propane+CO2.Atconstant
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Fundamentals of Supercritical Fluid Technology ��
temperature,forathree-componentsystem,theLLVequilibriumisonlyafunctionofpressure.Therefore,abinodalcurvecanbedrawnonatriangulardiagram,withtielineslinkingthetwoliquidphasecompositionsatspecifiedpressures(seetrian-gulardiagramonFigure1.14).Thebinodalcurvegives theboundaryof theLLVregion.ThediagramalsoshowstheminimumpressureforwhichLLimmiscibilityarisesatagiventemperature.Atlowerpressures(i.e.,lowerCO2composition),thesolvent has complete miscibility with the oil. However, there is also a minimumoperatingpressure toavoidvaporphaseflammabilitybecause, atpressures lowerthanthis,thepropanecontentofthevaporphaseistoohigh.ThefeasibleoperatingregioncanbeeasilydeterminedwiththehelpofFigure1.14.
1.5.4 suPercritical reactions
In general, gas-liquid catalyzed reactions are diffusion controlled. The use of anadequate supercritical SCF can bring the reactive mixture into homogeneous
0
10
20
30
40
50
60
70
80
0 0.2 0.4 0.6 0.8 1.0
0 0.2 0.4 0.6 0.8 1.0
CO2 Weight Fraction
CO2 Weight Fraction
Pres
sure
, (ba
r)
00.10.20.30.40.50.60.70.80.91.0
Prop
ane W
eigh
t Fra
ctio
n
Minimum CO2Content in theGaseous Phase
Liquid-Liquid-Vapor Region
Oil
CO2 + Propane at 308 K
CO2 + Oil + Propane at 308 K
MaximumOperating Pressure
MinimumOperating Pressure
Safe Operating Region
FIgure �.�� Safe operating extraction region at 308 K. Experimental data from Hegeletal.[48].
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�0 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
conditions,withtheconsequentreductionofthemasstransferresistancebyelimi-natingthegas-liquidinterfaceandbyincreasingthediffusivityofreactants.There-fore,thereactionrateandselectivitycanbegreatlyincreased.Härrödetal.[49]havestudiedexperimentallythehydrogenationofheavysubstratessuchasvegetableoilsandfattyestersundersupercriticalconditions.
The use of batch reactors is a common practice in bench scale experimentalstudiesonsupercriticalreactions.However,thecontrolofhomogeneousconditionsinthesereactorsisquitedifficult.Baikerandcoworkers[50]recommendtheuseofwindowsinthereactionvesselsinordertocontrolthephaseconditions.Eventhoughitispossibletohaveanindependentcontrolofprocessvariablesincontinuousreac-tors, the selection of pressure, temperature, and composition should be carefullydonetoobtainthedesiredhomogeneousstate.Knowledgeofthephasebehaviorofareactionprocesscanhelptounderstandtheresultsofexperimentalstudiesandtoplananddesignexperimentalruns.
Thesolvent tobeused ina supercritical reactionshouldbenonreactiveunderprocess conditions. The critical temperature of the solvent should be lower thanthe reaction temperature toassurecompletemiscibilityofallgaseous reactants inthesupercriticalsolvent.However,thecriticaltemperatureshouldnotbefarfromthereactiontemperaturetomaintainthefavorablepropertiesofthenear-criticalstate.
Toshowtheimportanceofmakinganadequatephaseequilibriumengineeringanalysis,weselectasupercritical reactioncarriedoutbyChouchietal. [51]asanexample.Chouchietal.havestudiedthehydrogenationofα-pineneundersupercriticalCO2 in abatch reactoroperatingat323Kand14MPawithaPd/Ccatalyst.Theauthorsshowedthatthereactionrateandconversionarelowwhenthereactoroper-atesunderhomogenousconditions.Onthecontrary,betterconversionswereachievedwhentheCO2pressurewasreduced,althoughthesystembecameheterogeneous.Aphaseequilibriumengineeringanalysisofthereactoroperatingconditionscangiveanexplanationtotheseseeminglycontradictoryresults.Thebatchreactorwasfirstfedwiththecatalyst,togetherwithaknownamountofα-pinene.Then,thesystemwaspressurizedwithCO2uptothedesiredpressure(8,9,10,or12MPa),and,finally,H2wasfeduntilatotalpressureof14MPawasreached.Theactualmolarcompositionofthereactingmixturewasunknown.Thiscompositionmaybeobtainedbyusingan equationof state suitable for densitypredictionsunder the reaction conditions.OnepossibilityistousetheMHV2[15,48]equationofstate.Thecomputationoftheactualmixturecompositionsrequiresaniterativeprocedureforestimatingthesystemcompressibilityfactor,theamountsofeachcomponentchargedintothecell,andtheevolutionofthereactorcompositionwithconversion.Thisanalysisindicatesthat,atthehigherCO2partialpressure,an important reduction inhydrogenconcentrationoccurs,whichislikelythereasonfortheobserveddecreaseinthereactionrates.
Phaseequilibriumengineeringanalysisofsupercriticalprocessesisoftheutmostimportance in developing new technologies that replace conventional solvents byhigh-pressuregasestoobtainenvironmentallyfriendlychemicalprocesses.Severalexamplesofprocessdevelopmentclearlydemonstratethatagoodunderstandingofphasebehaviorandapplicationofrigorousmodelingtoolsareessentialtoprocesssynthesesinwhichthefluidpropertiesareextremelydependentonpressure,tem-perature,andcomposition.
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Fundamentals of Supercritical Fluid Technology ��
reFerenCes
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recentresults,Fluid Phase Equilibria,8,93–105,1983. 3. Eckert,C.A.andChandler,K.,Tuningfluidsolventsforchemicalreaction,J. Supercrit.
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13. Ferreira,O.,Modelling of association effects by group contribution: Application to natural products,Ph.D.Thesis,Univ.dePorto,Portugal,2003.
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20. Ferreira,O.,Brignole,E.A.andMacedo,E.A.,Modelingofphaseequilibriaforasso-ciatingmixturesusinganequationofstate,J. Chem. Thermodynamics,36,1105–1117,2004.
21. Chapman,W.G.,Gubbins,K.E.,Jackson,G.andRadosz,M.,Newreferenceequationofstateforassociatingliquids,Ind. Eng. Chem. Res.,29,1709–1721,1990.
22. Holderbaum, T. and Gmehling, J., PSRK: A Group Contribution Equation of StateBasedonUNIFAC,Fluid Phase Equilibria,70,251–270,1991.
23. Espinosa,S.,Foco,G.,Bermudez,A.andFornari,T.,Revisionandextensionof thegroupcontributionequationofstatetonewsolventgroupsandhighermolecularweightalkanes,Fluid Phase Equilibria,172,129–143,2000.
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24. Michelsen,M.L.,Calculationofphaseenvelopesandcriticalpointsformulticompo-nentmixtures,Fluid Phase Equilibria,4,1–10,1980.
25. Kravanja,Z.andGrossmann,I.E.,Multilevel-hierarchicalMINLPsynthesisofprocessflowsheets,Comput. & Chem. Eng.,21,S421–S426,1997.
26. Gros,H.P.,Díaz,S.andBrignole,E.A.,Near-criticalseparationofaqueousazeotropicmixtures:Processsynthesisandoptimization,J. Supercrit. Fluids,12,69–84,1998.
27. Diaz,S.,Espinosa,S.andBrignole,E.A.,Citruspeeloildeterpenationwithsupercriticalfluids: Optimal process and solvent cycle design, J. Supercrit. Fluids, 35, 49–61,2005.
28. vanKonynenburg,P.H.andScott,R.L.,CriticallinesandphaseequilibriainbinaryvanderWaalsmixtures,Phil. Trans.,298,495–540,1980.
29. Lucks,K.D.,Theoccurrenceandmeasurementofmultiphaseequilibriabehavior,Fluid Phase Equilibria,29,209–224,1986.
30. Coorens, H.G.A., Peters, C.J. and De Swaan Arons, J., Phase equilibria in binarymixturesofpropaneandtripalmitin,Fluid Phase Equilibria,40,135–151,1988.
31. Peters,C.J.,Supercritical fluids: Fundamentals for application. Multiphase equilibria in near-critical solvents,KluwerAcademicPublisher.Editors:Kiran,E.,andLeveltSengers,M.H.,1994.
32. Peters,C.J.andGauter,K.,Occurrenceofholesinternaryfluidmultiphasesystemsofnear-criticalcarbondioxideandcertainsolutes,Chem. Rev.,99,419–431,1999.
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34. Horizoe,H.,Tanimoto,T.,Yamamoto,I.andKano,Y.,Phaseequilibriumstudyfortheseparation of ethanol-water solution using subcritical and supercritical hydrocarbonsolventextraction,Fluid Phase Equilibria,84,297–320,1993.
35. Brignole,E.A.,Andersen,P.M.andFredenslund,A.,Supercriticalfluidextractionofalcoholsfromwater,Ind. Eng. Chem. Res.,26,254–261,1987.
36. Wiebe,R.,Thebinarysystemcarbondioxide-waterunderpressure,Chem. Rev.,29,475–481,1941.
37. Coan, C.R. and King, A.D., Jr., Solubility of water in compressed carbon dioxide,nitrousoxide,andethane.,J. Am. Chem. Soc.,93,1857–1862,1971.
38. Kobayashi, R. and Katz, D., Vapor-liquid equilibria for binary hydrocarbon-watersystems,Ind. and Eng. Chem.,45,440–446,1953.
39. Zabaloy, M., Mabe, G., Bottini, S.B. and Brignole, E.A., The application of highwater-volatilitiesoversomeliquefiednear-criticalsolventsasameansofdehydratingoxychemicals,Fluid Phase Equilibria,5,186–191,1992.
40. Reverchon, E. and Adami, R., Nanomaterials and supercritical fluids, J. Supercrit. Fluids,37,1–22,2005.
41. Martin, A., Precipitation processes with supercritical carbon dioxide: mathematical modeling and experimental validation,Ph.D.Thesis,UniversidaddeValladolid,Spain,2005.
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43. Amaro-González, D., Mabe, G., Zabaloy, M. and Brignole, E.A., Gas antisolventcrystallizationoforganicsaltsfromaqueoussolutions,J. Supercrit. Fluids,17,249–258,2000.
44. Simoes,P.C.andBrunner,G.,Multicomponentphaseequilibriaofanextra-virginoliveoilinsupercriticalcarbondioxide,J. Supercrit. Fluids, 9,75–81,1996.
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45. Peter, S., Supercritical Fluid Technology in Oil and Lipid Chemistry. Chapter VI: Supercritical fractionation of lipids,Editors:King,J.W.andList,G.R.,AOCSPress,Illinois,65–100,1996.
46. Espinosa, S., Díaz, S. and Brignole, E.A., Thermodynamic modeling and processoptimizationofsupercriticalfluidfractionationoffishoilfattyacidethylesters.Ind. Eng. Chem. Res.,41,1516–1527,2002.
47. King, J.W. andList,G.R.,Supercritical fluid technology in oil and lipid chemistry,Editors:King,J.W.andList,G.R.,AOCSPress,Illinois,1996.
48. Hegel, P.E., Mabe, G.D.B., Pereda, S., Zabaloy, M.S. and Brignole, E.A., PhaseequilibriaofnearcriticalCO2+propanemixtureswithfixedoilsintheLV,LL,andLLVregion,J. Supercrit. Fluids,37,316–322,2006.
49. Härröd, M., van den Hark, S., Holmqvist, A. and Moller, P., Hydrogenation atsupercriticalsingle-phaseconditions,ISSAF - 4th International Symposium On High Pressure Process Technology And Chemical Engineering,Venice,Italy,2002.
50. Baiker,A.,Supercriticalfluids inheterogeneous catalysis,Chem. Rev., 99, 453–473,1999.
51. Chouchi,D.,Gourgouillon,D.,Courel,M.,Vital,J.andNunesdaPonte,M.,Theinflu-enceofphasebehavioronreactionsatsupercriticalconditions:Thehydrogenationofalfa-pinene,Ind. Eng. Chem. Res.,40,2551–2554,2001.
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25
2 Supercritical Extraction PlantsEquipment, Process, and Costs
Jose L. Martínez and Samuel W. Vance
Contents
2.1 Introduction...................................................................................................252.2 SupercriticalFluidExtraction:ProcessDescription.....................................26
2.2.1 SupercriticalFluidExtractionofCompoundsfromaSolidMatrix...282.2.1.1 ProcessingParametersintheSupercriticalExtraction
ofSolids................................................................................302.2.2 SupercriticalFluidExtractionofCompoundsfromaLiquidFeed... 31
2.3 SupercriticalFluidProcessingPlants:EquipmentDesign...........................342.3.1 Overview............................................................................................342.3.2 Vessels................................................................................................ 352.3.3 PumpsandCompressors.................................................................... 372.3.4 HeatExchangers................................................................................ 382.3.5 PipingandValves............................................................................... 392.3.6 ControlSystems................................................................................. 41
2.4 IndustrialProcessImplementation............................................................... 422.5 Conclusions...................................................................................................48References................................................................................................................48
2.1 IntroduCtIon
In the last decade, supercritical fluid technology has reemerged, mainly due toa dramatic rise in the research and development activities focused on innovativeapproachesaswellasnewtrendsinthepharmaceutical,food,andchemicalsectors.Inthefoodindustry, thesenewtrendsincludeanincreasedpreferencefornaturalproductsoversyntheticonesandregulationsrelatedtonutritionalandtoxicitylevelsoftheactiveingredients.Ontheotherhand,consumersaretakingamoreproactiveroleinmaintainingtheirhealth,whichhasdrivenanewgenerationofproductsonthemarketaddressingdiseaseprevention.Thesetrendshavemadesupercriticalfluidtechnologyaprimaryalternativetotraditionalsolventextractionfortheextractionandfractionationofactiveingredients.
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26 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
Theobjectiveofthischapteristoprovideareviewofsupercriticalfluidextraction,describingtheprocessanddiscussingtheinfluenceoftheprocessparameters.More-over,thischapterisintendedtogiveanoverviewofthemaincomponentsofasuper-criticalextractionplantaswellasthestepsinvolvedinprocesscommercialization.
2.2 superCrItICal FluId extraCtIon: proCess desCrIptIon
Asupercriticalfluidextractionprocessconsistsoftwosteps:extractionofthecom-ponentssolubleinasupercriticalsolventandseparationoftheextractedsolutesfromthesolvent.Theextractioncanbeappliedtoasolid,liquid,orviscousmatrix.Basedontheobjectiveoftheextraction,twodifferentscenarioscanbeconsidered:
1)Carriermaterialseparation.Inthiscase,thefeedmaterialconstitutesthefinal product after undesirable compounds are removed—for example,dealcoholizationofalcoholbeverages,removalofoff-flavors,ordecaffein-ationofcoffee.
2)Extract material separation. The compounds extracted from the feedmaterialconstitutethefinalproduct—forexample,essentialoiloranti-oxidantextraction.
Theseparationofsolublecompoundsfromthesupercriticalfluidcanbecarriedoutbymodifyingthethermodynamicpropertiesofthesupercriticalsolventorbyanexternalagent(Figure2.1).Inthefirstcase,thesolventpowerismodifiedbymanip-ulatingtheoperatingpressureortemperature.Inthesecondcase,theseparationcanbecarriedoutbyadsorptionorabsorption.Themorecommonmethoddecreasestheoperatingpressurebyanisoenthalpicexpansion,whichprovidesareductionofthefluiddensityandthereforeareductionofthesolventpower.Ifseparationtakesplacebymanipulatingthetemperature,twosituationsmayoccur,dependingonthesolubilityof thedissolvedcompounds. If solubility increaseswith temperature atconstantpressure,adecreaseintemperaturewilldecreasethesolubilityandseparatethecompoundsdissolvedinthesupercriticalsolvent.Ifsolubilitydecreaseswithanincreaseintemperatureatconstantpressure,anincreaseintemperaturewillseparatethecompoundsfromthesupercriticalfluidsolvent.Iftheseparationiscarriedoutbyanauxiliaryagent,suchasanadsorbent,nosignificantpressurechangeoccurs,sothedifferentialpressureacrossthepumpismuchlower.Thistypeofprocessimpliesloweroperating costs; however, the recoveryof the extract from the adsorbent isoften very difficult. To overcome this disadvantage of high losses of the extract,theadsorptionstepmaybereplacedbyanabsorptionstep.Theextractdissolvedinthesupercriticalsolventisabsorbedbyawashfluidinacountercurrentflowusingapackedcolumnorspraytowerunderpressure.Separationofsolutesbyadsorptionandabsorptionhasbeenappliedinthedecaffeinationofcoffee[1,2].
Oneofthemainadvantagesofsupercriticalfluidsistheabilitytomodifytheirselectivitybyvaryingthepressureandtemperature(i.e.,modifyingfluiddensity).Therefore,supercriticalfluidsareoftenusedtoextractselectivelyorseparatespecificcompounds from a mixture. One procedure is by a fractional extraction process.
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Supercritical Extraction Plants 27
In this case, the extraction is carried out in two stages. During the first stage, arelatively lowfluiddensity isselected,whichallowsextractionof thecompoundsthataresolubleatlowpressure.Then,theresidueisfurtherextractedathighfluiddensitytorecoverheaviercompounds(e.g.,dealcoholizationofcider[3]).Anotherexampleoffractionalextractionconsistsofremovalofnonpolarfractionsinthefirststagebyusingasupercriticalsolventandtheremovalofamorepolarfractionfromtheresidueinthesecondstagebyaddingacosolvent(e.g.,extractionofactiveingre-dientsfromgrapeseed[4]).
Anotherprocedure toselectivelyextractorseparatespecificcompoundsfromamixtureissequentialdepressurization[5].Inthiscase,bothfractions(lightandheavy)aresimultaneouslyextractedbyusinghigh-densityfluid.Thenthesupercriti-calsolventandtheextractpassthroughmultipledepressurizationsteps,allowingfrac-tionalseparation.Inthefirstdepressurizationstage,theheavierfractioniscollected;thevolatileorlightfractioniscollectedinthelaststage.Twodepressurizationstepsaregenerallyused,althoughinsomespecificcases,threeseparationstepshavebeenused.Thismethodiscommonlyusedintheextractionofspices,wherethesolubilityofoleoresinandessentialoil fractionsinasupercriticalsolventvarysignificantlywithpressureandtemperature.Generally, theextractiontakesplaceathighpres-sures(40to60MPa),sobothfractionsaresolubleinthesupercriticalsolvent.Theseparationorcollectionoftheoleoresinfractiontakesplaceinthefirstseparatorby
Group I. By Modifying the Thermodynamic Conditions
Group II. By External Agents
Extractor
Pump
Valve
Separator Extractor
Pump
Heat Exchanger
Heat Exchanger
Separator
Extractor Extractor Adsorption Vessel
Absorption Column
Pump
Pump Pump
FIgure 2.1 Basicschemeofsupercriticalextractionprocess.
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28 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
reducingtheextractionpressuretointermediatepressure(15to20MPa).Undersuchoperatingconditions,thearomaticfractionremainsinthesupercriticalphase.Afterleavingthefirstseparation,thepressureisfurtherreducedandtheessentialoilsarecollectedinthesecondseparator.Thistypeofprocesshasbeensuccessfullyappliedinmultipleproducts.Insomecases,bothfractionsaredesirable(e.g.,oleoresinandessentialoils,colorandpungentfraction),whereasinothers,onlyoneofthefrac-tionshascommercialinterest.
2.2.1 Supercritical Fluid extraction oF compoundS From a Solid matrix
Mostofthedevelopmentandindustrialimplementationinsupercriticalfluidextrac-tionhasbeenperformedonsolidfeedmaterials.Figure2.2illustratesageneralflowdiagramofasupercriticalextractionprocessfromsolids.Thesolventissubcooledpriortothepump,assuringaliquidphasetoavoidcavitationissues.Thepressurizedsolventisheatedaboveitscriticaltemperaturetotheextractiontemperaturepriortotheextractionvessel.Theextractionvessel,whichisfilledwiththefeedmaterial,iselectricallyorwaterheatedtotheextractiontemperature.Thesupercriticalsolventflowsthroughthefixedbedandthesolublecompoundsareextractedfromthecarriermaterial.Thesupercriticalfluidplustheextractleavestheextractionvesselfromthetop,throughapressurereductionvalve.Thesolventpowerdecreaseswithpressurereduction,sothecompoundsprecipitate.Toassuretotalprecipitation,thesupercriti-calsolventisheatedabovethesaturationtemperaturetoreachthegasphase.Underthoseconditions,thesolventpowerisnegligible.Thenthematerialiscollectedinaseparatorwhilethesolventingasphaseleavestheseparatorvesselfromthetopand
1 Extraction Vessel 6 Receiver2 Pressure Reduction Valve 7 Pre-cooler3 Vaporizer 8 Pump4 Separator 9 Pre-heater5 Condenser
1
9 8 7
2 4
5
3
6
FIgure 2.2 Flowdiagramofasupercriticalextractionprocessfromsolids.
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Supercritical Extraction Plants 29
isrecirculatedbacktotheextractionvessel.Oncetherawmaterialisfullyextracted,thefollowingstepsarerequiredintheextractionvessels:
DepressurizationOpeningoftheextractionvesselUnloadingthespentmaterialLoadingwithfreshmaterialClosingtheextractionvesselPressurizingtooperatingconditions
Oneofthemostdifficultaspectsisattainingcontinuousfeedofthesolidsandcontinuousdischargeatahighpressureextractionvessel.Generally,thesolidfeedmaterialishandledbyusingpreloadedbaskets.Fromanindustrialorcommercialpointofview,theuseofonlyoneextractionvessel,evenwithaquick-openingclosurethatallowsforrapidopeningandclosing,isnoteconomicallyviable.Therefore,multi-pleextractionsvesselsoperating inacountercurrentflowarerequired.Figure2.3showsageneralschemeofacascadeextractionwithfourextractionvessels.Inthiscase,oncetherawmaterialinthefirstextractionvesselisfullyextracted,thevesselistakenoutfromtheprocessbyvalving.Oncethevesselisdepressurized,emptied,andrefilled,itenterstheprocesslineasthelastextractionvessel.Thesecondextrac-toristhenextonetobeisolatedoftheprocessline.Operatingthisway,thefreshsupercriticalsolventextractsfirsttherawmaterialthatispartiallyexhaustedandinthefinalextractionstep,thesupercriticalsolventextractsfreshrawmaterial.Thisconfigurationprovideshigher solvent loading (amountofmaterial extract/amountofsolvent).Theobjectiveistomaximizethesolventloading(i.e., tomaintainthesupercriticalsolventsaturatedorclosetothesaturationpoint).
Sincetheextractorsareoperatedbatchwise,acriticalfactoristoshortenthecharge and discharge cycle times. Therefore, a cap automation mechanism with
••••••
From Pre-heater
To Pressure Control
FIgure 2.3 Scheme of cascade operation of multiple extraction vessels for extractionofsolids.
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30 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
quick opening closure, as well as fast depressurization, and unloading/loadingsequencearecriticalinthedesignofasupercriticalextractionplant.
2.2.1.1 processing parameters in the supercritical extraction of solids
ParametersaffectingthesupercriticalfluidextractionofsolidsarelistedinTable2.1.Theinfluenceoftheprocessparameterscanbesummarizedasfollows:
Solubilityofcompoundsincreasesbyincreasingtheextractionpressureatconstanttemperature.Atpressureclosetothecriticalpressure,solubilityofthecompoundsincreasesbydecreasingthetemperature.However,athighpressures,solubilityofcom-poundsincreasesbyincreasingthetemperature.Thiscrossovereffectisduetothecompetingeffectsofthereductioninsolventdensityandtheincreaseofthevaporpressure.Thelatterhasmarkedinfluenceathigherpressures.Thepressureatwhich thecrossovereffectoccursdependson the typeofcompoundstoextract.Thecrossoverrangeformostofthecompoundstakesplacebetween20and35MPa.The separation conditions depend on the solubility of the compounds atdifferentpressuresandtemperaturesaswellaswhetherafractionationofextract is carriedoutby sequential depressurization steps.Generally theseparationpressureiscarriedoutat5–6MPa.Foressentialoilsorvolatilefractions,theseparationtakesplaceat3to5MPaandlowtemperaturestomaximizetherecoveryofthetopnotescomponents.Foroils,theseparationcantakeplaceat15to20MPaduetotheirlowsolubilityinsupercriticalcarbondioxide(CO2)underthoseconditions.Thesolvent-feedratiodependsonmanyfactors,suchasconcentrationofthesoluteinthefeedmaterial,solubilityinthesupercriticalsolvent,type
•
•
•
•
table 2.1processing parameters in the extraction of solidsraw Material related
ParticlemorphologyandsizeMoistureChemicalreactionsforsettingfreetheextractcompoundsCelldestructionPelletization
•••••
operating Conditions
Extractionconditions:PressureTemperatureTimeSolventflowSolvent-feedratio
••••••
Extractionoperation:FractionalextractionConstantconditions
•••
Separationconditions:PressureTemperature
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Separationoperation:SinglestageFractionalseparation
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Supercritical Extraction Plants 31
of feed material, and distribution of the compound in the feed material.Lowsolvent-feedratiosimplyloweroperatingcostsandhigherproductioncapacity.Generally,theindustrialprocessestargetsolvent-feedratioslowerthan30.However,highersolvent-feedratiosare justifiedforhighadded-valueproducts.Inspecificcases,asolvent-feedratiohigherthan100:1hasbeenreachedforcommercialapplications.Highsolventflowrates implyhighoperatingandcapitalcosts.However,theycouldincreaseproductioncapacity.Thesolventflowrateortheresi-dencetimeofthesolventintheextractionvesselmustbeoptimized.Ahighresidencetimeimpliesalongbatchtime.Conversely,ashortresidencetimemayresultinshortercontacttimebetweenthesolventandsolute,resultingina loadingof the solventmuch lower than the saturationconcentrationat the selected operating conditions. Linear velocities ranging from 1 to5mm/sarecommonlyusedinthesupercriticalfluidextractionprocess.Thesizeandmorphologyofthesolidmaterialhaveadirecteffectonthemass transfer rate. In general, increasing the surface area increases theextractionrate.Therefore,smallerparticlesizeorgeometry(suchasflakes)generallyfavorshighermasstransfer,decreasingthebatchtimeaswellasdiffusioncontrolledprocess.Ifthesolublesubstancesarelocatedinrigidstructuresinsideofthesolidmatrix,thesizereductionbreaksthisstructuresoitwillbeeasilyaccessibleforthesolvent.However,verysmallparticlesfavorachannelingeffect,whichdecreasestheextractionrate.Particlesizeneedstobeevaluatedcasebycasebasedonthetypeofmaterialtobepro-cessed.Inthecaseofprocessingofspicesandseeds,particlesizeisgener-allybetween30and60Mesh.Similarlytoparticlesize,moisturecontentmustbeevaluatedcasebycase.High content of moisture is usually not desirable because moisture actsas amass transferbarrier.On theotherhand,moisture expands the cellstructure,facilitatingthemasstransferofthesolventandthesolutethroughthesolidmatrix (e.g., in seedsandbeans).For instance, the influenceofmoisturebetween3% to10%generallyhasno significant impacton themasstransferofedibleoilfromseeds.
2.2.2 Supercritical Fluid extraction oF compoundS From a liquid Feed
Whenfeedmaterialisinaliquidstate,extractionistypicallycarriedoutinacoun-tercurrentcolumn.Thedensematerial(liquid)isintroducedfromthemiddleorthetopofthecolumn,andthematerialwithlowerdensity(solvent)isintroducedfromthebottomofthecolumn.Thiscontinuousprocessleadstoloweroperatingcoststhanthoseincurredwithextractionfromasolidmatrix.Ageneralprocessflowdia-gramisshowninFigure2.4.Theseparationstepsandregenerationofthesolventissimilartotheextractionfromsolids.
Similartotheconventionalcountercurrentcolumnprocesses,thecontactbetweenphasesisfavoredbyrandomorstructuredpackingmaterial.Additionally,refluxofextractimprovesselectivityintheextractionprocess.Theextractandsolventleavethecolumnfromthetop,whiletheheaviermaterial,orraffinate,iscollectedfrom
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32 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
the bottom. The countercurrent column is heated electrically or with a hot waterjacketand theextractionprocesscan takeplaceatconstant temperatureorwithacontrolled temperaturegradient.Thelatterprocessprovidesan internalrefluxthatincreasesselectivity.
Processdesignisbasedonphaseequilibriumdata,whichdeterminethenum-berof theoretical stagesnecessary toperformaspecificseparation;heightof thecolumn,whichisrelatedtomasstransferorheightequivalenttoatheoreticalplate,anddiameterofthecolumn,whichdeterminesthecapacity.Thelatterparameterisrelatedtohydrodynamicbehaviorofthemixtureincontactwiththepacking.
In cases where the viscosity of the liquid is very high, the extraction processrequiresintensiveanduniformcontactbetweenthefeedandthesolvent.Thiscontactcanbecarriedoutbymechanicalmixingorbynebulizingtheviscousmaterialthroughanozzle.Inthecaseofmechanicalmixing,themixercanbemagneticallyormechani-cally coupled. In the latter case, thedriving shaft is actuatedoutsideof thevesseldirectlybyamotor,whereasinthefirstcase, it isactuatedbymagneticfields.Thetorquegeneratedbymagneticcouplingislower;however,thereisnorotarysealandthereisnoneedforlubrication.Directdrivecouplingsrotatetheouterhousingandthemagneticfieldthenrotatesthedrivenmagnetssecuredtothemixershaft.Whenthemixerismechanicallycoupled,ashaftdesignprovidinghightorqueathighpressures,withextendedlifetimeofbearingsandsealsisrequired.Additionallythemixersmustbeproperlydesignedbasedonthespecificapplication.Figure2.5showsamechanicalcouplingdevelopedbyTharTechnologiesthatoperatesat69MPa.
From Pre-heater
Extract
Feed
Raffinate
FIgure 2.4 Flowdiagramofasupercriticalextractionprocessfromliquids.
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Supercritical Extraction Plants 33
Inamechanicalmixingprocess,theviscousliquidormeltedproductispumpedinto the vessel. By adding the supercritical solvent, the viscosity of the productdecreases, which facilitates mixing and reduces the torque required. The super-criticalsolventflowsthroughtheviscousmaterialextractingthesolublecompounds.Thesupercriticalfluidandtheextractleavetheextractionvesselfromthetop.Oncetheextractioniscomplete,thematerialleftcanbedischargedfromthebottomoftheextractionvessel.Iftheviscosityoftheremainingmaterialisstillhigh,thesuper-criticalfluidassistsinremovingthematerialthroughtheopening.Thisprocesscanbeappliedtomaterialwithveryhighviscosityatatmosphericpressure.
Another alternative for processing viscous liquid material involves intensivecontactbetweenbothphases(i.e.,mixingandnebulizingthemixture).Inthiscase,theviscousmaterial and the supercritical solvent aremixedand sprayed throughanozzle.Thesupercriticalsolvent reduces theviscosityof the feedand thereforedecreasestheinterfacialtension.Bysprayingthroughanozzle,anatomizationtakesplace,creatingveryfinedropletswithaverylargesurfaceareaandahighcontactbetweenbothphases.Thesupercriticalsolventextractsthesolublematerialandtheinsolublesprecipitateinthebottomoftheextractionvessel.Thisprocessisfavoredwhen there is a significant difference in solubility between the compounds to beseparated.Thecriticalparametersinthisprocessarethecontactormixingdevices,spraying devices, vessel design, and solid removal from a pressurized vessel. Aprocessusingthisconcepthasbeensuccessfullydevelopedandindustriallyimple-mentedinthedeoilingofcrudelecithin[6].Thisisacontinuousprocessinwhich
FIgure 2.5 Mechanicalmixingusingamechanicalcoupling.Designpressure:69MPa(CourtesyofTharTechnologies,Pittsburgh).
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34 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
crudelecithinispumpedandmixedwithsupercriticalCO2andthensprayedthroughanozzleintoahighpressurevessel.TheneutrallipidsaresolubilizedintheCO2,while thepolar lipidsareprecipitated in thebottomof theextractionvessel.TheCO2 and the neutral lipids leave the extraction vessel and the oil is recovered inthe separator. The polar fraction in a powder form is continuously transferred toa storage tank (Figure2.6). Some work was done in the early 1980s; however, itwasnever industrially scalable,mainlybecause the solvent-feed ratiosusedwereextremelyhighandabatchprocesswasused,sotheproductioncapacitywasverylow.Therefore,theplantsizerequiredtosatisfycommercialdemandhadtobeverylarge,whichimpliedveryhighcapitalcost.Thenewprocessofferstwosignificantadvantages:(1)theprocessiscontinuousand(2)thesolvent-feedratiorequiredislow.Thisisanexampleofindustrialimplementationofasupercriticalprocessfora commodity product, so the operating costs must be comparable to that at con-ventionalprocessing.Theconventionalprocessisawell-establishedprocessintheoilindustryusingacetoneasasolvent.However,usingacetoneasasolventformsacetonederivatives(mesityloxide)withadverseeffectsforthedeoiledlecithinduetoitstoxicityandoffflavor.Theoilindustryhasbeensearchingforalternativemethodsbuthasnotfoundanalternativeprocessuntilnow.Asimilarprocesscanbeappliedtoremovalofresidualsolventsinthepharmaceuticalindustry[7].
2.3 superCrItICal FluId proCessIng plants: equIpMent desIgn
2.3.1 overview
Designandselectionofequipmentforasupercriticalfluidprocessing(SFP)systemrequiresconsiderationofsomeparametersandspecificationsthatareuniquetothis
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FIgure 2.6 Scheme foracontinuousprocess forde-oilingofcrude lecithin.1Tankofcrudelecithin,2Lecithinpump,3Preheater,4Mixer,5Recirculationpump,6Backpressureregulator,7Nozzle,8Extractionvessel,9Transfervessel,10CO2inlet.
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Supercritical Extraction Plants 35
typeofplant.Manyitemsthatwouldbeoff-the-shelfformostplantsaresimplynotavailableornoteasilyfoundforapplicationtotheoperationordesignconditionsoftheSFPenvironmentandprocess requirements.Forexample,manySFPsystemsrequiresanitarydesignforfood,nutraceutical,orpharmaceuticalproductsandoper-atingpressuressubstantiallyhigherthannormallyfoundinfood,nutraceutical,orpharmaceuticalplants.Meetingtheserequirementsentailsinvestigationofvendorswhocanprovideitemsthataresuitable.Uniqueconditionsmaybeencounteredinregularoperationoramajormalfunctionmayoccurthatrequiresspecialmaterialsof construction (e.g., metals can undergo brittle fracture in such environments).Systemcapitalcostmustbecloselycontrolledtobecompetitivewithothersystems.
2.3.2 veSSelS
Ingeneral,SFPvesselsaredesignedandmanufacturedinaccordancewithAmericanSocietyofMechanicalEngineers(ASME)SectionVIIIStandards.Inmanycases,theprocessrequiresone,orsometimestwo,full-diameterquick-openingclosuresforchargingfreshfeedstockordischargingspentfeedstock.Theclosuremechanismisoftenautomatedtominimizedowntimeofavesselwhenfillingoremptying.Thereareanumberofsuchclosuresproprietarytovesseldesignersorsuppliers.Consid-erationmustbegiventomethodsofcleaningvesselsbetweenchargesoremptyingvesselswhen the solidsplugorbridge.Vesselsare jacketedorelectrically tracedfor process temperature control. Vessel shape and aspect ratio must be carefullyevaluatedtominimizevesselcostswithoutaffectingperformance.Themostcriticalvesseldesignintheprocessisthatoftheextractionvessel.Itrequiresthemaximumdesignpressureandmaybethemostcriticalinselectionofmaterialsofconstruc-tion.Inmanycases,specialalloysofstainlesssteelorexoticmetalscanbeused,buttheactualselectionofalloysandthicknessesmayalsodependontheabilitytomachine,forge,andweldvesselload-bearingcomponents.Extractionvesselscanbefabricatedbyforging,machiningsolidbarstock,rollingandweldingofplate,multi-wallrollingandwelding,compositemultilayers,andcasting.
Fulldiameterquick-openingvessel closuresmayutilize self-energizing seals,segmentedrings,breechlocking,flanges,andthreadedcaps.Mostareproprietarydesigns.ExamplesofclosuresareshowninFigure2.7.
Vesselsotherthantheextractionvesselsareusuallydesignedforsubstantiallyloweroperatingpressuresandtemperatures,butthefunctionofeachvesselmustbecarefullyconsideredinselectingthesizeandshapeofseparationvesselsandprocessholdingvessels.Insomecases,thevesselsmayneedspecialdesignstokeepthemcleanandminimizepluggingandcontamination.
Atthebeginningandendofthebatchprocesscycle,thevesselsmayverywellbeatornearambientpressuresandtemperatures,asmaterialsarebeingtransportedtoorfromtheSFPsections.Butevenfortheseareas,thevesselsmaystillrequireadaptationforcleaning-in-place(CIP)orothercleaningmethods.
Inmanycases,thefeedstockisinparticulatesolidorpelletform.Theextrac-tionvesselwouldthenbedesignedfor(usually)batchchargingandemptyingoftheextraction feedstock. In this case, an extractionvesselwouldbefilledwith feed-stock,brought fromambient temperature andpressure to extractionpressure and
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36 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
temperature,extracteduntilthesolutehasbeenremoved,andthendepressurized,emptied,andrecharged.Thebalanceoftheplantwouldbeessentiallycontinuousinoperationwithclosedcyclesolventrecirculation.Multipleextractionvesselswouldbeusedtoapproachacontinuousoperation.
Insomecases,thefeedstockisliquidandtheextractionvesselmaybeapackedcolumnoperatingcontinuously(Figure2.8).Inthesesituations,atrulycontinuousoperationwouldbethenorm,withreductionofpressureonlydoneforproductchangeorsystemshutdownandoverhaulormaintenance.Somesystemsutilizesupercritical
FIgure 2.8 Countercurrent column (10 m) of a supercritical process extraction plant(CourtesyofTharex,Seoul).
(a) (b)
FIgure 2.7 Vesselclosuretypes.a)Automatedsegmentringclosure,b)Automatedclampclosure(CourtesyofTharTechnologies,Pittsburgh).
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Supercritical Extraction Plants 37
fluidchromatography(SFC)inapackedcolumntoachievetheseparationofcom-ponentstargetingveryhighpurities(95%to99%).Figure2.9showsaprocessscaleSFCusingadynamicaxialchromatographycolumn.
2.3.3 pumpS and compreSSorS
Thesecondmostcriticalequipmentitemsarethepumpsandcompressorsusedforbuildingsystempressuresandtemperaturestothesupercriticalregionsestablishedintheprocessdevelopmentstudies.SFPflowsarerelativelylowandpressuresarerela-tivelyhigh,rangingfrom5to120litersperminuteflowat6to65MPa.Theprocessrequiresclosecontroloftemperatures,pressures,andflows.Pressures,inparticular,requirecriticalcontrolbecausepressurefluctuationsmaymakesubstantialdiffer-enceinprocessingresultsandcanresultinoverpressuredevicesshuttingtheprocessdownandwastingbothsolutesandsupercriticalsolvents.Suchshutdownsresultinpoorproductionratesandunnecessarycostpenaltiesforthesystem.
Mosthigh-pressurepumpsaremultiplunger styles.Flowandpressurecontrolcommonly use some type of speed control, such as variable frequency speed. Afurtherdevelopmentincludesdiaphragmtypepumps,whichareactuatedbyplungersandmorenormalliquidsthatcausethedisplacementandflexingofthefinalpump-ingelement(thediaphragm).Thepumpshaveproprietarydesignfeaturestoprovidesuitableoperationforthepressuresandflowsrequired.Morestandardpumpscanbeusedforprovisionofmakeupsupercriticalfluidsandfinalproduct(extract)pumps.
Compressorsmayalsobereciprocatingpistons.Inraresituations,morestandardrotarycompressorscanbeused,ofteninrecoveryofotherwisewastedsupercriticalfluid.Economicsandprocessvariablesdetermine theextentof recoveryof spentsolventfluids.
Thefluidendsofplungerpumpsorcompressorsrequireclosetolerancemachin-ingof theplungerorpistonand thecylinder tominimize leakage.Nonlubricatedplungersareoftenchosen,withcarefulselectionofmaterialstoensurelowcoeffi-cients of friction and dimensional stability. Lubricants are avoided because they
FIgure 2.9 Dynamicaxialcolumn(30cmID)ofasupercriticalprocesschromatographyplant(CourtesyofTharTechnologies,Pittsburgh).
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38 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
contaminate theprocess solvent and solute.O-rings,gaskets, and seals for recip-rocatingandrotatingpartsmustbecarefullydesigned.Materialsmustbecompat-iblewiththesolventsandsolutes.AbsorptionofsolventintheO-ringmaypresentproblemswhenthesystemisdepressurizedbecausethesolventmayexpandwithintheO-ring,causingdisintegration,especiallyifdepressurizationisrapid.Unusualorexoticmaterials for theplungerandcylindermaybeusedascoatingsor solidsections.Proprietaryinformationisacquiredbysubstantialequipmentdevelopmentandiscarefullyprotectedbydesignersandfabricators.Figure2.10showsamulti-plungerpumpwithadesignpressureof96MPaandaflowrateof30kg/min.
2.3.4 Heat excHangerS
Heat exchange equipment also presents unique problems for supercritical fluidsystemsdue to thehighpressures required inkeypartsof theprocess.Althoughheatexchangersintheprocessindustriesareamatureandverycompetitivetech-nology,designsarenotreadilyavailableatthepressuresencountered.Also,specialconsiderationmustbegiventocleaningoftheprocesssideheatexchangesurfaceintheeventoffoulingwithsolutesandcleaninganddisassemblyoftheexchangerforchangeover toanotherproduct.Another specialconsideration in selectionofheatexchangertypesorstylesistheriskanalysisforheatexchangertubefailure.Aleakorcatastrophicfailuremaycreateadryicepluginthehighpressureside(forCO2asthesolvent),freezingoftheheatexchangefluid,andoverpressureofthelowpressureheattransferfluidpipingsystem.SelectionofoverpressuresafetydevicesmustbecarefullyinvestigatedbyprocessriskanalysisandHazardandOperability(HAZOP)studiestechniquesforthesystem.
Removable heat exchanger heads are often desirable. The supercritical fluidsolventmostoftenisonthetubesideofshell-and-tubeexchangersanddesigncom-promisesmustoftenbemadebetweenmultitubetubediameterandnumberoftubes.Thesmallerthediameterofthetube,themoretubesarerequiredtoprovidesuitable
FIgure 2.10 High-pressure multiplunger pump. Design pressure: 96 MPa, Flow rate:30kg/min(CourtesyofTharTechnologies,Pittsburgh).
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Supercritical Extraction Plants 39
fluidvelocitythroughtheexchangerbutthelesstubingandmetalrequiredfortheheat transfer area. Smaller diameter tubes present additional problems for clean-ingandforfasteningtothetubesheets(usuallybywelding).Largerdiametertubesimprovethefluidvelocityandtubenumbersbutalsomayresultinlongerexchangers.Asthetubelengthincreases,differentiallinearexpansionoftheshellandtubesmayrequireexpansionjointsorfloatingheads.Thesesituationscomplicatetheexchangerdesignandmayaddtothecostofdesignandfabrication.
In most process designs, ASME Section VIII Unfired Pressure Vessel CodeStandardsandCodeStampsarenecessary.Insomecases,asimplerdesignoftheexchangercanbeaccomplishedifthepossibilityofheatexchangesurfacefoulingandpluggingofthetubinginteriorcanbeminimizedbycarefullycontrollingpro-cessingconditionsinthesystem.
Typesofheatexchangers thatcanbeused includeshell-and-tubeexchangers,double-pipeandmulti-U-tubeexchangers,doublepipecoils,orsimplecoilsintanks.Anexampleofshell-and-tubeheatexchangerdesignisshowninFigure2.11.
2.3.5 piping and valveS
Selectionofpiping,fittings,andvalvesforSFPalsorequiresspecialdesignspecifi-cationsandcriteria.Thematerialtobeusedmustbenonreactivewiththesupercriti-calfluidsolventandsolutesintheprocess.Thepossibilityofreactionsbetweenthesolventandthepipingsurfacesmustbeevaluated.Thesolventmaybesubstantiallymoreaggressiveinthesupercriticalfluidregimethanwouldbetrueatlowerpres-sures,soadditionaltestingofmaterialsmayleadtomoreexpensivealloystomini-mize such reactions. Where possible, high strength alloys (with higher allowablestressthanthetypical300Seriesstainlesssteels)arethechoiceforoverallcostandprocess suitability.Sinceflow rates formost supercriticalfluid systemsaremuchlowerthaninmoreconventionalsystems,pipediametersandsuitablehigh-pressurefittings are smallerwhilemaintainingappropriateflowvelocities in the intercon-nectingpipingortubing.Pipingcostisthusminimized.However,theconventionalthreadedjointsor“standard”flangesarenotcosteffective.Inmostcases,specialhigh-pressurefittings,couplings,andthelikewillbetheselectionofchoice,bothforconvenienceandeconomicreasons.Specialhigh-pressurecouplingsareshowninFigure2.12.
Valving isanotheruniqueareafor theprocess.Twotypesofvalvingmustbeconsidered: (1) isolation valving and (2) flow control valving. Isolation valvingmost often includes plug valves or butterfly valves for leak-proof on-off service.Thesevalvescommonlyhavemetal-on-metalsealingsurfaceswherelowfrictionisdesired.Specialcoatings(sprayed,vapordeposition,orcomposition)maybeusedtoavoidseizingorgalling.Selectionofdissimilarmetalsormetaloxidesorcarbideswithhighhardnessvaluesandgoodmachiningproperties improvesperformance.However,pairingofmaterialsandselectionofdesigns for theoperatingenviron-mentisstillmoreartthanscience.Sospecialtyhigh-pressurevalvecompaniesarethevendorsofchoice.Flowcontrolvalvesarealsoaspecialtyitematsupercriticalfluidoperatingpressures. In somecases, pressuredrop through the control valvemay be at critical flow or with a phase change when passing through the valve.
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40 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
Theseconditionsrequirespecializedknowledgeoftheeffectsofthehighvelocitythroughthevalveorificeandthepossiblepresenceoftwo-phaseflowthroughthevalve.Again, inselectingmaterialsforpackingglandsandvalvestemseals,caremustbetakentoselectanappropriateelastomerorcompositethatwillnotabsorbhighpressuresolventduringoperationandthenfractureorfailwhenthepressureisreleased.ThesupercriticalfluidsolventmayvaporizeandexpandinthepackingorO-ring,withsubsequentdestructionoftheseal.Attherangeofpressuresundercon-sideration,dimensionalstabilityandeliminationofcreepflowarealsonecessary.
As SFP system throughputs become larger, automated control and isolationvalvesbecomemoreattractive.Pneumaticorhydraulicoperatorsand,occasionally,electricallyoperatedmodulatingoperatorsmayberequiredtominimizethedowntimefortheplant.Fail-safeoperationmustbetheorderoftheday.
FIgure 2.11 Shell-and-tubeheatexchanger(CourtesyofTharTechnologies,Pittsburgh).
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Supercritical Extraction Plants 41
2.3.6 control SyStemS
Asshouldbeobviousbythispoint,manyoftheareasofcontrolforSFPsystemsrequirespecialconsiderationbecauseofthehighoperatingpressures(withtheresult-inghighpotentialenergyintheprocess)andthepossiblehazardsforbothoperatingpersonnelandplantintegrity.Controlsystemfailuresinmoreconventionalprocess-ingplantswouldnotpresent thepossiblehazards tooperatorsanddamage to theequipmentandhazardsbeyondtheplantarea.Relativelysmallvariationsinprocessconditionscanbereflectedinsubstantialvariationsinsystempressuresandphasetransitions.Socontrol response to thesevariationsmustbe rapidandeffective indampingtheresultswithinthesystem.
Selectionofprimarysensorsmustbecarefullymadewithprovisionsforsensorfailure, leakage, or error. Redundancy must be considered and carefully thoughtthrough.Evenpressuregaugesandtemperatureelementsmustbeexamined.Pressuregaugesortransducersmayrequireliquidsealsorthermowellstopermitisolationandreplacementwhilethesystemisoperating.Temperaturesensorscanbethermocouplesorresistancetemperaturedetectors(RTD)sensors,butresponsetimemustbeweighedagainstthermowellisolation.Gaugesshouldhaveblowoutdiscs.Levelsensorsmustbeaccurateandreliable.Pumpandcompressorflowratesarecommonlymeasuredbymassflowmeters(Coriolismeters)andflowcontrolledbyfrequencymodulationoftheconnectedmotor.Overallsystemshutdowniscontrolledbydistributedcontrolwithcomputercapability.SystemconditionsatstartupandshutdownoftheprocessmustbethoroughlythoughtthroughwithaHAZOPreview.
Summarizing,theforegoingdescriptionofthefactorsthatmustbeconsideredinthespecificationandselectionofthehardwarethatgoesintoaSFPplantshowsthattheuniquemechanicalsystemrequirementsmustbecarefullymadeandreviewed.Eachsystemhassomecharacteristicsthatmustbeevaluatedbasedontheparticularenvironmentandprocesschemistryforthatproductorcommodity.Insomecases,the plant will be a multipurpose or multiproduct plant. Each purpose or productmustbeconsideredtoestablishthesingleproductthatwouldcontrolthemechanicalspecificationsofeachitemofequipmenttobeselected.
(a) (b)
FIgure 2.12 High-pressurecouplings:a)DUROLOK(CourtesyofBETEFogNozzle,Inc.,Greenfield,MA),b)Grayloc(CourtesyofGraylocProducts,Houston,TX).
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42 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
2.4 IndustrIal proCess IMpleMentatIon
Processdevelopmentrespondstoaspecificrequirementofacompanyortoamarketdemandforaspecificproduct.Insomecases,thecurrentprocessshowssignificantweaknessevenforawell-positionedproductinthemarket.So,theprocessingcom-panies that are aware of the limitations or constraints search for new processesto strengthen the product, resulting in a leading market position and projectinghigherrevenues.Inothercases,traditionaltechnologiesdonotofferasatisfactorysolutiontoaspecificproblem.Additionally,thereisacontinuoussearchtoreduceproductioncosts.
A general workflow for an industrial process implementation is illustrated inFigure2.13.Thefirststepistoprovethatthetechnologyiscapableofmeetingtheproduct specifications and process requirements defined by the customer or themarket.Intermsofproductspecifications,requirementsgenerallyareaminimumconcentrationorpurityofspecificcompoundsandminimumextractionorrecoveryefficiency. Regarding process requirements, the main constraints are maximumoperatingtemperatures,typeofpretreatmentormaterialconditioning,andaccept-ablecosolventstobeused.
In the case of supercritical fluid extraction, the first point to be addressedis if a compound tobe extracted is soluble in the supercritical solventor if thesolventwillbeselectivetofractionateorseparateamixtureofcompounds.Thethermodynamicdatarequiredare thesolubilityof thespecificcompoundin the
Supercritical FluidExtraction
CustomerRequirements
Lab scaleOptimization of
Process Parameters
Initial Cost Estimate
Scale-upPilot Plant Scale
Semi-industrial Scale
ProductSpecifications
ProcessRequirements
ConventionalTechnologies
Cost Calculation
CommercialPlant
CostComparison
FIgure 2.13 Workflowforindustrialprocessimplementation.
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Supercritical Extraction Plants 43
supercriticalfluidasafunctionofpressure,temperatureandsoluteconcentration,partitioncoefficients,andselectivityorseparationfactors.Averyextensivedata-baseofphaseequilibriaandsolubilitydataforbinarysystemshasbeengeneratedoverthelasttwodecadesandcanbeusedasareference.However,insomecases,thepublisheddataarequestionable.
Arapidwaytodeterminesolubilitydataorphasetransitionsforabinarymixture(specific compound and supercritical solvent) is by a phase equilibrium analyzer(Figure2.14).Thisisastaticmethodwherebythesoluteandsolventareloadedintoahigh-pressurevessel.Thisvesselconsistsofavariablevolumehigh-pressureviewcellwithanintegralstirrer,waterjacket,andvideosystem.Oncethemixtureiscom-pressedtoasinglephaseforaselectedtemperature,slowmovementofthecellpistonatacontrolledrateslowlydecreasesthepressureuntilasecondphaseappears.Byobservingthevideooutputofthesystem,itispossibletodeterminethecloudpointforthesampleatthecurrentpressureandtemperature.Additionalexperimentaldataareobtainedbymodifyingthetemperatureandrepeatingtheexperimentalprocedure.Themainadvantagesofthismethodarerapidgenerationofdataandvisibleconfir-mationofdissolution;inaddition,nosamplingisrequired,noextractionefficiencyisinvolved,andaminimumamountofsoluteisused.
Once the thermodynamic data are obtained, the next step is to evaluate theextractionofthatspecificcompoundfromtheoriginalsample,whichisgenerallyamulticomponentmixtureinasupercriticalfluidextractionsystematbenchscaletooptimizeprocessparameters.Theprocessparameterstooptimizearelistedhere:
FIgure 2.14 Phaseequilibriumanalyzer(CourtesyofTharTechnologies,Pittsburgh).
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44 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
Conditioningtherawmaterial:moisture,sizeandshape,etc.Kineticdata;pressure,temperature,andsolventflowrateeffect:
ExtractionyieldExtractiontimeQualityoftheextract
Fractionationconditions
Thelaborbenchsystemmustbeproperlydesignedtobeversatileandcoverawiderangeofoperatingconditions.Thesystemmustbeableto:
Cover a wide range of operating conditions: pressure, temperature, andflowratesUsecosolventsPerformsequentialdepressurizationbyusingatleasttwoseparationstagesContinuouslylogprocessparameterdata
Aninitialcostestimateisprovidedoncetheprocessdevelopmentsatisfiesthecustomerproductspecifications.Theinitialcostestimatediscalculatedusingscaleupmethodsbasedonthefollowinginformation:
Customerproductionrequirements(i.e.,amountofmaterialtoprocessperyear,workingdaysperyear,andworkinghoursperday)Rawmaterial(i.e.,particlesizeandshape,concentrationoftheproduct)Optimizedprocessparameters (i.e., extractionpressureand temperature,solventflowrate,residencetime,kineticsoftheprocess,bulkdensityofthefeedmaterial,andseparationpressureandtemperature)
Thiscostestimationprovidestothecustomerthefollowinginformation:operatingcosts ($/kg of feed, or $/kg of final product), plant size, and plant configuration.At thispoint, thecustomerdetermines if thesupercriticalprocesswillmeet theirbudgetandiftheinvestmentandoperatingcostsarecomparablewithorbetterthanconventional technologies. If so, the next step is to scale up the process to pilotplantorsemi-industrialscale.Theobjectivestoaccomplishinthisstage,meetingallproductsspecifications,are:
Verificationof theprocessparametersselected in the labscaleand theiroptimizationifrequiredOptimizationofutilityrequirementsOptimizationofrecirculationparametersofthesolventandaddressinganyissuesrelatedtomaterialhandling
Reducingoperatingcostsrequiresminimizingenergyrequirements,whichalsoimpliesareductionintheassociatedcapitalcostoftheauxiliaryequipment.Thesecostsaredirectlyrelatedtotherecirculatingcostsofthesolvent.Recyclingofthesolventdependsontheseparatingconditionsof thesubstancefromtheextractionfluid.Typically,recyclingisperformedatlowseparationpressuresandthesolvent
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Supercritical Extraction Plants 45
is recycled in the liquid state. Figure2.15 shows the solvent cycle in a pressure-enthalpydiagram.In thiscase, theseparationof thesolublecompoundsfromthesupercriticalsolventisachievedbyisoenthalpicthrottling(a-b)followedbyheating(b-c).Atc,thesolventisingasstate,sothesolventpowerisnegligible.Thenthesolventissubcooled(c-d)andpumpedtotheextractionpressure(d-e).Thesolventisheatedtotheextractiontemperature(e-a).Achillerunitisusedinordertocooldownandcondensetheextractionfluidbeforeitentersthepump.However,ifextractiontakesplaceatpressuresbelow30MPa,therecyclingofthesolventasgas,replac-ingthepumpbyacompressor,generallyresultsinenergysavings.Forinstance,inextractionofessentialoils,wheretheextractionconditionsaregenerallycarriedoutinthepressurerangeof8to20MPaandinthetemperaturerangeof35°Cto50°C,thesolventrecycledinagasstateismoreenergyefficient.
Aspreviouslymentioned, at pressureshigher than30MPa, solvent recyclinginliquidstageismoreefficient.Undertheseoperatingconditions,analternativetoprovideahigherenergyefficiencysolventcycleisadditionofacompressorintothesystemafterseparationoftheextractedcompounds[8].Afterexpandingthemixturetoformatwo-phaseregionandheatingthemixture,sothatthesolventbecomesasinglegasphase,thesolventiscompressedtoapressurehigherthancriticalpressurebyacompressor.Thenthesolventissubcooledbeforeenteringthepump.Twoadvan-tagesareobtainedusingthismethod.Thefirstisthat,insteadofusingachiller,thesolventcanbecooleddownwithwaterfromacoolingtower.Second,themechanicalenergyrequiredofthepumpislower.Theenergysavingsofrecyclingthesolventusingbothapumpandacompressor,comparedwiththemoretraditionalprocess,dependsonextractionconditionsbutcouldbeupto65%.
Incaseswherethesolubilityofthespecificcompoundintheextractionfluidisverylow,typicallylowerthan0.5%,atapressurehigherthanthecriticalpressureofthesolvent,thesolventcanberecycledinasupercriticalstateprovidingadditionalenergysavings.Forinstance,intheextractionofseedoilsusingsupercriticalCO2,theseparationoftheoilintheseparatorshouldbecarriedoutundersupercritical
100
–50,°C
–30,°C –10,°C 10,°C 30,°C 50,°C 70,°C 90,°C 110,°C 130,°C 150,°C 170,°C190,°C1000
100
10
200 300Enthalpy (kJ/kg)
Pres
sure
(bar
)
400 500 600
L-V
L V
SCF
a
bcd
e
FIgure 2.15 DiagramP-Hforalow-pressurerecirculationsolventusingpump.
7089_C002.indd 45 10/15/07 5:22:50 PM
46 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
conditionsatapressurelessthan20MPaandthesolventrecycledinsupercriticalstate.Thesolubilityoftheoilatpressuresbelow20MPaisgenerallylessthan0.3%.Additionally,thesupercriticalCO2couldberecompresseddirectlywithouttheneedofsubcoolingtheCO2.Thereisnotachangephasethatcouldcreatecavitationinthepump.Table2.2summarizesthesolventcycleatdifferentextractionconditionsbasedonthesolubilityoftheextract.However,becausethecompressibilityofsuper-criticalCO2ishigherthanthatofliquidCO2,itreducespumpcapacity.Figure2.16showsthesolventcycleofCO2regeneratedundersupercriticalconditions.
ChordiaandMartinez [8]describeanalternativemethodofprovidinghigherenergysavingsforrecyclingthesolventinthesupercriticalstate.Inthiscase,highpressurerecyclingisrealizedbyreplacingtheexpansionvalvewithaturbineandenergeticallycouplingtheturbinetothepump.Basedontheoperatingconditions,energy savings of up to 60% can be reached. Table2.2 summarizes the solvent
100
–50,°C
–30,°C 190,°C1000
100
10
200 300Enthalpy (kJ/kg)
Pres
sure
(bar
)
400 500 600
L-V
LV
SCF
a
bc
–10,°C 10,°C 30,°C 50,°C 70,°C 110,°C90,°C 130,°C 150,°C 170,°C
FIgure 2.16 DiagramP-Hforasupercriticalrecirculationsolvent.
table 2.2solvent Cycle at different operating Conditionssolubility of
solute at p > pc solvent Cycle
extraction pressure
solvent recycling state Main Components
>0.5%Lowpressure
recycling
Pext>30MPaLiquid Pump
Gas Compressor+pump
Pext<30MPaLiquid Pump
Gas Compressor
<0.5%Highpressure
recyclingPext>30MPa
Supercritical Expansionvalve+pump
SupercriticalTurbine+pump
(energeticallycoupling)
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Supercritical Extraction Plants 47
cyclerecommendedforenergysavingsatdifferentextractionconditionsbasedonsolubilityoftheextract.
An additional factor that must be considered to reduce operating costs is tominimize solvent losses. The losses take place from the extraction vessel duringthedepressurization for unloading the spent material, aswell as in the separatorwhenwithdrawing theextract.Mostof theCO2 lossesoccur in thedepressuriza-tionprocess.Generally,depressurizationinvolvestwosteps:a)Theextractionvesselisdepressurizeddowntothereceiverpressure(5to6MPa)andb)Therestofthesolvent isventedto theatmosphere.AnalternativetoreduceCO2losses is touseacompressorandcondenseadditionalCO2. In thiscase,once thepressure in theextractionvesselequals thepressure in thereceiver(5 to6MPa), thecompressorcompressestheCO2untiltheresidualpressureintheextractionvesselreaches0.2to0.5MPa.ThentheresidualCO2isreleasedintotheatmosphere.InplaceswhereCO2costishigh,investmentincapitalcostofadditionalequipmenttoreduceCO2lossesmakeseconomicsense.
An additional operating cost is labor. The personnel required to operate anindustrialSFEplantdependsonthesize,configurationof theplant,batchtime,andautomationoftheplant.Ingeneral,asupervisorandtwooperatorsarerequiredfor a fullyautomated largeplant.The laborcost, aswell as thecostofCO2, ishighlydependentonthegeographicallocation.Afterscaling-uptheprocessanddefinitionoftheoperatingparametersandconfiguration,thefinalcostestimateismade.Whiletheindustrialplantisbuilt,tollingisapreferablestepinmanycases.Thisstepisusedformultiplepurposes:formulationofdifferentproducts,marketevaluation,andlaunchingtheproductintothemarketwhiletheindustrialplantisunderconstruction.
ThecapitalcostofaSFEplantdependsonmanyfactors,suchasthenumberofvessels,designpressure,sizeofthevessels,flowrate,automation,andGoodManu-facturingPrice(GMP)compliance.Generally,thecapitalcostoftheSFEplantishigherthanthetraditionalorconventionalextractionplant,whiletheoperatingcostsarelower.However,tocompareproperlythecapitalcostsofSFEplantversustradi-tionalextractionprocess,isnecessarytotakeintoaccountalltheassociateequip-mentusedintheconventionalextractionprocess,suchasdistillationorevaporation
table 2.3Case study: Cost and revenue estimates
Flaxseed astaxanthin ginger
Amounttobeprocessed(MT/year) 3,000 50 3,000
Concentrationoftheproduct(%) 13 2.5 5
Numberofdays/year 300 300 300
Numberofhours/day 24 24 24
Estimatedequipmentcost($) 2,500,000 1,500,000 4,500,000
Operatingcosts1($/kgoffeed) 0.23 5.57 0.43
ROI 3.2 1.0 1.61 Includespowerconsumption,CO2losses,maintenance,andlabor
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48 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
systems for solvent recovery; aswell as associate costs inbuilding requirements,instrumentation and electrical connections to meet explosion proof. On the otherhand,thesellingpriceoftheproductsobtainedbySFEisalsohigher(bothextractandraffinate).
Table2.3illustratestheestimatedcostsandrevenuesobtainedforasupercriticalextraction plant used in three different industrial sectors: edible oil, spices, andalgae.Theoperating cost includespower consumption,CO2 losses,maintenance,andlabor.Inallcases,anextractionefficiencygreaterthan95%wasachievedandthe return of investment was less than 4 years. Based on the typical productionrequirementsselected,theplantsizevariessignificantlybetweenthethreeexamples.In thefirstcase, theexampleconsidered is therecoveryofflaxseedoil fromthecakeaftermechanicalpressing.Flaxseedoilisconsideredaspecialtyoil.Specialtyoilsaretypicallyprocessedbymechanicalpressingofseeds.However,thisprocess-ing techniquenormally leavesahighpercentageof residualoil (RO) in thecake(5 to15wt,%). Inmost cases, the cake is used as animal feed.Supercriticalfluidextractionprovidesasolvent-freemethodforrecoveringtheROfromthecake[9].TheoilextractedbysupercriticalfluidextractionhasahighercontentofphytosterolandvitaminEthantheoilobtainedbymechanicalpressing[10].Additionally,themealcanbefurtherprocessedtoobtainconcentratedorisolateproteins.Boththeoilandmealprocessedwithsupercriticalfluidextractionmeetthedemandsofthenutraceuticalandorganicmarkets.Similarprocessescanbeappliedtorecoverotherspecialtyoils,suchasborageandeveningprimrose.Thosespecialtyoilshavehighersalevalues,whichimplyshorterreturnoninvestment(ROI).
Inthesecondcase,thecasestudyistheextractionofastaxanthinfromamicro-algae (Hematococcus pluvialis). Astaxanthin content ranges from 1% to 3%. TheconcentrationofastaxanthinintheextractcanbemuchhigherbySFEthanbycon-ventional solvent (acetoneorhexane)bymanipulating the selectivityof the super-criticalsolvent.Extractswithastaxanthincontentrangingfrom5%to10%canbeobtained. Eventhoughtheoperatingcostsarehigh,theROIisshorterbecausethesalepriceoftheextractisveryhigh.
Inthethirdcase,thespiceselectedwasginger.Theextractionofgingeroleo-resinandessentialoiliscarriedoutbysequentialdepressurization.Inthiscase,thecombinedsalesofbothfractionsprovideaROIinlessthan2years.
2.5 ConClusIons
Supercriticalfluidtechnologyisconsideredbythenutraceuticalandpharmaceuticalsectors as a viable technology to satisfy customer demands by replacing conven-tional technologies as well as providing solutions that traditional technologiescannotprovide.Tosuccessfullyimplementthistechnologyontheindustrialscale,it isnecessarytounderstandthetechnology,focusingontheproperdesignoftheplantcomponentsandoptimizationoftheprocessparametersthatprovideminimumoperatingcosts.Someexampleshavebeenpresented,showingthatthistechnologyhas been successfully applied to commodity products. There is a future trend toimplement this technology as part of a process line combined with traditional
7089_C002.indd 48 10/15/07 5:22:53 PM
Supercritical Extraction Plants 49
processes—forexample,SFE+conventionalextraction+SFC,SFE+SFC,conven-tionalprocess+supercriticaldrying.
reFerenCes
1. Zosel,K.,U.S.Patent,3,806,619,1974. 2. Zosel,K.,U.S.Patent,4,247,570,1981. 3. Medina,I.andMartinez,J.L.,Dealcoholationofciderbysupercriticalextractionwith
carbondioxide,J. Chem. Tech. Biotech.,68(1),14,1997. 4. Martinez,J.L.,Ashraf-Khorassani,M.andChordia,L.,Supercriticalextractionprocess
ofgrape seedoil andphenoliccompounds,AICHEannualmeeting,SanFrancisco,2003.
5. Stahl, E., Quirein, K-W. and Gerar, D., Dense Gases for Extraction and Refining, Springer-Verlag,Berlin,1988.
6. Chordia,L.,Martinez,J.L.andDesai,B.,U.S.Patentapplication20050170063. 7. Martinez, J.L., Removal of residual solvents by supercritical fluids, AAPS 2004,
Baltimore,2004. 8. Chordia,L.andMartinez,J.L.,U.S.Patentapplication20050194313. 9. Chordia,L.andMartinez,J.L.,U.S.Patent7,091,366,2006. 10. Martinez,J.L.,Recovery of residual specialty oils after mechanical press using super-
critical fluid extraction,8thInternationalSymposiumonSupercriticalFluids,Kyoto,2006.
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51
3 Supercritical Fluid Extraction of Specialty Oils
Feral Temelli, Marleny D. A. Saldaña, Paul H. L. Moquin, and Mei Sun
CONTENTS
3.1 Introduction................................................................................................... 523.2 BioactivesinSpecialtyOils.......................................................................... 52
3.2.1 Carotenoids........................................................................................563.2.2 PolyunsaturatedFattyAcids(PUFAs)............................................... 573.2.3 Squalene............................................................................................. 583.2.4 Sterols................................................................................................ 583.2.5 Tocols................................................................................................. 59
3.3 ExtractionofDifferentTypesofSpecialtyOils........................................... 613.3.1 NutOils.............................................................................................. 62
3.3.1.1 FactorsAffectingExtractionYield...................................... 623.3.1.2 CharacterizationofProductsExtractedbySC-CO2............693.3.1.3 ComparisonwithConventionalMethods............................. 72
3.3.2 SeedOils............................................................................................ 723.3.2.1 FactorsAffectingExtractionYield...................................... 763.3.2.2 CharacterizationofProductsExtractedbySC-CO2............ 783.3.2.3 ComparisonwithConventionalMethods.............................80
3.3.3 CerealOils.........................................................................................803.3.3.1 FactorsAffectingExtractionYield......................................803.3.3.2 CharacterizationofProductsExtractedbySC-CO2............ 833.3.3.3 ComparisonwithConventionalMethods.............................84
3.3.4 FruitandVegetableOils....................................................................843.3.4.1 FactorsAffectingExtractionYield......................................863.3.4.2 CharacterizationofProductsExtractedbySC-CO2............ 893.3.4.3 ComparisonwithConventionalMethods.............................90
3.4 FutureTrends................................................................................................903.5 Conclusions................................................................................................... 91References................................................................................................................ 91
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52 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
3.1 INTrOduCTION
Some plant-based oils are classified as specialty oils due to their high concentra-tionsofbioactivecomponentswithdemonstratedhealthbenefits.Ingeneral,theyarecomprisedoftriacylglycerolswithafattyacidcompositionrichinunsaturatesandminorcomponentssuchastocols(tocopherolsandtocotrienols),carotenoids,sterols,andsqualene.Suchoilsincludenutoils(almond,hazelnut,peanut,pecan,pistachio,andwalnut),seedoils(borage,flax,eveningprimrose,grape,pumpkin,androsehip),cerealoils (amaranth, ricebran,andoatandwheatgerm),andfruitandvegetableoils (buriti fruit, carrot,olive, and tomato).Even though thedemand for specialtyoilsisgrowingatarapidpace,theyarestillconsideredanichemarketcomparedtothelarge-volumecommodityoils.Ingeneral,specialtyoilsaresoldintheformofcapsules,targetingthedietarysupplementmarket,aswellasgourmetoils.Similartocommodityoils,specialtyoilsarealsoproducedusingconventionalmethodsofmechanicalpressingand/orsolventextraction.Eventhoughcoldpressingattempera-tures below 60°C is used extensively in the specialty oil market, cold pressing islimitedintermsofoilrecoveryandthehighlevelsofresidualoilleftinthemeal.Ontheotherhand,conventionalsolventextractiondependsontheuseoforganicsolventssuchashexane,whichneeds tobe removedvia subsequent evaporation.Theheatappliedforsolventremovalmaybedetrimentaltoheat-labilebioactivecomponents.Inaddition,governmentregulationsontheuseoforganicsolventsaregettingstricterandthesafetyofresidualorganicsolventsinthefinalproductisbeingquestioned.
Supercriticalfluidextraction technologyisgrowingatarapidpacebecause itcanovercomemanyofthedisadvantagesassociatedwithconventionaltechnologiesand meet the consumer demand for “natural” products. The supercritical solventof choice for food applications has been supercritical carbon dioxide (SC-CO2).AdvantagesofprocessingwithSC-CO2includelowprocessingtemperatures;mini-malthermaldegradationoftheminorcomponentsofinterest;easeofseparationofextractionsolvent, resulting innosolvent residue left in theproduct;and the factthatprocessingintheCO2environmentminimizesundesirableoxidationreactions,whichisespeciallybeneficialforthesensitivebioactivecomponentsofspecialtyoilssuchassterols,tocols,carotenoids,andpolyunsaturatedfattyacids(PUFAs).
TheobjectivesofthischapteraretoreviewsomeoftherecentfindingsrelatedtothehealthbenefitsofbioactivecomponentspresentinspecialtyoilsandtheuseofSC-CO2extractiontechnologyfortherecoveryofspecialtyoilsfromdifferentplantsources,suchasnuts,seeds,cereals,fruits,andvegetables,withanemphasisontheeffects of various sample preparation and extraction parameters on theyield andcharacteristicsoftheoilsobtained.
3.2 BIOaCTIvES IN SpECIalTy OIlS
Ofthelargevarietyofbioactivecompoundspresentinnaturalsources,thischapterfocusesonlyonthecarotenoids,PUFAs,squalene,sterols,andtocols(tocopherolsand tocotrienols) foundmainly inspecialtyoils.ThemainchemicalandphysicalpropertiesofthesebioactivecomponentsaresummarizedinTable3.1[1,2].
7089_C003.indd 52 10/15/07 5:29:09 PM
Supercritical Fluid Extraction of Specialty Oils 53
TaB
lE 3
.1ph
ysic
al p
rope
rtie
s of
Bio
acti
ve C
ompo
unds
[1,
2]
Bio
acti
ve
Com
poun
dFo
rmul
aM
olec
ular
W
eigh
t
Mel
ting
po
int
(°C
)
Boi
ling
poin
t (°
C)
Solu
bilit
yaSt
ruct
ure
Car
oten
oids
β-
Car
oten
eC
40H
5653
6.87
183
—sl
EtO
H,c
hl;s
eth
.,ac
e,b
z
Ly
cope
neC
40H
5653
6.87
175
—sl
EtO
H,p
eth;
se
th;v
sbz
,chl
,C
S 2
L
utei
nC
40H
56O
256
8.87
196
—vs
bz,
eth
,EtO
H,p
eth
HO
H
OH
Toco
ls
sE
tOH
,eth
,ace
,chl
HO R2
R1
α-to
coph
erol
β-to
coph
erol
δ-
toco
pher
ol
γ-to
coph
erol
R1 CH
3C
H3
CH
3
CH
3H H
H HR2O
α-
Toco
pher
olC
29H
50O
243
0.71
3.0
210b
β-
Toco
pher
olC
28H
48O
241
6.68
—20
5b
δ-
Toco
pher
olC
27H
46O
240
2.65
—15
0c
γ-
Toco
pher
olC
28H
48O
241
6.68
–1.5
205b
cont
inue
d
7089_C003.indd 53 10/15/07 5:29:14 PM
54 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
TaB
lE 3
.1 (c
onti
nued
)ph
ysic
al p
rope
rtie
s of
Bio
acti
ve C
ompo
unds
[1,
2]
Bio
acti
ve
Com
poun
dFo
rmul
aM
olec
ular
W
eigh
t
Mel
ting
po
int
(°C
)
Boi
ling
poin
t (°
C)
Solu
bilit
yaSt
ruct
ure
Toco
ls (
cont
inue
d)
sE
tOH
,eth
.,ch
l,ac
e,o
il
HO R2
R1
α-tocotrieno
lβ-tocotrieno
lδ-tocotrieno
lγ-tocotrieno
l
R1 CH
3
O
CH
3
CH
3
CH
3H H
H HR2
α-
Toco
trie
nol
C29
H44
O2
424.
67—
—
β-
Toco
trie
nol
C28
H42
O2
410.
64
δ-
Toco
trie
nol
C28
H42
O2
410.
64
γ-
Toco
trie
nol
C27
H40
O2
396.
01
Ster
ols
C
ampe
ster
olC
28H
48O
400.
6815
7.5
——
HO
St
igm
aste
rol
C29
H48
O41
2.69
170
—vs
bz,
eth
,EtO
H
HO
7089_C003.indd 54 10/15/07 5:29:17 PM
Supercritical Fluid Extraction of Specialty Oils 55
β-
Sito
ster
olC
28H
50O
414.
7113
7—
sE
tOH
,eth
,HO
Ac
H
HO
Hyd
roca
rbon
Sq
uale
neC
30H
5041
0.72
–4.8
280d
slE
tOH
;se
th,a
ce,c
tc
Fatt
y a
cids
L
inol
eic
acid
C18
H32
O2
280.
45–7
229e
vsa
ce,b
z,e
th,E
tOH
O
OH
α-
Lin
olen
ica
cid
C18
H30
O2
278.
43–1
123
0–23
2fs
EtO
H,e
th;s
lbz
O
OH
γ-
Lin
olen
ica
cid
C18
H30
O2
278.
43—
——
OO
H
asl
:slig
htly
sol
uble
,s:s
olub
le,v
s:v
ery
solu
ble;
ace
:ace
tone
,bz:
ben
zene
,chl
:chl
orof
orm
,ctc
:car
bon
tetr
achl
orid
e,E
tOH
:eth
anol
,eth
:die
thyl
eth
er,H
OA
c:a
cetic
aci
d,
peth
:pet
role
ume
ther
,bB
oilin
gpo
inta
t0.0
133
kPa,
cB
oilin
gpo
inta
t0.0
0013
3kP
a,d
Boi
ngp
oint
at2
.266
kPa
,eB
oilin
gpo
inta
t2.1
33k
Pa,f B
oilin
gpo
inta
t0.1
33k
Pa.
7089_C003.indd 55 10/15/07 5:29:21 PM
56 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
3.2.1 Carotenoids
Carotenoidsrepresentagroupofover600fat-solublepigments[3].Thesepigmentsareresponsibleforthebrightyellow,orange,andredcolorsoffruits,roots,flowers,fish, invertebrates, birds, algae, bacteria, molds, and yeast. Some carotenoids arealso present in green vegetables, where their color is masked by chlorophyll [4].Carotenoidsaretypicallydividedintotwoclasses:carotenes,whichareC40poly-unsaturatedhydrocarbons, andxanthophylls,oxygenatedderivativesofcarotenes.Carotenoid compounds are colored due to their high level of conjugated doublebonds,whichalsomakesthemquiteunstable.Indeed,eachconjugateddoublebondcanundergoisomerizationtoproducevarioustrans/cisisomers,particularlyduringfoodprocessingandstorage[5].About10%ofcarotenoidsarecalled“provitaminA,”indicatingthattheypossessatleastoneunsubstitutedβ-iononeringthatcanbecon-vertedintovitaminA[4].Thetwomaincarotenoidsthathavebeenheavilystudiedare β-carotene and lycopene. In terms of specialty oils, carotenoids are mainlypresentinburitifruit,carrot,rosehip,tomato,andwheatgermoils.
Of all carotenoids,β-carotene has the highest provitamin A activity, approxi-mately twice thatofα-andγ-carotene[4]. In theearly1980s,evidencesupportedβ-carotene as a chemopreventive agent [6]. Thus,β-carotene was the subject of anumberofstudies,suchastheAlpha-TocopherolBeta-CaroteneCancerPrevention(ATBC),β-CaroteneandRetinolEfficacyTrial(CARET),andthePhysician’sHealthStudy. The ATBC trial concluded that β-carotene supplementation did not helpsmokerswhopreviouslyhadaheartattack;infact,theirriskoffatalcoronaryheartdiseaseactuallyincreased[7,8].TheCARETstudy[9]showedthatsupplementationwithβ-caroteneandvitaminAhadnobenefitforcurrentandrecentex-smokersandmale asbestos-exposedworkers and that itmay increase the incidenceand riskofdeathdue to lungcancer,cardiovasculardisease,andanyothercause.Finally, thePhysician’sHealthStudyconcludedthatβ-caroteneintakerenderedneitherbenefitnorharmintermsofcancer,cardiovasculardisease,stroke,oroverallmortality[10].
Lycopene, although lackingprovitaminAactivity, isknown tobeoneof themost potent antioxidants among the digestible carotenoids. Its highly conjugatedmolecularstructureisresponsibleforthebrightredcolorofripetomatoesaswellasthepigmentationofwatermelons,pinkgrapefruits,apricots,andpapayas[3,11].Anumberofstudieshaveshownthat lycopenecouldplayaprotectiverole in thedevelopmentofatherosclerosis[12,13].Aswell, in vivoand in vitrostudieshaveshown that it has a hypocholesterolemic effect, thereby suggesting that lycopenecould attenuate atherogenesis and reduce the risk of cardiovascular disease [14].Somestudieshavefoundthatlycopeneintakecouldlowertheriskofprostatecancer,whileothers reportednoprotectiveeffect [3].However,acasestudyshowed thathigh consumption of tomatoes and tomato-based food products reduced stomachcancers[15].AccordingtoOmoniandAluko[14],thecomplexinteractionamongthepotentiallybeneficial compounds found in tomatoesmight contribute to theiranticancer properties. According to Rao and Shen [16], the recommended dailyintakeoflycopeneis5to10mg/day.Itisinterestingtonotethatapproximately80%ofdietarylycopenecomesfromtomatoesandthatprocessedtomatoeshaveahigherleveloflycopenethanrawtomatoesbecauseheattreatmentandhomogenizationof
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Supercritical Fluid Extraction of Specialty Oils 57
tomatoesenhance theavailabilityof lycopene[17–19].Cohnetal. [20]comparedthe consumption of synthetic lycopene and lycopene in processed tomatoes andfound that theavailabilitywas thesame.Concerninghealthclaims, inNovember2005,theU.S.FoodandDrugAdministration(FDA)allowedthefollowingqualifiedhealthclaim:“Verylimitedandpreliminaryscientificresearchsuggeststhateatingone-halftoonecupoftomatoesand/ortomatosauceaweekmayreducetheriskofprostatecancer.FDAconcludesthatthereislittlescientificevidencesupportingthisclaim”[21].
3.2.2 Polyunsaturated Fatty aCids (PuFas)
PUFAsarefattyacidsthatcontaintwoormoredoublebondsinthecarbonchain.MostPUFAsareessentialfattyacidsandhavetobeprovidedtothebodythroughthediet.Theyareusuallyclassifiedasω-3andω-6,dependingonthepositionofthefirstdoublebondfromthemethylendofthecarbonchain.α-linolenic(ALA),eicosapentaenoic(EPA),docosapentaenoicacid(DPA),anddocosahexaenoic(DHA)acidsareexamplesofω-3PUFAs,whereaslinoleicacid(LA)andγ-linolenicacid(GLA)areexamplesofω-6PUFAs.ThemainsourceofEPA,DPA,andDHAarefishoils(seeChapter5).Becausethefocusofthischapterisplant-derivedspecialtyoils,onlyPUFAssuchasLA,ALA,andGLAwillbediscussed.With regard tospecialtyoils,PUFAsarefoundmainlyinalmond,apricot,hazelnut,peanut,walnut,borage,eveningprimrose,pumpkin,andricebranoils.
LA,ALA,andGLAareessentialfattyacidsthathumanenzymescantransforminto the PUFAs required by the body [22]. For instance, LA is converted by thehumanbodyintoarachidonicacid.Thelatter is transformedintoeicosanoidsandprostaglandins,whichareimportantmediatorsincardiovasculardisease[22].AlackofLAcanleadtofattyliver,skinlesions,andreproductivefailure[23].ALA,ontheotherhand,isconvertedtoEPAandDHA[23].DHAisamajorcomponentofthephospholipidmembranesofthebrainandretinas;therefore,alackofDHAcausesabnormalfunction[24].Whenthebodyexperiencesalackofω-3inthedietalongwithanincreaseinω-6,thelackofω-3tendstobeaccentuated,whichmayleadtoinhibitionofthesynthesisofDHAfromALA[25].Thus,itisimportanttokeeptheratioofω-6toω-3balancedinthediet.Somestudiesreportareductionincardio-vascular disease risk associated with higher ALA intake [26–28]. However, someinvestigatorsarestilltryingtoproveotherwise.SuchdebateisclearlyillustratedinthemultipleletterspublishedintheAmerican Journal of Clinical Nutrition [29,30].
It has been well established that intake of LA and GLA increases the tissuebiosynthesisof1-seriesprostaglandins,whichinturnsuppressesinflammation[31].ClinicalstudieshavealsoshownthatadministrationofGLAcanreducepainandswelling in rheumatoidarthritis [32,33].GLA is also said tobea“conditionallyessential fatty acid for the skin” [34]. Furthermore, a diet rich in EPA and GLAwasdeemedbeneficialforpatientswithacutelunginjury[35].BasedontheLyonDietHeartStudy,dietaryintakeofALAshouldbeabout1.8to2g/day[22].ThebestsourcesofALAarecanolaoilandalgae.NutsareagoodsourceofnotonlyALAbutalsoLA.DuetothecloseassociationbetweenALAandLA,careshouldbetakennottoconsumelargeamountsofLA-richoils,suchassoybean,sunflower,
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58 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
andwalnutoils[22].ThedoseofGLAfedduringastudyonrheumatoidarthritiswas 1.4g/day, which was reported to be well tolerated by the patients [32]. TheaveragerecommendedintakeofPUFAsis7%oftotalenergyintake[36].However,consumingexcessivelevelsofPUFAswithoutproperintakeofantioxidantsisnotrecommendedsincePUFAsarepronetooxidation,whichmayplayaroleincarcino-genesis[37]andotherdiseases.
3.2.3 squalene
Squaleneisalipidthatwasoriginallyobtainedfromsharkliveroil.Itisalsofoundinolive,palm,andwheatgermoils[38].Anumberofanimalstudiesshowedthatdietarysqualenehasdistinctanticarcinogeniceffects. Itwasshownthatsqualenepresentsinhibitoryactionincarcinogenesismodelsofskin[39,40],colon[41],andlung[42]cancer.However,itdoesnotpresentchemopreventiveactivity[43].Onereportedsideeffectofsqualeneisthatofa51-year-oldmanwithesophagealcancerwhodevelopedsevereexogenouslipoidpneumoniaaftereatinglargedosesofsqualene[44].
Besides its anticarcinogenic activity, squalene prevents lipid peroxidation inhumanskinsurface[45]andisusefulintreatingconditionsresultingfrominadequateimmuneresponse[46].Itisalsousefulasacytoprotectant(medicationthatcombatsulcersbyincreasingmucosalprotection)incyclophosphamide-inducedtoxicities[47]andlow-dosesqualene(860mg)coadministrationwithlow-dosepravastatin(10mg)furtherenhancestheefficacyofpravastatinasacholesterol-loweringdrug[48].
Inthehumanbody,squaleneistheprecursortoimportantsterolssuchascholes-terol[49,50].Thus,itwasoriginallythoughtthatincreasedsqualeneconsumptionwouldactuallyincreasebloodcholesterollevels.Thisdoesnotseemtobethecasewhen0.5gofsqualeneisconsumedperday.Indeed,MiettinenandVanhanen[51]observedanincreaseintotalbloodcholesterolconcentrationsinmalesubjectsafteradietarysupplementationof1g/dayofsqualenefor9weeks,butwhenthedosewasreducedto0.5g/day,thebloodsterollevelswentbacktonormal.Itistruethat60%to80%ofdietarysqualeneisabsorbedthroughtheintestine[52]andthatasubstan-tialamountofthissqualeneisconvertedtocholesterolinthehumanbody.However,areasonableincreaseinsqualeneconsumptionappearstosignificantlyincreasethefecalexcretionofcholesterol[53].
3.2.4 sterols
Themainsterolsinplantmaterialsaresitosterol,campesterol,andstigmasterol[54].Theyaremainlyfoundinthespecialtyoilsofacorn,hazelnut,walnut,cherry,grape,pumpkin,andricebran.Overtheyears,ithasbeenwellestablishedthatahighdietaryintakeofphytosterolslowersbloodcholesterollevelsbycompetingwithdietaryandbiliarycholesterolduringintestinalabsorption[55–57].However,recently,PlatandMensink[58]speculatedthatbecausephytosterolsaremorereadilyoxidizedbyfreeradicalsthancholesterol,theycouldincreasethelevelofoxidizedlow-densitylipo-proteins(LDLs),whichformatheroscleroticplaquesinarteries.Atthistime,littleinformationisavailabletoprovesuchaclaim.Ontheotherhand,phytosterolsarenotrecommendedforindividualswhoaresufferingfromsitosterolemia,aninher-itable disorder that increases the absorption of cholesterol and phytosterols [55].
7089_C003.indd 58 10/15/07 5:29:24 PM
Supercritical Fluid Extraction of Specialty Oils 59
Fortunately,thisdisorderisquiterare;Björkhemetal.[59]knewof45patientswiththisdiseasein1998.
Phytosterolshavealsobeenthesubjectofmuchinvestigationforpropertiesotherthantheirabilitytolowercholesterol.Inthe1980s,consumptionofsitosterolwasshowntoreducecoloncancerinrats[60];however,thereisstillnostrongandcon-sistentevidencethatthesamewouldbetrueinhumans[61].Somestudiesreportthatsterolsmayhaveaneffectonimmunefunctionandthattheycouldpreventthesubtleimmunosuppression experienced by marathon runners [62, 63]. Finally, animalstudiesreportedthatphytosterolscouldinhibittheinflammatoryresponse[64]andthattheycouldcauseinsulin-releasingpropertiesfordiabetics[65].AccordingtotheScientificCommitteeonFood,theaverageamountofphytosterolsintheWesterndietis150to400mg[66].However,therecommendeddoseofphytosterolstoreduceplasmaLDL-cholesterollevelsby5%to15%is1.3to2g/day[67,68].InordertoachievesuchlevelsandcomplywiththeFDA-approvedhealthclaimontheroleofplantsterolorplantstanolestersinreducingtheriskofcoronaryheartdisease[69],foodmanufacturershave introducedvarious functional foodproductswithaddedphytosterols. In those countries where such products are currently marketed, thesuccessissogreatthatauthoritiesarenowworriedaboutconsumerseatingtoomuchphytosterols.Theconcernislegitimatebecauseastudyshowedthatdailyconsump-tionof3.8to4.0gofplantsterolesterscansignificantlylowerserumconcentrationsofvariouscarotenoidsandtocopherols[70].
3.2.5 toCols
TocopherolsandtocotrienolsmakeupthetocolsfamilyofvitaminEcompounds,whichmustbeobtainedfromthedietbecausehumanscannotsynthesizethem[71].Tocolsarefoundinalmond,hazelnut,pecan,walnut,flax,buritifruit,tomato,ricebran,andwheatgermoils.Thedifferencebetweentocopherolsandtocotrienolsliesin thephytylchainattached toachromanol ring: thephytylchain issaturated intocopherols,whereasthephytylchainintocotrienolshasthreedoublebonds[72].Thesecompoundsrepresentagroupoffourisomerswithvaryingnumbersandposi-tionofmethylgroupsonthechromanolring:α-,β-,γ-,andδ-tocopherolandα-,β-,γ-,andδ-tocotrienol.
Althoughallof these tocol isomers are absorbed through the intestine in thehumanbody, it isbelieved thatonlyα-tocopherolcontributes towardmeeting thehumanvitaminErequirement[73].Thereasoningbehindthisclaimisthatintra-cellular vitamin E content and distribution are regulated by different proteinsbindingspecificallytoα-tocopherol[74].Anotherfactorcontributingtothisschoolofthoughtisthatα-tocopherolisthemostpotentnaturallyoccurringscavengerofreactive oxygen and nitrogen species [72]. However, evidence is building on theimportanceofconsumingamixtureofthewholefamilyofvitaminEcompoundssincetheymayhaveadditiveandsynergisticactivitiesthatsupportbroaderbenefi-cialbiologicalfunctions[75].Theymayalsoactsynergisticallywithothernaturallyoccurringcompoundscommonlyfoundinfruitsandvegetables[72].Anoverviewofthecurrentscientificliteraturerevealstheimportanceoftocopherolsandtocotrienolsasthemajorfat-solubleantioxidants[73].Indeed,thesemoleculescanscavengefree
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60 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
radicalsinthebody,therebypreventingthemfromdamagingcellmembranesandgeneticmaterialandchangingthecharacteroffatsandproteins[76].OneexampleistheprotectionthatvitaminEgrantstoPUFAs,whichareespeciallyvulnerabletodestructiveoxidation[73].EventhoughvitaminEisviewedasapotentantioxidant,in vitro studies have shown that vitamin E may have pro-oxidant effects at highdosages [77,78]. Interestingly, itappears thatα-tocopherolalonecanhaveapro-oxidanteffect[79];however,inthepresenceofγ-andδ-tocopherol,thepro-oxidanteffectofα-tocopherolseemstodiminish[75].
Theantioxidantpropertiesofmixedisomersoftocolscouldalsobebeneficialinpreventingtheonsetofatherosclerosis[80,81].ThismedicalconditionisinpartduetofreeradicalsoxidizingLDLs,whichinturnformatheroscleroticplaquesonthesurfacesofarterywalls.Byscavengingfreeradicals,tocopherolsblockthisprocess[80].Tocotrienolsarealsobelievedtobeusefulinthepreventionandtreatmentofatherosclerosisinpeoplewithtype2diabetes[82].Besidesitsantioxidantproper-ties,α-tocopherol acts as a regulator of gene expression that lowers the build-upofoxidizedLDLsinarteries[83,84].Unliketheliteratureonatherosclerosis,thescientificliteratureontheeffectsofvitaminEoncardiovasculardiseaseisdivided.SomeplainlystatethatthereisnoconcreteevidencethatvitaminEreducescardio-vascular-relatedmortality,particularlyinhigh-riskindividuals[85],whereasothersshow a reduction in mortality [86, 87] or no effect on cardiovascular events anddeath[75,88–92].
TheapplicationofvitaminEincancertreatmenthasalsobeenstudied.AlthoughanimalstudiesweresuccessfulinshowingthatvitaminEinhibitedcarcinogenesisandultraviolet-induceddeoxyribonucleicaciddamage[93–96]andpreclinicaldatarevealedthatitmightstimulateanantitumorimmuneresponse[97],clinicaltrialshavegivenmixedresults[72].SomestudiesonpatientswithstageIandIIheadandneckcancerwhowerefedsupplementsofα-tocopherolshowedahigherincidenceofsecondprimarycancersandalowerdegreeofcancer-freesurvival[98].Also,afewstudiesshowedthatα-tocopherolwasoflittletonobenefitinpreventinglungcancer[99].However,α-tocopherylsuccinatewasfoundtobetumorspecificinprostateandbreasttissues[100].Thereisalsoevidencethatγ-tocopherolmayhelppreventandtreatcolon[101]andprostate[102]cancer.
Anumberofstudiesreport thatvitaminEcanbenefit individualswithosteo-arthritis[103–106].Supplementationof400mg/dayofvitaminEhadabeneficialanalgesicandanti-inflammatoryeffect,withalowincidenceofsideeffects[107].Aswell,supplementationwithmixedisomersofvitaminEwasadvantageous[108].Some research has shown that α-tocopherol supplementation delays or preventsAlzheimer’sdisease(AD)diagnosisofelderlyindividualswithsignsofmildcogni-tiveimpairment[109];however,othersreportnoclearbenefitsofvitaminEinthetreatmentofpeoplewithAD[110–112].UnrelatedtoAD,astudyonnursinghomeresidentsshowedthatsupplementalvitaminEreducedtheincidenceanddurationofrespiratoryinfections[113].
The FDA has approved the following health claim for dietary supplementscontaining vitamin E and/or vitamin C [114]: “Some scientific evidence sug-gests that consumption of antioxidant vitamins may reduce the risk of certainformsofcancer.However,FDAhasdeterminedthatthisevidenceislimitedand
7089_C003.indd 60 10/15/07 5:29:25 PM
Supercritical Fluid Extraction of Specialty Oils 61
notconclusive.”Someworkhasalsobeenperformed toachievequalifiedhealthclaims for vitaminE supplements against cardiovascular disease. However, thishealthclaimwasrefusedbasedoninsufficientevidence[115,116].Theoptimumconsumption levelofvitaminE for thegeneralpopulation is an interestingcon-sideration that is stillopen todebate.According toHealthandWelfareCanada,the Recommended Daily Intake (RDI) for vitamin E should be 5 to 10 mg; theAmericanDieteticAssociationsetstheRecommendedDietaryAllowance(RDA)at15mg/dayofα-tocopherolforadultsand19mg/dayforwomenwhoarebreast-feeding[73].BecausenotallsourcesofvitaminEhavethesamebiologicalactivity,one has to keep in mind that 1international unit (IU) of natural vitamin E cor-responds to0.67mgofα-tocopherol,whereas1IUofsyntheticvitaminEcorre-spondsto0.45mgofα-tocopherol[73].Inaddition,thegrowingevidenceshowingthebenefitsofmixed isomersof tocopherolsand tocotrienols shouldprobablybereflectedintheRDIandRDA.AlthoughvitaminEwasconsideredtobenontoxicformanyyears, it isnowknownthatanoverdoseofvitaminEcaninterferewithbloodclotting,especiallywhentakenalongwithanticoagulantmedicationorwithacetylsalicylicacid[117].Furthermore,arecentmeta-analysisincluding19studieswithmorethan135,000patientsshowedthatmorethan400IU(270mg)perdayofvitaminEsupplementationtopatients(ages47to84years)whomostlyhadchronicdiseasesmighthaveincreasedall-causemortality[118].
3.3 ExTraCTION OF dIFFErENT TypES OF SpECIalTy OIlS
The specialty oils rich in bioactives can be extracted from many plant sources.Theseextractedoilsaremainlymixturesoftriglycerides,freefattyacids(includingPUFAs),monoglycerides,diglycerides,andotherminorcomponents,suchastocols,carotenoids, sterols, and squalene. For specialty oils to be extracted from differ-entplantmaterialsusingSC-CO2,theyhavetobesolubleinSC-CO2.Thesolubil-itybehaviorofmajorandminorlipidcomponentsinSC-CO2hasbeenpreviouslyreviewedandcorrelated[119,120].SolubilityisastrongfunctionofSC-CO2densityandthepropertiesofthesolute,suchasmolecularweight,polarity,andvaporpressure.AllthelipidcomponentsofinterestinspecialtyoilsaresolubleinSC-CO2todiffer-entextents,dependingontemperatureandpressureconditions.Generally,solubilityof lipids inSC-CO2decreaseswithan increase inpolarityandmolecularweight,thusfollowingtheorder:fattyacidesters,fattyacids,andtriglycerides[120].
SC-CO2extractionofspecialtyoilsfromvarioussourceshasbeenstudiedquiteextensively. The extraction efficiency and the characteristics of the products areaffectedbyseveralparameters,suchasparticlesizeandmoisturecontentofthefeedmaterial,extractiontemperatureandpressure,solventflowrate,extractiontime,andtheuseofacosolvent.Therefore,thefollowingdiscussionemphasizestheimpactoftheseprocessingparametersontheyieldandcompositionofspecialtyoilsobtainedfromnuts,seeds,cereals,fruits,andvegetables.Eventhoughsomenutsareseedswithin the fruit of the plant, they are classified in this chapter as nuts based onconsumption;forexample,almondisclassifiedasanutbecauseitisconsumedasasnackuponroasting,whereasapricotkernelisclassifiedasaseed.Inaddition,some
7089_C003.indd 61 10/15/07 5:29:26 PM
62 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
fruitsandvegetablesliketomatoesandgrapesarealsoincludedinthediscussionofseedsbecausetheirseedswereusedinextractionstudies.
3.3.1 nut oils
Nutsprovideprotein,fiber,essentialfattyacids,andvitamins.Theirpleasantflavorand aroma and unique texture lead to their popularity as snacks. However, theirhighfatcontentlimitstheirconsumptionduetoconsumerconcernsabouttheirhighcaloric content. Besides, nuts are prone to oxidation due to the presence of highlevelsofPUFAs.Eventhoughtheyhavelowoxidationstability,nutoilshavebeencommercializedinsomecountriesashighlynutritiousspecialtyoils.Defattednutsalsohavemarketvalueaslow-fatproducts;therefore,thepropertreatmentofnutsbeforeandafterextractionneeds tobeconsidered.NumerousstudiesonSC-CO2extraction of nuts, including acorn [121], almond [122–125], hazelnut [126–128],peanut[129–133],pecan[134–136],pistachio[137],andwalnut[138–140],havebeenreportedandaresummarizedinTable3.2.
3.3.1.1 Factors affecting Extraction yield
3.3.1.1.1 Sample PreparationMost of the nuts were extracted fresh, but some were roasted [122, 141]. Feme-niaetal. [122]reported that theroastednutshadhigheroilcontentdue towaterlossandpartialdegradationofproteinandprobablypectinaswellduringroasting.However,itwasdifficult toextracttheoilfromroastedalmonds,possiblyduetotheformationofnewlinksamongcellwallpolymers,thusreducingporosityandstrengtheningthewallstructure[122].
a) Particlesize:Thenutsaregenerallygroundtosmallparticlestoincreasesurfaceareaandshortenthepathlengthsoverwhichthesolutesmusttraveltoreachthebulkfluidphaseandthereforefacilitatetheextractionofthenutoil.Thus,particlesizeimpactsextractionkineticsoftheoil,whichispres-entasreleasedoilonthesurfaceoftheparticlesaswellasunreleasedoilinsidetheparticles.TheextractionrateisdictatedinitiallybythesolubilityofthefreeoilinSC-CO2(fastextractionperiod)andlaterbythediffusionofoilinsidetheparticles(slowextractionperiod).Ingeneral,whenfreshsolventcomesincontactwiththefeedmaterial,thefreeoilonthesurfaceisquicklysolubilizedandextracted.Extractionrateisfastandlimitedbyequi-libriumsolubility,asrepresentedbytheinitiallinearportionoftheextrac-tioncurve.Whentheoilonthesurfaceoftheparticlesisdepleted,SC-CO2hastodiffuseintotheparticlesandsolubilizetheoilandSC-CO2+oilhastodiffuseout,whichisaslowprocessdrivenbytheoilconcentrationgradi-ent;thustheextractioncurveapproachesaconstantvalueasymptotically.Özkaletal.[126]reportedayieldof0.51goil/ghazelnutattheendofthefastextractionperiodandonly0.01goil/ghazelnutduringtheslowextrac-tionperiod.Therefore,theextractioncouldbestoppedafterthefastextrac-tionperiod.Theoilyieldobtainedattheendofthefastextractionperiod
7089_C003.indd 62 10/15/07 5:29:27 PM
Supercritical Fluid Extraction of Specialty Oils 63
TaB
lE 3
.2Ex
trac
tion
of B
ioac
tive
Com
poun
ds fr
om N
uts
usi
ng S
C-C
O2
raw
M
ater
ial
Feed
(g)
Sam
ple
prep
arat
ion
Bio
acti
ve
Com
poun
d
Extr
acti
on C
ondi
tion
sr
ecov
erya
(%)
ref
.pa
rtic
le S
ize
(mm
)H
2O (
%)
T (°
C)
p (M
pa)
Flow
rat
eTi
me
(min
)C
osol
vent
Aco
rnn.
i.0.
27n.
i.O
leic
aci
d,
linol
eic
acid
,β-
sito
ster
ol,
stig
mas
tero
l,ca
mpe
ster
ol,
toco
pher
ols
4018
1.5
×1
0–2 m
/min
bn.
i.N
one
n.i.
121
Alm
ond
1500
–200
0n.
i.n.
i.U
nsat
urat
edo
il50
3333
3.3–
666.
7g/
min
n.i.
Non
en.
i.12
2
4000
n.i.
n.i.
Uns
atur
ated
oil
6048
.2n.
i.n.
i.N
one
n.i.
123
3000
–400
0M
illed
,bro
ken,
w
hole
n.i.
Toco
pher
ols,
ol
eic
acid
,lin
olei
cac
id
35,4
0,5
035
,45,
55
166.
7,3
33.3
,50
0g/
min
n.i.
Non
en.
i.12
4
n.i.
0.3,
0.7
,1.9
n.i.
Uns
atur
ated
oil
4035
12,2
3.8
g/m
inn.
i.N
one
n.i.
125
Haz
elnu
t4
<0
.85
3O
leic
aci
d,
linol
eic
acid
40,5
0,6
030
,37.
5,4
51,
3,5
g/m
in10
Non
e34
126
51–
23
Ole
ica
cid,
lin
olei
cac
id40
,50,
60
30,4
5,6
02
×1
0–3L
/min
250–
300
Non
e59
127
500.
7n.
i.U
nsat
urat
edo
il,
β-si
tost
erol
,α-
toco
pher
ol
35–4
818
–23.
42.
7–4.
3×
10–2
m/m
in24
0N
one
>95
128
cont
inue
d
7089_C003.indd 63 10/15/07 5:29:28 PM
64 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
TaB
lE 3
.2 (c
onti
nued
)Ex
trac
tion
of B
ioac
tive
Com
poun
ds fr
om N
uts
usi
ng S
C-C
O2
raw
M
ater
ial
Feed
(g)
Sam
ple
prep
arat
ion
Bio
acti
ve
Com
poun
d
Extr
acti
on C
ondi
tion
sr
ecov
erya
(%)
ref
.pa
rtic
le S
ize
(mm
)H
2O (
%)
T (°
C)
p (M
pa)
Flow
rat
eTi
me
(min
)C
osol
vent
Pean
ut10
00(
h.e.
),
500
(v.e
.)h.
e.:0
.86–
1.68
v.
e.:0
.86–
1.19
,3.
35–4
.75
n.i.
Uns
atur
ated
oil
h.e.
:25–
100;
v.e
.:25
–120
h.e.
:27.
5–69
;v.
e.:1
4–55
h.e.
:20
L/m
in;
v.e.
:40
L/m
inv.
e.:1
80N
one
h.e.
:50;
v.
e.:9
912
9
5,57
90.
864–
1.18
,1.
18–1
.7,1
.7–2
.36,
2.
36–3
.35,
3.
35–4
.75
5,9
,15
Uns
atur
ated
oil
25,5
5,7
5,
9527
.5,4
1.5,
55
40a
nd6
0L
/min
180
Non
e99
130
50,8
60G
roun
d:0
.864
–1.1
8,
1.18
–1.7
,1.7
–2.3
6,
2.36
–3.3
5,
3.35
–4.7
5Fl
akes
:1.2
7
n.i.
Uns
atur
ated
oil
2555
40L
/min
180
Non
en.
i.13
1
1n.
i.n.
i.U
nsat
urat
edo
il40
–80
13.8
–55.
1n.
i.n.
i.N
one
n.i.
132
n.i.
n.i.
4.2–
5.1
Uns
atur
ated
oil
50–6
535
–50
n.i.
n.i.
Non
en.
i.13
3
Peca
n5–
6H
alve
s4
Uns
atur
ated
oil,
to
coph
erol
s40
,80
41.3
,55.
1,
68.9
n.i.
160
Non
e77
134
20H
alve
s4.
9,6
.4,7
.4,
11U
nsat
urat
edo
il75
623
L/m
in60
Non
en.
i.13
5
90H
alve
s,p
iece
sH
alve
s:4
.8;
Piec
es:4
.1U
nsat
urat
edo
il45
,62,
75
41.3
,55.
1,
62,6
6.8
1,1
.5,2
,2.5
,3,4
,7.
5L
/min
60N
one
n.i.
136
Pist
achi
o10
1–1.
68n.
i.U
nsat
urat
edo
il50
,60,
70
20.7
,27.
6,
34.5
2.6
g/m
inn.
i.10
%E
tOH
+66
.1c
137
7089_C003.indd 64 10/15/07 5:29:28 PM
Supercritical Fluid Extraction of Specialty Oils 65
Wal
nut
n.i.
0.01
,0.0
5,0
.1,0
.5n.
i.L
inol
eic
acid
,ol
eic
acid
,lin
olen
ica
cid,
β-
sito
ster
ol,
cam
pest
erol
,α-
toco
pher
ol
35,4
0,4
5,
4818
,20,
22,
23
.4n.
i.n.
i.N
one
9513
8
n.i.
Piec
es3
Lin
olen
ica
cid,
un
satu
rate
doi
l80
68.9
150
g/m
inn.
i.N
one
n.i.
139
n.i.
Piec
esn.
i.U
nsat
urat
edo
il80
68.9
150
g/m
inn.
i.N
one
n.i.
140
T:t
empe
ratu
re,P
:pre
ssur
e;n
.i.:n
otin
dica
ted;
h.e
.:ho
rizo
ntal
ext
ract
ion,
v.e
.:ve
rtic
ale
xtra
ctio
n,a
Rec
over
y(g
ext
ract
/go
ilin
fee
dm
ater
ial×
100
),b s
uper
ficia
lvel
ocity
,c yie
ld(
g/10
0g
feed
mat
eria
l).
+
Cos
olve
nta
dded
con
tinuo
usly
into
SC
-CO
2at
the
leve
l(%
,w/w
)in
dica
ted.
7089_C003.indd 65 10/15/07 5:29:29 PM
66 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
wasdependentonparticlesize[126].Forexample,theoilyieldforhazelnutwithaparticlesizeof1.5mmwasabouthalf(0.22goil/gnut)ofthatfromparticleslessthan0.85mm(0.51g/g).Inanotherstudy,usingwhole,bro-ken(4to8mm),andmilled(0.5to3mm)almond,Leoetal.[124]observedthattheextractionyieldincreasedwithreducingparticlesize(50,350,and800g/kg,respectively)undersimilaroperatingconditions.Inaddition,oilrecoveryincreasedfrom36%to82%whentheparticlesizeofpeanutwasreduced from 3.35 and 4.75 mm to 0.86 and 1.19 mm [130]. There is apracticallimittogrindingtosmallerparticlesduetotheoilynutparticlesstickingonthesieves[130].
b) Moisture and equilibration time: Moisture has a great impact on oilextractionbecausethekernelexpandswithmoistureabsorption,resultingin a more permeable cell membrane, allowing both oil and CO2 to passmorereadily.However,excesswatercanalsoimpedethediffusionofoiland have a negative effect on oil accessibility. The amount of pecan oilextractedafter48hoursofmoistureequilibrationwasapproximately30%higher thanthatobtainedafter1hour[135].Waterwascoextractedwiththeoiland increased linearlywith the initialmoisturecontentofpecans[135]. The moisture content of the extracted oil was 0.7% and 11.7% atinitial moisture levels of 3.5% and 12%, respectively, in the pecan. Theoilobtainedwascloudywithayellowishcolor.Ahigh levelofwater inthe extracted oil is not desirable since it negatively impacts its stability.Moistureaffectsnotonlyextractionefficiencyandyieldbutalsothephysi-calstructureofthenuts[135].Breakageofthekernelsduringtheextrac-tioncanbeavoidedbyadjustingthemoisturecontenttoacertainlevel,forexample,8%to11%(w/w)forpeanuts[133].Passeyetal.[133]testedsoak-ing,steaming,andhumidificationaspretreatmentmethodsforpeanutsandfoundthatsoakingandsteamingwereaseffectiveashumidificationinpre-ventingthebreakageofthekernels,buttheycausedbrowning,lossofwatersolubles, and low rate of extraction. Moisture content also affects pecanbreakage[135].Withmoistureabsorption,thekernelbecomessoftandpli-able,inpartbecausethemoistureaffectstheplasticizationofproteinsandcarbohydratesandaltersthephysicalpropertiesofthetissues.Extractionofpecanafter48hoursequilibrationproducedlessbreakagethanextractionat1houratmoisturecontentsof6.1%and7.7%[135].Thiswasduetothewaterinthekernelbeingmoreevenlydistributedafteralongerequilibra-tiontime.However,theeffectoftheequilibrationtimedecreasedat8.5%moistureandwasnegligibleat11.6%[135].Thiscanbeexplainedbytheosmoticpressurecausedbythewateraroundthekernel.
3.3.1.1.2 Extraction Parameters a) Temperature and pressure: Most of the nut oil extractions were per-
formed at a temperature range of 35°C to 100°C and a pressure rangeof9to70MPa.Thesolubilityofoil inSC-CO2ismainlydeterminedbytheSC-CO2density and thevolatilityof theoil components. Ingeneral,
7089_C003.indd 66 10/15/07 5:29:29 PM
Supercritical Fluid Extraction of Specialty Oils 67
SC-CO2 density increases with pressure at constant temperature anddecreaseswithtemperatureatconstantpressure,wherethedensitydecreasebecomessmallerathigherpressures.Ontheotherhand, thevolatilityofoilcomponentsincreaseswithtemperature.Thesetwoopposingeffectsoftemperatureondensityandvolatilityleadtothewell-establishedcrossoverbehaviorof solubility isotherms.A temperature increasemayalsocausebreakdown of cell structure and increase the diffusion rate of the oil intheparticles,thereforeacceleratingtheextractionprocess[130].Itisalsoimportanttoconsiderthatoilcompositionvarieswidelyamongthediffer-enttypesofnutsandthusdifferencesinfunctionalgroupsandfattyacidcompositionare responsible for thedifferences involatility, solubility inSC-CO2,andcrossoverpressure.Solubilityincreaseswithtemperatureandpressureabovethecrossoverpressure.Thesegeneraltrendsoftheeffectsoftemperatureandpressureonoilsolubilityandyieldarereflectedinsomestudies.Forexample,thesolubilityofpeanutoilinSC-CO2decreasedwithtemperatureatpressuresbelow35MPaandincreasedathigherpressures[130].Increasingthetemperaturefrom29°Cto91°Cincreasedtheinitialextractionratefrom15to129mgpeanutoil/LCO2at55MPa;however,at27.5MPa, increasingthe temperaturefrom27°Cto100°Cdecreasedtheinitialextraction rate from7.6 to0.5mgoil/LCO2[130]. Increasing thepressurefrom41.3to55.1MPaincreasedthepecanoilyieldfrom14.3%to 21.3% at 45°C and from 17.5% to 31.5% at 75°C. However, a furtherincrease inpressure to66.8MPaonly slightly increased theoil yield to21.5%and32.4%at45°Cand75°C,respectively[136].Thepositiveeffectofpressureonoilyieldwasalsoobservedathightemperatures.Similarly,raisingthepressurefrom17.7to68.9MPayielded100%and200%moreoilfrompecansat40°Cand80°C,respectively[134].
The rate of depressurization following an extraction affects thebreakage of the nut kernel, as demonstrated for the pecan kernel [135].When the vessel was depressurized from 62 MPa in 20 min there wasnobreakage,whereasasignificantamountofbreakageoccurredduringthe10-mintest.Duringthe20mindepressurization,thepressureintheextractionvesselwasdropped from62MPa toabout7MPawithin thefirstminuteandwasaround2MPaafter10minand0MPaafter20min.This suggested that the final stages of depressurization were crucial incausingpecanbreakage.When theextractionvesselwasopened imme-diatelyafterdepressurizingthereactor,theparticlesjumpedaroundandpoppingsoundswereheard,suggestingthatmostofthebreak-upoccurredastheCO2-saturatedparticlesweredepressurized[130].Gradualpressur-izationanddepressurizationwerealsonecessarytominimizedamagetothewalnutpieces[139].TheruptureorbreakageofthecellsoccursduetothephasechangeofCO2.ArapiddepressurizationtoatmosphericpressureformsliquidCO2aswellasdryice.Therefore,theCO2trappedinsidethesolidmatrixexpands,causingbreakageinthecells.Slowdepressurizationwith appropriate level ofheating toovercome the Joule-Thomsoneffect
7089_C003.indd 67 10/15/07 5:29:30 PM
68 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
andtomaintainthetemperatureabove31°CassuresthattheCO2willbeingasphaseandavoidanybreakage.
b) Flowrateandflowdirection: Increasing solventflowrate results inanincreasedsolvent-to-feedratioanddecreasedmasstransferresistance.Theextractedpecanoilyieldincreasedfrom8.8%to21.5%withanincreaseinflowratefrom1to4L/min(measuredatstandardtemperatureandpressure,STP);however,withafurtherincreaseto7.5L/min,theoilyieldchangedonlyfrom21.5%to21.7%[136].Theeffectofflowratewasalsostudiedfortheextractionofhazelnutoil[127].Atalowpressure(15MPa),anincreaseinflowratefrom0.5to2mL/min(measuredatextractorpressureand10°C)didnotcauseasignificantdifferenceintheextractionyieldofhazelnutoil;however, at a highpressure (30MPa), theoil yield increasedmore thanthree-fold[127].ThismightbeduetothelowsolubilityofoilinSC-CO2atlowpressures.AnotherimportantfactorthataffectsextractionyieldisthedirectionoftheCO2flow.Theverticalextractorproducedahighertotaloilrecoveryandamoreuniformlyextractedpeanutmealsamplethanthehorizontal extractor [130]. This might be caused by the meal settling inthehorizontalextractorandleavingalowerresistanceflowpathalongthetop of the meal, leading to channeling and insufficient contact betweenthe fresh solvent and peanut meal. Similarly, the flow direction greatlyaffected thesolubilityof thepeanutoil inSC-CO2atboth55MPa/75°Cand55MPa/95°C,withthedownwardflowresultinginahighersolubility[130].Thisisprobablybecauseofthelargetemperaturegradientbetweenthetopandbottomoftheextractorintheupwardsystem,whichmightbedue to the incoming fresh CO2 cooling the inside of the extractor. Withdownwardflow,itwaspossibletomaintainthetemperaturethroughouttheexperiment,whichwasattributedtothebalancingofdensity-inducedcon-vectioneffectswithdownwardflow[130].Thus,itwaspossibletomaintaintheconstantextractionrateforalongerperiod.
c) Extractiontime:Duetothephysicalstructureofthenut,thepenetrationof thesolventand thediffusionof theunreleasedoil in theparticlesareveryslow.Therefore,extractiontimeisusuallylimitedtothefastextrac-tionperiodsincetheamountofoilrecoveredintheslowextractionperiodis negligible. The duration of the fast extraction period is also inverselyrelated to particle size. However, the extraction rate and the duration ofthefastextractionperiodarealsoaffectedbytemperature,pressure,flowrate,andcosolventaddition.Forexample,thedurationsofthefastextrac-tionperiodforthehazelnutoilextractionsconductedat50°Cand45MPawere50and60minforparticlesizesoflessthan0.85mmand1.5mm,respectively[126].Whentheextractionconditionswerechangedto40°C,37.5MPa,and5g/minflowrate,thefastextractionperiodwas50minforthenutparticlesoflessthan0.85mminsize[126].Fastextractionperioddecreasedfrom183to64and32minwithapressureincreasefrom30to45and60MPaat40°C.Ontheotherhand,itdecreasedfrom64to33minwithatemperatureincreasefrom40°Cto50°Cat45MPa[127].
7089_C003.indd 68 10/15/07 5:29:31 PM
Supercritical Fluid Extraction of Specialty Oils 69
d) Useofcosolvent:Ahigheryieldcanbeobtainedbyaddingasmallamountofcosolvent,suchasethanol.Forexample,when10%ethanolwasaddedtoCO2(w/w),theextractionyieldofpistachiooilat60°Cincreasedby230%at34.5MPaandby750%at20.7MPacomparedtothatobtainedwithCO2alone[137].Inaddition,theextractionyieldofpistachiooilusing10%etha-nolasacosolventat60°Cwashigherat34.5MPathanthatat20.7MPa.
3.3.1.2 Characterization of products Extracted by SC-CO2
3.3.1.2.1 Chemical CompositionThefattyacidcompositionofthenutoilsispresentedinTable3.3.Themainfattyacidsintheextractednutoilsarelinolenic(Ln),linoleic(L),oleic(O),andpalmitic(P)acidsformingtriglycerideslikeLLL,OLL,LLLn,OOO,andPOO,theamountsofwhicharedependentonthetypeofnut.ThefattyacidcompositionofSC-CO2-extractedoilsexhibitedminordifferencesincomparisontooilsobtainedfromthefeedmaterial.However,asmallincreaseinthepercentageofoleicandstearicacidswasdetectedwhenabout65%ofthealmondoilwasextracted[122],indicatingthatSC-CO2 extraction may result in minor modifications of the fatty acid profile oftheextractedoils.Ontheotherhand,nofractionationwasdetectedduringextrac-tionsofhazelnutoil,asthefattyacidcompositionofthethreehazelnutoilfactionsobtainedduringthefirst30min,between70and120min,andafter120minwassimilartothatoftheoilextractedwithhexane[127].Therewasalsonosignificantdifferenceinthefattyacidcompositionofthepecanoilsobtainedatextractiontimesbetween15and480min[136].Thetocopherolcontentofthefat-reduced(25%and40%)walnutswassignificantlylowerthanthat inthefull-fatnuts[139].Thenutsafterextractionhadincreasedprotein,mineral,andcarbohydratecontentduetothereductionintheiroilcontent.Theextractedalmondflakeswith86.5%oilremovalhad approximately twice as much protein, carbohydrates, and minerals as rawalmonds[123].Theproteincontentof25%and40%fat-reducedwalnutsincreasedfrom14%inthefull-fatnutto21%and27%,respectively[139].Thecellstructureofthealmondwasgraduallymodifiedastheextractionprogressed[122].When15%oftheoilwasextracted,thepectinwasaffectedwithnomodificationofcelluloseandhemicelluloses.When35%oftheoilwasextracted,markedchangescouldbeobservedinbothpectinandhemicelluloses;whileat57%and64%oilextraction,the sample exhibited major cell wall disruption [122]. Similar observations werereportedforwalnuts,wherelipidextractionbeyond40%resultedincollapsedcellwallsandfractureandpowderingofwalnutpieces.Mineralssuchascalciumandmagnesiumwerealsoaffectedbytheextraction,especiallyafter65%oftheoilwasextracted.Whenthesedivalentcationswereremovedfromthecalcium-pectincom-plex,thecross-linksbetweenthegalacturonicacidunitsofadjacentpectinchainsorbetweenthepectinsandotherpolymersweredestroyed.Amassbalancelossof2%to20%wasreportedbyGoodrumandKilgo[130],whoattributedittothelossofvolatileorganicsandwatervaporintheexhaustCO2stream.Ingroundpeanuts,thehigherthemoisturecontent,temperature,andpressure,thegreaterwasthemassloss[130].Asexpected,agreatermassofvolatilecomponentswaslostintheexhaust
7089_C003.indd 69 10/15/07 5:29:31 PM
70 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
TaB
lE 3
.3N
ut F
at C
onte
nt a
nd F
atty
aci
d C
ompo
siti
ona o
f Nut
Oils
raw
M
ater
ial
Fat
Con
tent
(%
, w/w
)
Fatt
y a
cid
Con
tent
a–d
C16
:0C
16:1
C18
:0C
18:1
C18
:2C
18:3
ref
.
Aco
rn
12.1
13.4
2–13
.44a
0.07
–0.0
82.
564
.81–
65.4
216
.43–
17.0
70.
52–0
.57
121
Alm
ond
57.0
7.87
–8.4
8b0.
56–0
.63
1.58
–1.6
869
.25–
70.3
119
.65–
20.1
6—
122
54.5
6.60
–7.1
0b0.
50–0
.60
1.7–
2.2
68–7
317
.7–2
2—
124
Haz
elnu
t66
.25.
27–6
.01c
0.17
–0.2
02.
19–2
.45
82.6
5–85
.18
6.27
–8.4
20.
08–0
.09
128
56.0
5.86
–5.9
9c—
2.14
–2.1
779
.34–
79.6
211
.37–
11.4
5—
127
Peca
n58
.56.
20–1
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c—
2.9–
3.2
60.4
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723
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5.5
1–3.
313
6
Wal
nut
69.0
e6.
90–8
.10d
—1.
5–2.
016
.5–1
6.7
60.9
–61.
212
.5–1
3.7
140
71.0
6.08
–6.4
9a0.
072.
1–2.
1320
.98–
21.2
256
.46–
56.8
813
.16–
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113
8
Palm
itic
acid
(C
16:0
),P
alm
itole
ica
cid
(C16
:1),
Ste
aric
aci
d(C
18:0
),O
leic
aci
d(C
18:1
),L
inol
eic
acid
(C
18:2
),L
inol
enic
aci
d(C
18:3
).a m
ol%
,b wt%
,c not
indi
cate
d,d G
CA
rea
%,e F
atc
onte
ntr
epor
ted
in[
139]
.
7089_C003.indd 70 10/15/07 5:29:32 PM
Supercritical Fluid Extraction of Specialty Oils 71
CO2streamathighertemperatures.Aswell,lossesduetoinefficienciesinthecollec-tionofsolutespriortoCO2exhaustshouldnotbeoverlooked.
3.3.1.2.2 Other Quality AttributesThe color of the residual meal and the extracted oil are affected by the degreeof extraction. With higher oil removal, the color of the pecan and peanut kernelresidueswaslighter,asmostofthepigmentsarefatsoluble[134,141].TheL-value(ameasureoflightness)ofthefat-reducedwalnutswashigherthanthatofthefull-fatnuts, indicatingthat thefat-reducedwalnutshadawhiterappearance[139].Inanotherexample,agradualcolorchangeontheresidualmealwasobserved,withmeallocatedatthesolventoutletbeingdarkerandhavingmoreoil[130].Thecolorofmealresidueisalsoaffectedbytemperatureandcosolventaddition.Thepeanutmealbecamedarkerwith temperature increaseatconstantpressure.At75°C, themeallocatedatthereactorinletchangedfromchalkwhitetolightbrownandfinallytodarkbrownwhentemperaturereached95°C[130].Pecankernelsappearedmorered and less yellow after extraction, and this trend increased with temperature[134].WhenpureSC-CO2wasused,onlyasmallchangeinoriginalpistachiocolorwasobserved.However,whenethanolwasaddedasacosolvent,analmostwhitepistachioresiduewasproducedduetotheextractionofchlorophyll[137].Coloroftheresidueisalsoaffectedbypretreatmentofthesample.Comparedwithsoakedpeanuts,humidifiedpeanutshadtheclosestcolorofoilandpeanuts[133].
Thecoloroftheextractedoilisaffectedbytemperature,pressure,andcosolventaddition.PalazogluandBalaban [137] showed increasingcolor intensities for theextractedpistachiooilswithanincreaseinpressure.Thecoloroftheoilextractedat60°Cand34.5MPawasdarkyellow,whereasthatobtainedat50°Cand27.5MPawas lighter. Oil extracted at 70°C and 20.7 MPa with 5% ethanol addition wasyellowishgreenandthatat70°Cand34.5MPawasgreen.Thecrudepeanutoilcolorrangedfromyellowtodarkbrownasextractiontemperatureincreasedfrom25°Cto120°C[130].Theextractedpecanandwalnutoilswereallamberincolor[134,139],whereasthealmondoilwasyellow[124].
Thetexture(hardness)ofthepistachionutchangedsignificantlyafterSC-CO2extraction.Asensorycrunchiness test indicated that theharder thepistachio, thecrunchieritis[137].Theshear-compressionforceofpeanutsincreasedwithextrac-tion time,which indicates that thedefattedpeanuthasacrispy texture[141].Thehardnessofthewalnutdecreasedwiththefatcontentofthenut(fullfat,25%and40%fat-reduced)[139].Also,flavorintensitywasreducedafterSC-CO2extraction,whichmightbeattributedtotheremovaloftheflavorcompounds.Thearomaandflavorintensity,fracturability,andmoistnessofpeanutsalldecreasedwithincreas-ingextractiontime[141].
Thepeanutmealvolumedecreasedafterextractionandthedecreaseinvolumeincreasedwithtemperatureandpressure.Thisbulkvolumereductionwasattributedtothecompactionofthemealbyhighpressureandtheeliminationofoilfromthemeal.At25°C,themealcrumbledwhentouched.At100°Cand55MPa,themealwasacoherentmass,whichwasdifficulttofracturebyhand[130].Thebulkdensityoftheextractedalmondswasconsiderablylessthanthatoftherawmaterials,about41%lessforthedicedalmondsand54%to59%lessfortheflakedalmonds[123].
7089_C003.indd 71 10/15/07 5:29:33 PM
72 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
Fewstudiesevaluated theoxidative stabilityofnuts afterSC-CO2extraction.Thefat-reducedwalnuts(25%and40%)hadalowerperoxidevalue(PV)thanthatofthefull-fatwalnutswhenstoredat25°Cand40°Cfor8weeks.After5weeksat40°C,the25%fat-reducedwalnutshadahigherPVthanthe40%fat-reducednuts,buttherewasnosignificantdifferencethroughoutthe25°Cstoragecondition[139].
3.3.1.3 Comparison with Conventional Methods
Nutoils are traditionally recoveredmechanical pressingof organic solvents.Themechanicalpressingprocesscausedconsiderablesplitting(12%to43%)andbreak-age(3.6%to46%)ofpeanuts[133].Furthermore,about5%ofwater-solublesugarsandproteinswerelostinthesoakingstepfollowingthepressing,whichcanexpandthepeanutbacktoitsoriginalsizeandshape.Similarly,aconsiderableamountoffat-solublevitaminsandothervaluableconstituents,suchasphospholipids,areremovedduringhexaneextraction[134].Ithasbeendemonstratedthatpolarphospholipidsplay an important role in lipid stability. Organic solvent extraction also leavesundesirablesolventresidueinthefinalproducts[134,139].Ontheotherhand,thewalnutoilextractedbySC-CO2hadahigheramountoftocopherols(405.7μg/goil)comparedwiththeoilextractedwithhexane(303.2μg/goil)[138].TheoilextractedbySC-CO2wasalsoclearerthanthatobtainedbyhexane,indicatingtheneedforlessrefining[121,128,138].However,theSC-CO2-extractedoilshowedgreatersus-ceptibilitytooxidation.
SC-CO2extraction isalsoused toobtainsheanutoil.The traditionalprocessinvolvespouringhotmoltensheaoilinto10%Fuller’searthcontaininghotacetone,coolingandprecipitatingthepolyisoprenoidgumontotheearth,andthenfilteringtoremovetheearthandgum[142].However,withSC-CO2extraction,acleanandhigh-qualityoilcanbeobtainedwiththehigh-molecular-weightpolyisoprenoidsleftintheextractionvesselasarubber-likemass[142].Inaddition,theextractedoilhaslowlevelsoffreefattyacids,monoglycerides,diglycerides,iron,triterpeneacetate,andtriterpenecinnamate,whichisamajoradvantageforitsuseintheconfectionaryindustryasacocoabuttersubstitute.
3.3.2 seed oils
Numerous studies on the extraction of specialty oils from seeds, such as apricot[143–145],borage[146–148],cherry[149],echium[148],eveningprimrose[150–152],flax[153],grape[154–158],hiprose[159],Hybrid hibiscus[160],milkthistle[161],munch [162],pumpkin [163], rosehip [164–168], seabuckthorn [169–171], sesame[172],andtomato[173],usingSC-CO2havebeenreportedandaresummarizedinTable3.4.InadditiontoSC-CO2,somestudiesalsousedpropaneasthehigh-pressuresolvent.Forexample,Silybum marianumseedoilextractionrateusingpropanewasfoundtobehigherthanthatobtainedwithCO2,whileusing10timeslesspropanethanCO2[161].However,thetocopherolcontentoftheoilobtainedbyCO2(0.085%)washigherthanthatobtainedbypropane(0.02%)[161].Thus,propanemightnotbeanappropriatesolvent for tocopherolextraction.Toattaincompleteoilextractionfromrosehipseeds,asolvent-to-feedratioof3wasusedforpropane/CO2mixtureat
7089_C003.indd 72 10/15/07 5:29:34 PM
Supercritical Fluid Extraction of Specialty Oils 73
TaB
lE 3
.4Ex
trac
tion
of B
ioac
tive
Com
poun
ds fr
om S
eeds
usi
ng S
C-C
O2
raw
M
ater
ial
Feed
(g)
Sam
ple
prep
arat
ion
Bio
acti
ve
Com
poun
d
Extr
acti
on C
ondi
tion
s
rec
over
ya
(%)
ref
.pa
rtic
le S
ize
(mm
)H
2O (
%)
T (°
C)
p (M
pa)
Flow
rat
eTi
me
(min
)C
osol
vent
Apr
icot
5<
0.8
53.
9O
leic
aci
d,
linol
eic
acid
40,5
0,6
015
,30,
45,
52
.5,6
0<
0.5
g/m
inn.
i.N
one
n.i.
143
5n.
i.3.
9O
leic
aci
d,
linol
eic
acid
40,5
0,6
030
,37.
5,4
52,
3,4
g/m
in15
0,1
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%
EtO
H+
8514
4
5<
0.4
25,
<0
.85,
0.9
2,
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3.9
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olei
cac
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,50,
60,
70
30,3
7.5,
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52
.5,6
01,
2,3
,4,5
g/m
inn.
i.0,
0.5
,1,
1.5,
3%
E
tOH
+
n.i.
145
Bor
age
10n.
i.n.
i.L
inol
enic
aci
d,
stea
rido
nic
acid
4010
–35
0.5
L/m
inn.
i.0,
0.5
–2%
ca
pryl
ic
acid
met
hyl
este
r+
51.5
c14
6
400.
5,0
.75,
1,
1.5
0,1
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enic
aci
d10
,40,
60
5–35
0.5–
2L
/min
180
Non
e29
c14
7
150
0.65
n.i.
Lin
olen
ica
cid
10,2
5,4
0,5
56,
10,
20,
30
0.03
–0.2
g/m
inn.
i.N
one
n.i.
148
Che
rry
n.i.
n.i.
10.8
8L
inol
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acid
,st
erol
s40
,60
18,2
0,2
21.
2,3
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4.8×
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m/m
inb
n.i.
Non
en.
i.14
9
Ech
ium
150
0.65
n.i.
Stea
rido
nic
acid
,lin
olen
ica
cid;
10
,25,
40,
55
6,1
0,2
0,3
00.
03–0
.2g
/min
n.i.
Non
en.
i.14
8
Eve
ning
pr
imro
se0.
8<
0.5
n.i.
γ-L
inol
enic
aci
d35
–60
8–71
1×
10–3
L/m
in10
0N
one
9515
0
50<
0.35
5n.
i.γ-
Lin
olen
ica
cid
40,5
0,6
020
,30,
50,
70
18g
/min
n.i.
Non
e>
95
151
cont
inue
d
7089_C003.indd 73 10/15/07 5:29:35 PM
74 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
TaB
lE 3
.4 (c
onti
nued
)Ex
trac
tion
of B
ioac
tive
Com
poun
ds fr
om S
eeds
usi
ng S
C-C
O2
raw
M
ater
ial
Feed
(g)
Sam
ple
prep
arat
ion
Bio
acti
ve
Com
poun
d
Extr
acti
on C
ondi
tion
s
rec
over
ya
(%)
ref
.pa
rtic
le S
ize
(mm
)H
2O (
%)
T (°
C)
p (M
pa)
Flow
rat
eTi
me
(min
)C
osol
vent
50<
0.35
5n.
i.γ-
Lin
olen
ica
cid
40,5
0,6
020
,30,
50,
70
9,1
8,2
7g/
min
Non
en.
i.15
2
Flax
5n.
i.n.
i.L
inol
enic
aci
d,
toco
pher
ols
50,7
021
,35,
55
1,3
L/m
in18
0N
one
7415
3
Gra
pe17
6<
1.25
n.i.
Lin
olei
cac
id,
toco
pher
ols,
ph
ytos
tero
ls,
squa
lene
6537
60g
/min
360
Non
e13
.6c
154
20n.
i.6.
8n.
i.40
280.
5–1
L/m
inn.
i.N
one
n.i.
155
400.
35,0
.75,
1.
5,2
.83
0.3,
1.1
,2.
4,6
.3U
nsat
urat
edo
il10
,40,
60
5,1
0,2
0,3
00.
5,1
,1.5
,2L
/min
300
Non
e92
156
3n.
i.n.
i.Ph
enol
ics
4020
,30
n.i.
n.i.
Non
en.
i.15
7
4,5
000.
25–0
.42,
0.
42–0
.841
,0.
841–
2
n.i.
Uns
atur
ated
oil
(lin
olei
cac
id)
35,4
0,4
520
,25,
30,
40
0.4
mL
/min
;4
L/m
in12
0,2
10,
300
Non
e10
015
8
Hip
rose
130.
42,0
.79,
1.
03n.
i.L
inol
eic
acid
40,5
0,7
010
.3,2
0.6,
41
.3,6
8.9
1,2
,4,6
g/m
inn.
i.N
one
7.4c
159
Hyb
rid
hibi
scus
50.
1n.
i.Ph
ytos
tero
ls80
53.7
2×
10–3
mL
/min
50N
one
20c
160
Milk
this
tle30
n.i.
n.i.
Toco
pher
ols
25,4
0,6
0,8
010
,20,
30
n.i.
n.i.
Non
e20
.5c
161
Mun
ch15
00.
05–0
.25
n.i.
β-D
imor
phec
olic
ac
id(
DA
)
45
30n.
i.n.
i.N
one
>9
516
2
7089_C003.indd 74 10/15/07 5:29:36 PM
Supercritical Fluid Extraction of Specialty Oils 75
Pum
pkin
200.
36n.
i.PU
FAs,
ste
rols
,to
coph
erol
s35
–45
18–2
00.
03–0
.1m
/min
b
120
Non
en.
i.16
3
Ros
ehip
10n.
i.n.
i.C
arot
enoi
ds,
unsa
tura
ted
oil
28,3
510
,25
1–1.
5L
/min
n.i.
Prop
ane+
n.i.
164
260.
85–2
.36,
0.
425–
0.85
,0.
15–0
.425
n.i.
Uns
atur
ated
oil
40
3011
.4g
/min
n.i.
Non
e39
.2c
165
100
n.i.
n.i.
Uns
atur
ated
oil,
fla
vono
ids,
ca
rote
noid
s
40,5
0,6
030
,40,
50
21g
/min
n.i.
Non
e7.
1c16
6
210
n.i.
n.i.
Lin
olei
cac
id,
α-lin
olen
ica
cid
40,6
0,8
030
,50,
70
n.i.
n.i.
Non
en.
i.16
7
26n.
i.n.
i.U
nsat
urat
edo
il40
,50
30,4
04,
8,1
2,1
8,
24g
/min
60–9
0N
one
n.i.
168
Sea buck
thor
n3–
40n.
i.n.
i.U
nsat
urat
edo
il25
,40,
60
9.6,
17.
4,2
71
L/m
inn.
i.N
one
n.i.
169
120
<0.
491,
0.
491–
0.64
3,
0.64
3–1.
033,
>
1.03
3
n.i.
Lin
olei
cac
id30
,35,
40,
45
15,2
0,2
5,3
00.
83–3
.33
L/m
in27
0N
one
n.i.
170
n.i.
0.5–
113
.1L
inol
eic
acid
30,3
5,4
0,5
015
,20,
25,
30
1.7,
3.3
,5,6
.7L
/min
300
Non
en.
i.17
1
Sesa
me
10n.
i.n.
i.O
leic
aci
d,
linol
eic
acid
50,6
0,7
020
.7,2
7.6,
34
.5n.
i.n.
i.0,
5,
10%
EtO
H+
89.4
172
Tom
ato
4.5
0.27
n.i.
Uns
atur
ated
oil
40,5
5,7
010
.8,1
7.6,
24
.52.
8g/
min
480
Non
en.
i.17
3
aR
ecov
ery
(ge
xtra
ct/g
oil
infe
edm
ater
ial×
100
),b s
uper
ficia
lvel
ocity
,c yie
ld(g
/100
gfe
edm
ater
ial)
,+ cos
olve
nta
dded
con
tinuo
usly
into
SC
-CO
2ath
ele
vel(
%,w
/w)i
ndic
ated
.
7089_C003.indd 75 10/15/07 5:29:37 PM
76 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
28°Cand10.0MPa,whilearatioofonly1wassufficientforpropanealoneat25°Cand5.0MPa[174].
3.3.2.1 Factors affecting Extraction yield
3.3.2.1.1 Sample PreparationUnlikeextractingoilfromnuts,whichhavesphericalandelongatedhexagonalcellstructurescontainingoil,itisnearlyimpossibletoextractoilfromuncrackedseedsduetotheiruniquephysicalstructures.Theoil innutsiseasilyaccessiblebydif-fusioninto thecellulosicstructure;however, theseedcoat isalmost impermeable[155].Theoilintherosehiporhiproseseeds,forexample,iscontainedintheoil-bearingstructures,whichareenclosedinathickandhighlylignifiedtesta[159,165].Thehiprosemaybecontainedin longmicroscopicchannels,whichareprotectedby a lignin structure that is probably too compact to alloweffective diffusion ofthesupercriticalfluid ina reasonable time[159].Therefore,grinding theseeds isanimportantstepofsamplepreparation,whichcanbreaktheintactchannelsandexposetheoilinthechannelstotheextractionsolvent.
a) Particlesize:Asexpected,theoilyieldincreasedwithdecreasedparticlesize.Forexample,whenhiproseseedparticlesrangingfrom1.03to0.79and0.42mmwereused, theoil yield increased from4.9% to5.2%and7.4%,respectively[159].Similarparticlesizeeffectswereobservedwhengrape[156]andborage[147]oilswereextractedfromtheirseeds.Particleswithdiameters less than0.35mmweresuggested forgrapeseedextrac-tion[156].Withadecreaseintheparticlesizeofseabuckthornseedsfrombetween0.50–2.36to0.43–1.00mm,thedurationoftheextractionprocesswasreducedfrom6to3hours[175].
b) Moistureandequilibrationtime:Themoisturecontentofthegrapeseed(6.3%,2.4%,1.1%,0.3%)wasmodifiedbydryingthegroundsamplefordif-ferentlengthsoftime(0,2,4,6.3hours)[156].Theextractionyieldwasnotsignificantlyaffectedbythemoisturecontentofthegrapeseeds.However,the sample exposed to the longer drying time (6.3 hours) had a slightlyloweroilyield,whichmightbeduetotheevaporationofvolatileconstitu-ents[156].Similarly, theextractionyieldof theborageseedoilwasalsonotsignificantlyaffectedbythedryingprocess(partiallyandalmostfullydehydrated);however,themoisturecontentoftheborageseed(0%,1.8%,7.4%)hadanegativeimpactontheextractionyield,withthehighermois-turecontentsamplesresultinginlowerextractionyields[147].
3.3.2.1.2 Extraction Parameters a) Temperature and pressure: In general, extraction yield increased with
pressure at constant temperature. For example, increasing the pressurefrom10to15,20,and25MPaat40°Cresultedinanincreaseinborageoilyieldfrom0.1%to5.6%,15.2%and21.9%,respectively.However,onlyaslightincreaseintheyield(21.6%to24.3%)wasobservedwithapres-sureincreasefrom30to35MPa[146].Asimilartrend(34.4%to91.3%,
7089_C003.indd 76 10/15/07 5:29:37 PM
Supercritical Fluid Extraction of Specialty Oils 77
respectively) was observed during extraction of evening primrose seedsat60°Candpressuresof20and30MPa[151].Atconstant temperature,increasingthepressureto50MPaimprovedtheoilyieldslightlyto97.2%,whileafurtherincreaseinpressureto70MPahadalmostnoeffectontheyield (97.7%) [151]. Not only the extraction yield but also the extractionrateincreasedwithpressure.Inaddition,withanincreaseinpressurefrom20.6to41.2and68.9MPa,theextractiontimeforhiprosedecreasedfromabout30to15and5min,respectively[159],sincetriglyceridesaresolubi-lizedtoagreaterextentathigherpressuresinSC-CO2.Morethan94%oftheavailableoilfromeveningprimroseseedswasextractedinonly14min[151].AsdiscussedinSection3.3.1.1.2,temperaturehasanegativeeffectonsolubilityandextractionyieldatlowpressuresbutapositiveeffectathigherpressuresdue to thecrossoverof the solubility isotherms.Moreover, thenegativeeffectoftemperatureatlowpressuresseemstobegreaterthanthepositiveeffectathighpressures.Forexample,withanincreaseintempera-turefrom40°Cto50°Cat20MPa,theeveningprimroseoilyielddroppedfrom66.1%to59.6%,withafurtherdropto34.4%at60°C.However,atahigherpressureof50MPa, theoilyield increasedfrom96.8%to97.5%withatemperatureincreasefrom40°Cto50°Candthenslightlydroppedto 97.2% at 60°C [151]. A similar trend was reported for the extractionof Silybum marianum oil using SC-CO2. In this case, the seed oil yielddecreasedfrom19.9%to5.2%withatemperatureincreasefrom25°Cto80°Cat20MPabut increasedfrom15.3%to20.5%at30MPa[161]. Infact,thepressureincreaseseemstohaveamoresubstantialeffectontheoil yield than the temperature increase. Sesame oil yield increased onlyfrom5.4%to11.8%whentemperaturewasincreasedfrom50°Cto70°Cat27.6MPa;however,theyieldincreasedfrom3.6%to31.3%whenpressurewasincreasedfrom20.7to34.5MPa[172].
b) Flowrateandflowdirection:Theextractionyieldofborageoilincreasedwithflowrate[147].Similarly,66%and74%offlaxoilwasobtainedataflowrateof1and3L/min(measuredatSTP),respectively[153].Similartothenutoils,flowdirectionwasreportedtoaffecttheextraction,withdown-wardflowbeingmorefavorable.Sovovaetal.[155]reportedthatgrapeseedoilextractionwasretardedwhenthesupercriticalsolventflowwasupwardthroughthebed(duetonaturalconvection).Itisthereforeadvantageoustooperatelaboratoryextractionunitsinthedownwardflowmode.
c) Extractiontime:Thedurationofthefastextractionperioddependsontheplanttypeandvariety.Forexample,twovarietiesofgrapeseedsresultedinlowoilyields(5.9%and6.1%)throughoutthefastextractionperiodof60 min with a very little amount of additional oil extracted at extendedextractiontimes[154].Theotherfourgrapevarietiescontainingintermedi-ate levelsofoil (9.4%to10.7%)werenearlycompletelyextractedwithin60min,whereasthehighoilcontentvariety(13.6%)required120min.
d) Useofcosolvent:Additionof10%ethanolasacosolventgreatlyenhancedthe extraction yield of sesame oil. At 27.6 MPa and 50°C, the recoveryincreasedfrom5.4%to74.1%uponadditionofethanolintoSC-CO2,whereas
7089_C003.indd 77 10/15/07 5:29:38 PM
78 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
at70°C, theyield increased from11.8% to89.4%, respectively [172].At40°C,whencaprylicacidmethylesterwasusedasacosolvent(0%,0.5%,1%,and2%),theborageoilyieldincreasedfrom0.1%to2.9%,3.1%and6.7%at10MPa;from15.2%to20.7%,30.0%and36.6%at20MPa;andfrom21.6%to30.7%,38.2%and51.5%at30MPa[146].Atalowpressureof10MPa,theamountofcoextractedsolventwashigh,rangingfrom51.1%to73.0%and79.7%ofthetotalfattyacidmethylesterat0.5,1%,and2%additionofcaprylicacidmethylester[146].Whenpressurewasincreasedto20MPa,theamountofcoextractedsolventwasreducedto11.9%,20.8%,and39.8%ofthetotalfattyacidmethylesterat0.5%,1%,and2%,respec-tively,due to thehigher recoveryof triglycerides. Itwasshown that thiscosolventcanbeeasilyremovedfromthefinalextractatlowpressures.
3.3.2.2 Characterization of products Extracted by SC-CO2
3.3.2.2.1 Chemical CompositionThefattyacidcompositionoftheseedoilsissummarizedinTable3.5;wheretheunsaturatedfattyacids(mainlyC18:1,C18:2,andC18:3)accountedformorethan90%ofthetotalfattyacids.FattyacidcompositionoftheoilobtainedbySoxhletextractionwassimilartothatoftheSC-CO2extractforgrapeseed[154].Inaddi-tion, fatty acidprofilesof thegrape seedoilsobtainedatdifferent temperatures(35°C and 40°C) and pressures (30 and 40 MPa) were also similar [158]. How-ever,Szentmihalyietal.[164]foundthatthelowertemperatureSC-CO2extractionresultedinhigherlevelsofoleicandlinoleicacidsintherosehipoilcomparedtothoseintheSoxhletextract.Dauksasetal.[146]foundthatthelinolenicacidcon-tentoftheborageseedoilincreasedfrom16.2%to20.1%withapressureincreasefrom10to20MPa;however,itslightlydecreasedto18.5%withafurtherincreaseto 35MPa.The fatty acid compositionof theborageoil obtainedusingvariousconcentrationsofcaprylicacidmethylesterasacosolventatdifferentpressureswasdifferent;however,nocleartrendswereestablished[146].Thefattyacidcomposi-tionsaswellastheratiosbetweenthesaturatedandunsaturatedfattyacids(11:89)of the Hippophae rhamnoides L. seed oils extracted using SC-CO2 and hexaneweresimilar[177].Althoughtherewasnodifferencebetweenthefattyacidcompo-sitionsofSC-CO2-andhexane-extractedgrapeseedoils,SC-CO2-extractedfrac-tionsobtainedat30,60,120and180minweredifferent[156].Theα-linolenicacidcontentoftheSC-CO2-extractedflaxoilwashigherthanthatofsolvent-extractedoil; however, the saturated and monounsaturated fatty acids were higher in thesolventextract[153].
3.3.2.2.2 Other Quality AttributesThe SC-CO2-extracted evening primrose oil had a yellow color and its intensityincreasedwithpressure,approachingthedeepyellowcolorofthehexane-extractedoil[151].Theyellowintensityoftheborageoilextractsalsoincreasedwithpressure[146].Similarly,theSC-CO2extractedseabuckthornseedoilwasaclear,yellow-brownliquidatroomtemperature,whereasthepulpflakeoilwasredandsemisolid[175].OdabasiandBalaban[172]foundthatthesesameoilfromSC-CO2extractionappearedclear.However,when5%ethanolwasusedasacosolvent,theoilextract
7089_C003.indd 78 10/15/07 5:29:39 PM
Supercritical Fluid Extraction of Specialty Oils 79
TaB
lE 3
.5Se
ed F
at C
onte
nt a
nd F
atty
aci
d C
ompo
siti
on o
f See
d O
ils
raw
Mat
eria
lFa
t C
onte
nt
(%, w
/w)
Fatt
y a
cidy
Con
tent
a–d
C16
:0C
16:1
C18
:0C
18:1
C18
:2C
18:3
ref
.
Apr
icot
48.1
5.22
–5.7
1c0.
6–0.
781–
1.3
67.3
7–68
.07
24.8
4–25
.11
––14
4
Bor
age
29.0
13.3
2c0.
194.
5819
.78
39.5
722
.56
147
Che
rry
8.5
5.26
a0.
272.
1532
.64
40.8
41.
114
9
Ech
ium
e30
.06–
8b––
3–5
15–1
914
–18
37–4
514
8
Eve
ning
pri
mro
se27
.54.
75–6
.7c
––1.
46–1
.87
4.85
–5.4
75.2
7–77
.07
(γ-)
9.2
2–10
.36;
(α
-)0
.13–
0.16
150
Flax
38.0
5.7–
5.8d
––4.
0–4.
214
.1–1
4.4
12.8
–13.
460
.5–6
115
3
Gra
pe10
–15
6.28
–8.2
6b0.
06–0
.15
3.6–
5.22
12.7
1–18
.47
67.5
6–73
.223
0.44
–1.1
215
4
Hyb
rid
hibi
scus
8.5–
2014
.8–2
7.0b
0–0.
70–
4.1
17.8
–47.
342
.6–6
40–
0.7
160
Lun
aria
e37
.01–
3b––
1–2
16–2
08–
102–
414
8
Mun
ch22
.81.
9–2.
4b––
1.5–
1.9
17.5
–22.
112
.3–1
4.9
0.7–
116
2
Nee
me
45.0
13–1
5c––
14–1
950
–60
8–16
––17
6
Pum
pkin
43.5
14.9
c0.
253.
1913
.559
.91.
6416
3
Ros
ehip
8.0
3.6–
7.87
d––
2.45
–3.2
716
.25–
22.1
135
.94–
54.7
520
.29–
26.4
816
4
18.5
8.85
b––
2.42
21.7
540
.09
––17
7
Sesa
me
––9.
23–1
0.53
c––
4.17
–5.3
37.6
–38.
4844
.17–
45.8
5––
172
Tom
ato
2910
.9–1
5.79
b1.
15–3
.81
4.97
–11.
9819
.9–2
4.13
39.6
9–53
.24
4.58
–5.5
173
Palm
itic
acid
(C
16:0
),P
alm
itole
ica
cid
(C16
:1),
Ste
aric
aci
d(C
18:0
),O
leic
aci
d(C
18:1
),L
inol
eic
acid
(C
18:2
),L
inol
enic
aci
d(C
18:3
).a m
ol%
,b wt%
,c not
indi
cate
d,d G
C%
Are
a,a
nde F
atty
aci
dco
mpo
sitio
nre
port
edf
orth
ene
wm
ater
ial.
7089_C003.indd 79 10/15/07 5:29:40 PM
80 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
wasclearatlowtemperaturebutcloudyathightemperature.With10%ethanoladdi-tion,allsampleswerecloudyregardlessofthetemperature.Thiscouldbeattributedtothecoextractionofphospholipids,waxes,andpigmentsthatoccurswithethanoladdition.Therefore, theselectionandconcentrationofcosolventadditionmustbeoptimizedbasedondesirableproductqualityattributes.
3.3.2.3 Comparison with Conventional Methods
HexaneextractiontendstogiveahigheroilyieldthanSC-CO2extractionduetotheextractionofundesirablecompounds thatmustbe removedduring refining [147].Gomezandde laOssa [156] reported that thegrape seedoilyield fromSC-CO2extractionwas6.9%,while thatwithhexanewas7.5%.This isdue to thehexaneextraction being nonselective for triglycerides since hexane can also extract freefattyacids,phospholipids,pigments,andunsaponifiables.BozanandTemelli[153]alsofoundthattheoilyieldobtainedfromflaxseedwithSC-CO2aftera3hextrac-tionwas21–25%,whereasthepetroleumetherextractiongavea38%yield.Inaddi-tion,theSC-CO2-extractedoilsfrompumpkinandHippophae rhamnoides L.seedswere clearer than those extracted by hexane [163]. Bernardo-Gil et al. [149] alsoreported that cherry seedoil extractedwithSC-CO2wasclearer than thehexaneextract,minimizingtheneedforfurtherrefining.Nevertheless,thepumpkinseedoilextractedwithhexanewasbetterprotectedagainstoxidation(inductiontime=8.3h)comparedwiththeSC-CO2–extractedoil(inductiontime=4.2h)[163].
3.3.3 Cereal oils
Todate,thecerealoilsthathavebeenstudiedareamaranth[178–180],oat[181],ricebran[182–187],wheatgerm[188–191],andwheatplumule[192]oil,whicharesum-marizedinTable3.6.
3.3.3.1 Factors affecting Extraction yield
3.3.3.1.1 Sample preparationCereal grains, which in general have a moisture content of 3% to 12%, may notrequireadryingsteppriortoextraction.
a) Particlesize:Extractionyieldsobtainedusingoriginal(0.75mm)andthemilled (0.3 mm) wheat germ were similar [188]. However, according toPanfilietal. [190],wheatgermoil recovery increasedfrom57%to92%whentheparticlesizewasreducedfrom0.5to0.35mm.Asimilartrendwas reported for the SC-CO2 extraction of oil from Amaranthus grain,whereverylittleoilwasextractedfromwholegrains[179].
b) Moisture:InthecaseofAmaranthusgrain,moisturecontentsof0%,5%,and10%hadnosignificanteffectontheoilandsqualeneextractionyields[179]. As the maximum moisture content of the harvested Amaranthusgrain is 10%, drying is not a necessary step. However, in wheat germextraction, the tocopherol yield increased with a decrease in moisturecontentfrom11.5%to8.2%and5.1%[189],butdecreasedwithafurther
7089_C003.indd 80 10/15/07 5:29:40 PM
Supercritical Fluid Extraction of Specialty Oils 81
cont
inue
d
TaB
lE 3
.6Ex
trac
tion
of B
ioac
tive
Com
poun
ds fr
om C
erea
ls u
sing
SC
-CO
2
raw
Mat
eria
lFe
ed (
g)
Sam
ple
prep
arat
ion
Bio
acti
ve
Com
poun
d
Extr
acti
on C
ondi
tion
s
rec
over
ya
(%)
ref
.pa
rtic
le S
ize
(mm
)H
2O (
%)
T (°
C)
p (M
pa)
Flow
rat
eTi
me
(min
)C
osol
vent
Am
aran
th
(Am
aran
thus
cr
uent
us)
40n.
i.n.
i.L
inol
eic
acid
,ol
eic
acid
40,4
5,5
010
,20,
25,
30
3.3,
6.8
,8.5
g/m
inn.
i.N
one
n.i.
178
60n.
i.n.
i.Sq
uale
ne40
,50,
60,
70
15,2
0,2
5,3
01,
2,3
,5L
/min
120
Non
e4.
8c17
9
(Am
aran
thus
ca
udat
us)
5<
0.2
n.i.
Toco
pher
ols,
sq
uale
ne40
20.2
,40.
52
L/m
in15
Non
en.
i.18
0
Oat
1500
,185
n.i.
n.i.
Ole
ica
cid,
lin
olei
cac
id40
25,3
5n.
i.30
020
%
EtO
H+
+
n.i.
181
Ric
ebr
an20
n.i.
3.1
Toco
pher
ols
4014
.7–3
4.3
n.i.
n.i.
Non
e22
c18
2
300
n.i.
10.1
Uns
atur
ated
oil,
to
coph
erol
s,
ster
ols,
ory
zano
l
0,2
0,4
0,6
017
,24,
31
41.7
g/m
in36
0N
one
96.8
183
300
n.i.
8.48
Uns
atur
ated
oil,
to
coph
erol
s,
ster
ols,
ory
zano
l
40,4
5,5
08.
6,9
.9,1
1.2
58.3
g/m
in24
0N
one
4.1c
184
300.
5n.
i.PU
FAs,
to
coph
erol
s,
squa
lene
40,5
0,7
020
.7,2
7.6,
34
.5,4
1.3
n.i.
480
Non
e80
185
20n.
i.n.
i.α-
Toco
pher
ol,
γ-or
yzan
ol
40,6
0,8
034
.5,5
1.7,
68
.91.
1g/
min
n.i.
Non
e24
.7c
186
7089_C003.indd 81 10/15/07 5:29:41 PM
82 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
TaB
lE 3
.6 (c
onti
nued
)Ex
trac
tion
of B
ioac
tive
Com
poun
ds fr
om C
erea
ls u
sing
SC
-CO
2
raw
Mat
eria
lFe
ed (
g)
Sam
ple
prep
arat
ion
Bio
acti
ve
Com
poun
d
Extr
acti
on C
ondi
tion
s
rec
over
ya
(%)
ref
.pa
rtic
le S
ize
(mm
)H
2O (
%)
T (°
C)
p (M
pa)
Flow
rat
eTi
me
(min
)C
osol
vent
Ric
ebr
an80
00.
25,0
.5,0
.75
n.i.
Lin
olei
can
dpa
lmiti
cac
ids,
si
tost
erol
and
ca
mpe
ster
ol
50,6
010
,20,
30,
40
300
g/m
in18
0N
one
20c
187
Whe
atg
erm
250.
3,0
.75
n.i.
Toco
pher
ols,
lin
olei
cac
id10
–60
5–30
0.5–
2L
/min
180
Non
e>
95c
188
5n.
i.n.
i.To
coph
erol
s35
,40,
45,
50
13.7
,20.
7,
27.6
,34.
5,
41.3
1–3×
10–3
L/m
in90
Non
e2.
2c18
9
600–
700
0.35
–0.5
n.i.
Lin
olen
ic,l
inol
eic
acid
s,β
-car
oten
e,
lute
in,a
nd
zeax
anth
in,
toco
pher
ols,
to
cotr
ieno
ls
5525
,38
0.5
L/m
in18
0N
one
9219
0
20Fl
akes
:2.1
n.
i.To
coph
erol
s10
,25,
40,
50
20.2
300–
400
L/m
inn.
i.N
one
9519
1
Whe
at
plum
ule
n.i.
n.i.
n.i.
PUFA
s35
,45,
55,
65
10,1
5,2
0,3
01.
2,1
.8,2
.4,
3L
/min
60–4
80N
one
n.i.
192
T:t
empe
ratu
re,P
:pre
ssur
e,n
.i.:n
otin
dica
ted.
aR
ecov
ery
(ge
xtra
ct/g
oil
infe
edm
ater
ial×
100
),b s
uper
ficia
lvel
ocity
,c yie
ld(g
/100
gfe
edm
ater
ial)
,++C
osol
vent
add
edto
sam
ple
befo
ree
xtra
ctio
nat
the
leve
l(%
,w/w
)ind
icat
ed.
7089_C003.indd 82 10/15/07 5:29:42 PM
Supercritical Fluid Extraction of Specialty Oils 83
reductioninmoistureto4.3%.Thismightbeattributedtotheshrinkingofthegermparticles.
3.3.3.1.2 Extraction Parameters a) Temperature and pressure: During wheat germ extraction, tocopherol
yieldincreasedslightlywithapressureincreasefrom13.8to27.6MPaat40°C, while a further increase in pressure to 34.5 and 41.4 MPa did notimprove theyieldsignificantly [189].On theotherhand, tocopherolyielddecreased with temperature at pressures below 26.2 MPa and increasedwithtemperatureatpressuresabove26.2MPa.Similarly,amaranthoilyieldincreasedwithpressureintherangeof15to25MPa.Also,temperaturehadanadverseeffecton theamaranthoilyieldat thepressure rangeof15to25MPabuthadapositiveeffectat30MPa[179].Similarly,anotherstudyonamaranthseedoilshowedthatextractionyieldandratedecreasedwithtemperatureat10MPaandincreasedwithtemperatureat20and30MPa[178].Althoughtheamaranthoilyieldvariedsignificantlywithpressure,theyieldsofsqualeneintheamaranthoilat40°Canddifferentpressureswereveryclose,rangingfrom0.24to0.27g/100ggrain[179].Ontheotherhand,theamountofsqualeneextractedfromricebrandecreasedwithCO2den-sityandthemaximumamountwas2.9%at70°Cand20.7MPa[185].TheSC-CO2densityhasadifferenteffectonfattyacids.Asthereduceddensitywasincreasedatconstanttemperature,theamountoflinoleicacidextractedincreasedbutthatofoleicaciddecreased[185].
b) Flowrate:Theamaranthoilyieldandinitialextractionratebothincreasedwithflowratefrom1to2L/min,buttherewerenodifferencesatflowratesof2,3and5L/min(measuredatSTP)underthesameextractionconditions[179].Atflowratesabove2L/min,theextractionratewasreducedmarkedlyafter1hofextractionandonlyasmallamountofoilwasextractedinthefollowing2h.Similarly,inthewheatgermoilextractions,theoilyieldandextractionratebothincreasedwithflowratefrom0.5to1and1.5L/min(measuredatSTP),whileafurtherincreaseofflowrateto2L/mindidnotchangeeithertheoilyieldortheextractionrate[188].
c) Extraction time: Not many studies involving cereal oils evaluated theextractionyieldasfunctionofextractiontime.Asexpected,theextractionyieldincreasedwithextractiontimeforwheatplumuleoil[192].
d) Useofcosolvent:Theadditionofethanolasacosolventslightlydecreasedthe free fatty acid content from 5.6% to 4% in the case of oat oil, butincreasedthephosphoruscontentfromlessthan1ppmto80ppmprobablyduetotherecoveryofpolarphospholipids[181].
3.3.3.2 Characterization of products Extracted by SC-CO2
3.3.3.2.1 Chemical CompositionFattyacidcompositionoftheSC-CO2extractedcerealoilsispresentedinTable3.7.Themainfattyacids in thericebranandwheatgermoilsareoleic, linoleic,andpalmiticacids[185,187,188].ThefattyacidcompositionsofSC-CO2-andhexane-extractedoilsweresimilarandtherewerenodifferencesamongtheextractsobtained
7089_C003.indd 83 10/15/07 5:29:42 PM
84 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
atdifferentextractionconditions[188].Crudericebranoilcontains70%triglycer-ides,7%freefattyacids,3.6%fattyacidesters,andsomeminorcomponents,suchasoryzanol[193],α-tocopherol,andβ-sitosterol.Theα-tocopherolcontentofricebranoilwasreportedtobe284mg/kg[182]and1050.8to1279.3mg/kg[186].Inaddition,Shenetal.[184]foundthattheSC-CO2-extractedricebranoilcontained0.23mg/goilofα-tocopherol and18.5mg/goilofβ-sitosterol.Tocopherol andcarotenoidsaretwoimportantminorcomponentsinwheatgerm.α-Tocopherolwasreportedtobethemostabundanttocopherolisomerat1329mg/100gwheatgerm[189]andat2123mg/kgwheatgermoil[190].Luteinandzeaxanthinwerethemostabundantcarotenoidsinwheatgerm,at47.7and37.3mg/kgoil,respectively[190].Squalene,animportantminorcompoundinamaranthoil,wasreportedtobepresentat5.27%(40°C,25MPa)to15.3%(50°C,20MPa)intheextractsobtained[179].
3.3.3.2.2 Other Quality AttributesTheSC-CO2-extractedricebranandwheatgermoilsbothhadlightcolorcomparedwiththoseextractedbyhexane[182,191].However,theSC-CO2-extractedoilwasveryunstablecomparedwithhexane-extractedoil[182].
3.3.3.3 Comparison with Conventional Methods
Because solvent extraction is less selective than SC-CO2 extraction, it generallyresultsinahightotalextractyield,leadingtoreducedconcentrationsofdesirablebioactives.Forexample,wheatgermoilyieldfromSC-CO2extractionwasslightlylower(7.3%to8.0%)thanthatobtainedwithhexane(8.6%),whereasthetocopherolcontentoftheSC-CO2-extractedoilwashigher[188].TheyieldoftotaltocopherolsfromwheatgermobtainedbySC-CO2washigher thanthoseobtainedbysolventextraction [189]. Extraction of vitamin E by hexane and chloroform/methanoltook about 960 and 140 min, respectively, whereas SC-CO2 extraction requiredonly90min.Theoperating temperature forSC-CO2extractionwas lower (40°C)than thoseofhexaneandchloroform/methanol (70°Cand65°C)extraction [189].By choosing suitable extraction conditions, some compounds can be selectivelyextractedbySC-CO2,butnotbysolventextraction.SqualenecanbeextractedfromricebranbySC-CO2butnotbyusingchloroformandmethanol[185].About80%ofPUFAswasextractedbySC-CO2,whereasonly60%recoverywasobtainedwithsolventextraction[185].Ahighsqualeneyield(0.31g/100gAmaranthusgrain)andconcentration(15.3%inextract)wasobtainedat50°Cand20MPausingSC-CO2,whilethesqualeneconcentrationinthesolventextractwasonly6%[179].
3.3.4 Fruit and Vegetable oils
Buritifruit[194],carrot[195-198],cloudberry[199],hiprosefruit[174],olivehusks[200],tomato[201-207]arethemainfruitsandvegetablesstudied(Table3.8).Lyco-pene is the dominant carotenoid (85% to 90% of total carotenoids) in the tomatoextract[207],whereasβ-carotene(60%to80%)isthemajoroneincarrotextract.Squalene,tocopherol,andsterolsarethemainbioactivecomponentsfoundinolives.
7089_C003.indd 84 10/15/07 5:29:43 PM
Supercritical Fluid Extraction of Specialty Oils 85
TaB
lE 3
.7Fa
tty
aci
d C
ompo
siti
on o
f Cer
eal O
ils
raw
Mat
eria
l
Fatt
y a
cid
Con
tent
a
C14
:0C
16:0
C16
:1C
18:0
C18
:1C
18:2
C18
:3C
20:0
C20
:1r
ef.
Am
aran
th—
12.3
2–17
.94
—2.
71–4
.66
23.8
5–32
.88
43.6
6–47
.48
—0.
38–1
.54
—18
0
Oat
0.1–
0.2
13.6
–15.
7—
1.4–
1.6
38.3
–43.
739
.1–4
2.2
1.0–
1.5
—0.
8–0.
918
1
Ric
ebr
an—
16.5
–17.
9—
1.1–
1.4
38.8
–41.
437
.8–4
0.4
1.5–
1.7
0.3–
0.6
0.4–
0.6
183
Whe
atg
erm
—18
.09–
18.9
50.
22–0
.24
0.49
–0.7
313
.69–
16.5
257
.1–5
8.99
6.46
–8.5
1—
—18
8
Palm
itic
acid
(C
16:0
),P
alm
itole
ica
cid
(C16
:1),
Ste
aric
aci
d(C
18:0
),O
leic
aci
d(C
18:1
),L
inol
eic
acid
(C
18:2
),L
inol
enic
aci
d(C
18:3
),A
rach
idic
ac
id(
C20
:0),
Eic
osen
oic
acid
(C
20:1
).a
Uni
tsn
otin
dica
ted.
7089_C003.indd 85 10/15/07 5:29:44 PM
86 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
3.3.4.1 Factors affecting Extraction yield
3.3.4.1.1 Sample PreparationDryingofsamplespriortoextractionisnecessary,ascarrotsandtomatoescontain80%to95%moisture.Grindingisalsoneededtoachievesmallparticlesize.Olivepomaceandhuskareby-productsofoliveoilproduction.Tofurtherrecoverthevalu-ablebioactivecompoundsbySC-CO2extraction,pretreatmentofsuchby-productstheconventionalfruitandvegetableprocessingindustryisnecessary.
a) Particle size:Duringextractionof freeze-driedcarrots, ahigherextrac-tionyieldwasobtainedwithsmallercarrotparticles[196,198].Thetotalcarotenoid yield increased from 1109.9 to 1369.6 and 1503.8 μg/g drycarrotwhentheparticlesizewasdecreasedfrom1–2mmto0.5–1mmand0.25–0.5mm,respectively[198].
b) Moisture: Moisture had different effects on the carotenoids yield. Theα- andβ-carotene yields increased with decreasing level of moisture inthefeedmaterial,whiletheluteinyielddecreased[198].Theluteinyielddecreasedfrom55.3to29.9,19.3and13.0μg/gdrycarrotwithadecreaseinmoisturefrom84.6to48.3,17.5and0.8%,whiletheα-andβ-caroteneyields increased from 184.1 to 323.0, 442.3 and 599.0 μg/g, and from354.2to547.8,668.3and891.7μg/gdrycarrot,respectively[198].Ontheotherhand,onlytraceamountsoflycopenewereextractedwhenthetomatofeedmaterialcontained50%to60%moisture[207].Thiscanbeexplainedbythefactthatwatercanactasacosolventfortheextractionofrelativelypolarcompounds,likelutein,whereasthepresenceofwaterisnotfavorablefortherelativelynonpolarlycopeneandcarotenes.
3.3.4.1.2 Extraction Parameters a) Temperature and pressure: Lycopene extraction yield increased with
pressurefrom33.5MPato45MPaataconstanttemperatureof66°Candincreasedwith temperaturefrom45°Cto66°Cataconstantpressureof45MPa[207],becauseSC-CO2densityincreaseswithpressureatconstanttemperatureandsolubilityincreaseswithtemperatureabovethecrossoverpressure.Temperaturegreatlyaffectstheextractionrateatpressuresabovethecross-overpressure.Usingtomatoskin[202],theextractionrateandyieldweregreatlyincreasedat110°C,resultingin96%lycopenerecoveryin40minand100%recoveryin50min.However,onlyabout20%and30%recoverywereachieved in80minat60°Cand85°C, respectively.Pressurealsoaffectedthecompositionoftheextractsastherecoveryoftrans-lycopene increased and that of cis-lycopene decreased with CO2density [205]. Therefore, the fractionation of trans-lycopene is possiblewhenoptimumCO2densityischosenasthelycopeneisomershavediffer-entsolubilitiesinSC-CO2.
b) Flowrate:Totalcarotenoidsyieldincreasedwithflowrate[198],rangingfrom934.8to1332.3µg/gand1973.6µg/gdrycarrotatCO2flowratesof0.5,1,and2L/min(measuredatSTP),respectively.However,thelycopene
7089_C003.indd 86 10/15/07 5:29:44 PM
Supercritical Fluid Extraction of Specialty Oils 87
TaB
lE 3
.8Ex
trac
tion
of C
ompo
unds
from
Fru
its
and
vege
tabl
es u
sing
SC
-CO
2
raw
M
ater
ial
Feed
(g)
Sam
ple
prep
arat
ion
Bio
acti
ve
Com
poun
d
Extr
acti
on C
ondi
tion
sr
ecov
erya
(%)
ref
.pa
rtic
le S
ize
(mm
)H
2O (
%)
T (°
C)
p (M
pa)
Flow
rat
eTi
me
(min
)C
osol
vent
Bur
itif
ruit
n.i.
n.i.
11C
arot
enoi
ds,
toco
pher
ols
40,5
520
,30
18.6
,25
.8g
/min
n.i.
Non
e7.
8c19
4
Car
rot
20.
5–1
0.8
α-,β
-Car
oten
e,
lute
in
40,5
012
–33
1.2
L/m
in48
0N
one
n.i.
195
n.i.
0.26
,0.4
7,1
.12
n.i.
Car
oten
oids
40,5
0,6
07.
8–29
.4n.
i.n.
i.1,
3,5
%E
tOH
+n.
i.19
6
2000
n.i.
n.i.
caro
tene
s,
phen
olic
s,
phyt
oste
rols
,lin
olen
ica
cid
45–5
035
–38
n.i.
120–
180
Non
en.
i.19
7
20.
25–0
.5,0
.5–1
,1–
20.
8,1
7.5,
48
.7,8
4.6
α-,β
-car
oten
e,
lute
in
40,5
5,7
027
.6,4
1.3,
55
.10.
5,1
,2L
/min
240
0,2
.5,5
%
cano
lao
il+
0.2c
198
Clo
udbe
rry
42n.
i.n.
i.U
nsat
urat
edo
il,
β-ca
rote
ne,
toco
pher
ols
40,6
09,
10,
12,
15
,30
n.i.
n.i
Non
en.
i.19
9
Hip
rose
fru
itn.
i.0.
36n.
i.To
coph
erol
s,
caro
teno
ids
3525
1–1.
5L
/min
n.i.
Non
e10
017
4
Oliv
ehu
sks
n.i.
0.4
n.i.
Uns
atur
ated
oil
(ole
ica
cid)
35–5
710
.4–1
8n.
i.n.
i.N
one
n.i.
200
cont
inue
d
7089_C003.indd 87 10/15/07 5:29:45 PM
88 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
TaB
lE 3
.8 (c
onti
nued
)Ex
trac
tion
of B
ioac
tive
Com
poun
ds fr
om F
ruit
s an
d ve
geta
bles
usi
ng S
C-C
O2
raw
M
ater
ial
Feed
(g)
Sam
ple
prep
arat
ion
Bio
acti
ve
Com
poun
d
Extr
acti
on C
ondi
tion
sr
ecov
erya
(%)
ref
.pa
rtic
le S
ize
(mm
)H
2O (
%)
T (°
C)
p (M
pa)
Flow
rat
eTi
me
(min
)C
osol
vent
Tom
ato
0.5
0.05
–0.2
5n.
i.Ph
ytoe
ne,
phyt
oflue
ne,
ξ-ca
rote
ne,
β-ca
rote
ne,
lyco
pene
40,5
0,6
08–
264×
10–3
L/m
in30
Non
en.
i.20
1
0.3
n.i.
n.i.
Lyco
pene
60,8
5,1
1040
.51.
5×
10–3
L
/min
50A
ceto
ne,M
eOH
,E
tOH
,hex
ane,
di
chlo
rom
etha
ne,
wat
er+
+
100
202
n.i.
n.i.
n.i.
Lyco
pene
45–8
035
–38
n.i.
120–
180
Non
e55
203
3n.
i.n.
i.Ly
cope
ne,
toco
pher
ols
32–8
613
.8–4
8.3
2.5
×1
0–3
L/m
in
n.i.
Non
e61
204
0.5
n.i.
n.i.
Lyco
pene
408–
284
×1
0–3
L/m
in
n.i.
Non
en.
i.20
5
20n.
i.n.
i.Ly
cope
ne40
32n.
i.n.
i.N
one
n.i.
206
3000
1n.
i.Ly
cope
ne45
–70
33.5
–45
133.
3–33
3.3
g/m
in12
0–48
01–
20%
ha
zeln
uto
il++
6020
7
T:t
empe
ratu
re,P
:pre
ssur
e,n
.i.:n
otin
dica
ted.
aR
ecov
ery
(ge
xtra
ct/g
oil
inf
eed
mat
eria
l×1
00),
b sup
erfic
ialv
eloc
ity,c y
ield
(g/
100
gfe
edm
ater
ial)
,+co
solv
enta
dded
tos
ampl
ebe
fore
ext
ract
ion
atth
ele
vel(
%,w
/w)
indi
cate
d,+
+co
solv
enta
dded
tos
ampl
ebe
fore
ext
ract
ion
atth
ele
vel(
%,w
/w)
indi
cate
d.
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Supercritical Fluid Extraction of Specialty Oils 89
yielddecreasedasflowratewasincreasedfrom2.5to15mL/min(measuredatextractiontemperatureandpressure)[204].Comparedwiththelycopenerecoveryof38.8%(oryieldof4.59μg/grawmaterial)obtainedataflowrateof2.5mL/min,only8%recovery(~1μg/gorlessyield)wasobtainedataflowrategreaterthan10mL/min(measuredatextractiontemperatureandpressure)[204].Withflowratesfrom0.875to1.25L/min(measuredatSTP),theolivehuskoilyieldincreased,whereastheyielddecreasedathigherflowrates[208].ThedecreasemaybeattributedtotheshortresidencetimeofCO2intheextractorandthereforetheCO2leavingtheextractornotbeingsaturatedwithoil.A lowflowrate (1.8g/min)producedasmalleramountofsqualenebutatahigherconcentration,whereasahighflowrate(5.4g/min)producedahigheramountofsqualeneatalowerconcentrationintheextract[209].
c) Useofcosolvent:Acetone,ethanol,methanol,hexane,dichloromethane,and water have been compared as cosolvents in SC-CO2 by mixing thecosolventwiththesamplepriortoextraction[202]anditwasshownthatall cosolvents tested except water increased lycopene recovery. In fact,water showed a negative effect, decreasing lycopene recovery to 2%.Ethanol increased recovery but decreased extraction rate. All the othercosolventsstudiednotonlyincreasedthelycopeneyieldbutalsoimprovedtheextractionratetovaryingdegrees[202].Theuseofvegetableoilsasacosolventfortherecoveryofcarotenoidsfromvegetableswasrecentlydeveloped [198,203, 207]. For example, hazelnut oil was chosen byVasapolloetal. [207]becauseof its lowacidity,whichcanprevent thedegradationoflycopeneduringextraction.Lycopeneyieldincreasedwithhazelnutoiladditionasacosolvent,buttheextractwasmoredilutedathigheramountsofoil[207].For theextractionwithoutcosolventaddi-tion, the lycopene recovery was practically maintained below 10%from2to5hoursextractiontime,whileinthepresenceofhazelnutoil,the lycopene recovery increased to about 20% in 5 hours and 30% in8hours.SunandTemelli[198]addedcanolaoilinacontinuousmannerintoSC-CO2fortherecoveryofcarotenoidsfromcarrot.TheextractionyieldwithSC-CO2withoutcanolaoiladditionforα-carotenewas137to330.4μg/gandβ-carotenewas171.7to386.6μg/gfeedmaterialatdif-ferent temperatures and pressures, while the yields more than doubleto288.0–846.7μg/gand333.8–900.0μg/g feed forα-andβ-carotene,respectively,uponadditionofcanolaoil.Themajoradvantageofusingvegetableoilsascosolvents is theeliminationoforganic solventaddi-tion,whichneedstoberemovedlater,andthefactthattheoilenrichedinbioactivescanbeusedasisinavarietyofproductapplications.
3.3.4.2 Characterization of products Extracted by SC-CO2
3.3.4.2.1 Chemical CompositionFattyacidcompositionofoilsextractedfromvariousfruitsandvegetablesisshowninTable3.9.Trilinolein(LLL)isthemaintriglyceridepresentincarrotoilfollowed
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90 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
byLLP,LLO,POL,andOOP[197].Linoleicacidisthemainfattyacid,followedbypalmiticacidinbothcarrotandtomatooils[197,204].ThefattyacidcompositionoftomatoextractobtainedbySC-CO2wassimilartothatofchloroformextract.ButtheextractsobtainedbySC-CO2atdifferenttemperatureandpressureconditionshaddifferentfattyacidcompositions,whichwereduetothedifferencesinthesolubili-tiesoflinoleicandpalmiticacidsatdifferentconditions[204].
3.3.4.2.2 Other Quality AttributesTheyellow-orangecolorofcarrotoilwasmainlycontributedbythecarotenes,whicharefat-solublepigments[197].
3.3.4.3 Comparison with Conventional Methods
CarrotoilextractedbySC-CO2hadhighercarotenes(1,850mg/kg)thanthatofcom-mercialcarrotoil(170mg/kg)[197].Italsohadahighsterolcontent(30.2mg/kg),which was 17-fold higher than that in commercial carrot oil (1.7 mg/kg). ThesqualeneconcentrationofoliveoilintheSC-CO2extractwas10timeshigherthanthatobtainedwithsolventextraction.However, thisenrichmentwasaccompaniedbyadropintheoverallextractedsqualenequantities[209].SC-CO2extractionpro-duced superiorolivehuskoil in termsofoil acidity,PV,andphosphoruscontent[208];therefore,asimplerrefiningprocesswouldberequired.
3.4 FuTurE TrENdS
TheliteraturereviewedinthischapterdemonstratesthefeasibilityofusingSC-CO2fortherecoveryofspecialtyoilsfromavarietyofplantmaterials.Asshown,it isessentialtostudyeachplantmaterialindividuallybecausethepretreatmentoffeedmaterialandoptimumextractionconditionsaredependentonthestructureandcom-positionofthespecificplantmaterial.Themajorityofthesestudieshavebeencar-riedoutat laboratoryscale,andpilot-scaleSC-CO2extractionstudiesare lacking.Even thoughsomeapplicationshavealready reachedcommercial scale,additionalpilot-scalestudieswouldprovideimportantdatanecessaryforscale-upandeconomicfeasibilityassessment.ForSC-CO2technologytobeadoptedmorewidely,itseco-nomic viability and advantages over conventional techniques must be proven foreachapplication.Pilot-scalestudiesmayshowthatdespiteinitialhighcapitalcosts,
TaBlE 3.9Fatty acid Composition of vegetable Oils
raw Material
Fatty acid Conent
C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 C20:1 ref.
Carrot 0.4 16.6 1.8 1.8 11.6 60.1 4.9 0.4 197
Tomato 1.0b 1.5 0.6 69.0 5.8 1.3 — 11.4 206
1.2c 3.8 3.5 18.6 4.4 3.4 — 18.9 206
Myristicacid(C14:0),Palmiticacid(C16:0),Palmitoleicacid(C16:1),Stearicacid(C18:0),Oleicacid(C18:1),Linoleicacid(C18:2),Linolenicacid(C18:3),Eicosenoicacid(C20:1).a wt%,bSeparationvessel1,cSeparationvessel2.
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Supercritical Fluid Extraction of Specialty Oils 91
operatingcostswouldbelowerandtheoverallfeasibilitycanbeprovenatcertainscalesofoperation.Inaddition,supercriticaltechnologyallowsthepossibilityofcou-plinganextractionoperationwithcolumnfractionationundersupercriticalconditionstofurtherconcentratethebioactivecomponentsofinterest.Aswell,theresidualmealfollowingextractionofspecialtyoilscanbeevaluatedforotherhigh-valueendusessincedegradationofmealisminimizedwhenSC-CO2isusedasthesolvent.Ontheotherhand,moreresearchisneededtoinvestigatethequalityattributesofSC-CO2-extractedspecialtyoils,suchasoxidativestability,chemicalcomposition,stabilityofbioactivecomponents throughoutextractionandstorage,andtheflavorprofileandconsumeracceptabilityofsuchoils.TheadvantagesofSC-CO2extractionovercon-ventionalsolventextractionneedtobebettercommunicatedtoconsumers.
3.5 CONCluSIONS
Specialtyoilsaretraditionallyrecoveredbymechanicalpressingorextractionusingorganic solvents.Thedisadvantagesof theseconventional techniquesare thehighlevelofresidualoilinthepressedmeal,undesirablesolventresidueleftintheproduct,anddegradationoffat-solublebioactivecomponents.SC-CO2extractionisaprom-ising technology that overcomes these disadvantages for the recovery of specialtyoilsrichinbioactivecomponentssuchascarotenoids,PUFAs,squalene,sterols,andtocolsfromdifferentplantsources.Extensiveresearchcarriedoutwithalargevarietyofplantmaterials—suchasnuts,seeds,cereals,fruits,andvegetables—hasshownthatSC-CO2 is effective in recovering specialtyoils rich inbioactive compounds.Theextractionefficiencyintermsofyieldandrecoveryaswellasthecompositionofspecialtyoilsareaffectedbydifferentfactors,suchassamplepreparation(particlesizeandmoisturecontent)andextractionparameters(temperature,pressure,solventflow rate, extraction time, anduseof a cosolvent).Theseparametersalsohaveanimpactonvariousqualityattributes,suchascolor,flavor,andoxidativestabilityoftheextractedoilandtextureoftheresidualmeal.Ingeneral,thecoloroftheresidualmealbecamelighterasmoreoilwasremovedbecausemostofthepigmentsarefatsolu-ble.SC-CO2extractionproducedsuperioroilwithrespecttooilacidityandperoxidevalue. However, more research on quality attributes like oxidative stability wouldbebeneficialtobetterelucidatetheeffectoftheuseofSC-CO2ontheextractionofspecialtyoils.Ethanolhasbeenusedasacosolventinnumerousstudiestoenhancetheefficiencyofextraction;however,thefactthatadditionalheattreatmentisneededtoremoveethanolfromthefinalproductshouldnotbeoverlookedbecauseheattreat-mentcanbedetrimentaltothesensitivebioactivecomponentsofinterest.
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103
4 Extraction and Purification of Natural Tocopherols by Supercritical CO2
Tao Fang, Motonobu Goto, Mitsuru Sasaki, and Dalang Yang
Contents
4.1 Background................................................................................................. 1044.1.1 ProblemswithConcentratingTocopherolsUsingMolecular
Distillation....................................................................................... 1044.1.2 PretreatmentbeforeConcentratingTocopherol............................... 1054.1.3 FundamentalResearchonConcentratingTocopherols................... 106
4.2 MainExperimentalMaterials..................................................................... 1074.3 AnalyticalMethods..................................................................................... 1074.4 CorrelationforExperimentalData............................................................. 1074.5 BinaryPhaseEquilibria.............................................................................. 107
4.5.1 ApparatusandProcedure................................................................. 1074.5.2 HighPressureViewCell.................................................................. 1104.5.3 PhaseEquilibriumProperties.......................................................... 1104.5.4 Solubility.......................................................................................... 1134.5.5 DistributionCoefficient................................................................... 113
4.6 TernaryPhaseEquilibria............................................................................ 1184.6.1 ApparatusandProcedure................................................................. 1184.6.2 InfluencesofPressureandTemperatureonPhaseEquilibrium...... 1204.6.3 SeparationFactorbetweenTocopherolandMethylOleate............. 1224.6.4 EquilibriumLines............................................................................ 1234.6.5 PhaseBehaviorofME-DOD...........................................................124
4.7 SeparationwithSupercriticalCO2Fractionation........................................ 1264.7.1 FractionationApparatusandProcedure.......................................... 1274.7.2 PretreatmentResultandCompositionofME-DOD........................ 1294.7.3 EffectoftheInitialPressure............................................................ 1314.7.4 EffectoftheFinalPressure............................................................. 1324.7.5 CompositionofTocopherolConcentrate......................................... 134
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104 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
4.7.6 ViscosityComparison...................................................................... 1344.7.7 ApplicationinCommercialProduction........................................... 136
4.8 Conclusions................................................................................................. 136References.............................................................................................................. 138
4.1 BaCkground
Tocopherols,commonlyknownasvitaminE,areknownforantioxidativeactivitywhich has been widely applied in the fields of food, medicine, and cosmetics.Figure4.1 illustrates the molecular structures of tocopherols. The main source ofnaturaltocopherolsisdeodorizerdistillate(DOD),abyproductoftheedibleoilrefin-ingprocessthatisrichintocopherolsandsterols[1].
4.1.1 Problems with ConCentrating toCoPherols Using moleCUlar Distillation
Molecular distillation, also called short-path distillation, has been applied to thecommercialproductionoftocopherolsfromDOD[2–5].Itischaracterizedbyhighvacuuminthedistillationspace,shortexposureofthedistilledliquidtotheoper-ating temperatures, and short distancebetween the evaporator and the condenser(20to70mm)[3].
However,onthebasisofprojectinvestigation,wefoundsomeproblemsinthecommercialproductionoftocopherolsusingmoleculardistillation.First,theprocessof molecular distillation is generally performed with multistage distillators (3 to5units)athighvacuum(0.1to10Pa)andhightemperature(433to503K).Notice-ably,ahighqualityvacuumpumpisabsolutelyindispensableforensuringenoughvacuumconditionforeachdistillator.Also,itisobviousthatthehighqualitypumpusedforcommercialproduction,thedistillator,withfinestructureandstrictopera-tionconditions,leadstorelativelyhighequipmentinvestmentandoperationcost.
O CH3
CH3
CH3 CH3CH3
CH3
HO
R1
R23'
4'2
7'8'
11'
Substituents R1 R2 Notation
CH3 CH3 α-tocopherolCH3 H β-tocopherol
H CH3 γ-tocopherolH H δ-tocopherol
Figure 4.1 Molecularstructureoftocopherols.(FromBrunner,G.,Gas Extraction: An Introduction to Fundamentals of Supercritical Fluids and the Application to Separation Processes, Darmstadt:Sternkopff,Springer,NewYork,1994.Withpermission.)
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Extraction and Purification of Natural Tocopherols by Supercritical CO2 105
Second, according to our communication with some companies employingmoleculardistillation,itisgenerallydifficulttokeepallpressuresinthedistillatorconstantat low levels (0.1 to10Pa)during the longoperation.Consequently, theoperationtemperaturehastobeincreasedtocompensateforthedecreaseinvacuumdegreewithaviewtostabilizingtheconcentrationofthefractions.Asaresult,suchfluctuationsintemperatureandpressureleadtoanunstablequalityofthetocoph-erolproduct. Inaddition, thedefinitionofmoleculardistillation isnotveryaccu-ratebecausethereisnoeffectofrectificationcausedbyfractioncondensationandrefluence. As we know, the effect of rectification is the main difference betweendistillationandvaporization.Furthermore,thephenomenonofentrainmentprobablyoccurswithoutrectificationandinfluencesontheseparationselectivity.
Third,thegeneralopinionisthattheshortcontactorresidencetimeoftheproductathightemperaturesduringmoleculardistillationdoesnotcauseanydegradationoftheproductanddoesnotaffectthequalityoftheproduct.However,Mau[5]researchedonconcentratingtocopherolsbymoleculardistillationandreportedtheexistenceoftocopheroldimmersandotherdegradationproductsat433to493K,eventhoughpres-surewaslowerthan0.133Pa,andthetotalrecoveryoftocopherolsinallfractionsandresiduewasonlyabout75.16%oftheinitialamountoftocopherolsinfeed.
Finally, in the commercial operation of molecular distillation with three tofivedistillationunits,theresidencetimeoftocopherols,themaincompoundinthesecondorthirdfraction,isnolessthan1hour,whichisnotashorttimeforasepara-tionoperation.
Becausethermaldegradationoftocopherolsiscommonlycausedbyhighprocess-ingtemperature[6],developmentofnewalternativeisolationtechniques,includingsupercriticalfluidextraction(SFE),hasbeendesired.
4.1.2 Pretreatment before ConCentrating toCoPherol
Tomodifythecompositionofrawmaterial,aprocessofpretreatmentisgenerallynecessary. Pretreatment involves two steps (esterification and methanolysis) thatconvert free fatty acids (FFAs) and glycerides (Gly.), respectively, into fatty acidmethylesters(FAMEs).ThemainobjectiveistomodifythecompositionofDODandtoincreasethesolubilityofsoybeanDODinsupercriticalcarbondioxide(CO2)extraction.InthepublishedliteratureonchemicalmodificationofDOD,esterifica-tionwascarriedoutwiththecatalystssulfuricacid(H2SO4)[5,7–9,12],hydrochloricacid(HCL)[10],orNa[11].Additionally,someresearchersaddedasecondreactionofmethanolysiswiththecatalystssodiummethoxide(NaOCH3)[8,12]andsodiumhydroxide(NaOH)[9].Inourlabwork,H2SO4andNaOCH3wereselectedasthecatalystsofmethylesterificationandmethanolysis, respectively.Figure4.2 showsthepretreatmentprocessofDOD.Aftereachreaction,themixturewaswashedwithhot water until it became neutral. Finally, the mixture was held at low tempera-tureand,asaresult,mostofthesterolscrystallizedandwereremovedbyfiltration.Afterpretreatment,theoil,methylesterifiedDOD(ME-DOD),wasobtained,whichcontainsmainlyFAMEs(70%to80%),tocopherols(10%to15%),andimpurities(suchasresidualsterols,glycerides,squalene,pigments,andlongchainparaffins,comprisingintotalabout10%to15%).
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106 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
4.1.3 fUnDamental researCh on ConCentrating toCoPherols
SupercriticalCO2isrelativelysuitableasaseparatingsolventforextractingsomefat-solublecomponents.AlthoughsomeresearcherstriedtoconcentratetocopherolsfromDODbysupercriticalCO2[6–16],theoperationparameters,especiallypressure,vary.Forexample,Zhaoetal.[7]concentrated75%tocopherolsat12MPa,whereasLee et al. [10] reported that 40 MPa could be used to obtain 40% extract. Kingetal. [14] combined SFE with supercritical fluid chromatography (SFC) for con-centratingtocopherols,andtheoptimizedconditionswere25MPa/353KforSFEand 25MPa/313 K for SFC. Generally, if pressure is low, satisfactory selectivitycanbeobtainedbutproductivityislow.Contrarily,higherpressureleadstolowerselectivityandhigherproductivity.Tofindmore reasonableoperatingconditions,phaseequilibriaofME-DOD+CO2mustbeclarified.
WhenconcentratingnaturaltocopherolsfromME-DOD,theimportantstepistoremoveFAMEs,whichcontributemorethan70%ofME-DOD.Toexplorethereasonableoperation conditions for this step, the complex systemofME-DOD+CO2wasinitiallyregardedasapseudo-ternary(methyloleate+tocopherol+CO2)system.ThereasonforchoosingmethyloleateisthattheME-DODappliedinour
Methanolysis (Reflux at 343 K for 120 min )
H2SO4(3 wt.% oil), methanol (65 wt.% oil)
DOD(FFA, glycerides, sterols, sterol esters, tocopherols and squalene)
Methyl Esterification (Reflux at 333 K for 120 min)
Oil Phase, MEDOD (FAMEs, Tocopherols, Squalene, Residual Sterols and Glycerides)
Solid Phase (Crude Sterols)
Standing Separation and Washing with Hot Water (353 K) Until Neutral (pH=7)
Upper, Oil Phase (partly methyl esterified DOD)
Lower, Water Phase
NaOCH3 (1 wt.% oil), Methanol (55 wt.% oil)
Standing Separation, Neutralization with HCL and Washing with Hot Water (353 K)
Lower, Water Phase
Chilling at 277 K, Crystallization and Filtration
Upper, Oil Phase
Figure 4.2 Pretreatment process for preparing ME-DOD from DOD. (From Fang, T.,Goto,M.,Wang,X.,Ding,X.,Geng,J.,Sasaki,M.andHirose,T.,J.Supercritical Fluids, 40,50, 2007.Withpermission.)
7089_C004.indd 106 10/8/07 11:50:34 AM
Extraction and Purification of Natural Tocopherols by Supercritical CO2 107
researchisfromthesoybeanoilindustryandthemainFAMEsaremethyloleateandmethyllinoleate(about75%to80%ofallFAMEs);thelatterissimilartomethyloleateinphysicochemicalproperties[17].
Asfarasphaseequilibriumisconcerned,twobinarysystemsofα-tocopherol+CO2andmethyloleate+CO2weremeasuredandcorrelated.Thentheternarysystem(methyloleate+tocopherol+CO2)andtherealisticsystem(ME-DOD+CO2)wereinvestigated.Finally,thephaseequilibriumdatawereanalyzedandaccordinglytheseparationofnaturaltocopherolsfromME-DODwascarriedoutwithsupercriticalCO2fractionation.
4.2 Main experiMental Materials
CO2wassuppliedbytheUchimuraSansoCo.,Ltd.(Osaka,Japan),withapurityof99.97%.MethyloleateandDl-α-tocopherolwereobtainedfromWakoPureChemicalIndustries,Inc.(Tokyo,Japan)withpuritiesof≥ 98%.DOD(9.23%tocopherols)wassuppliedbyKaidiFineChemicalIndustrialCo.,Ltd.(Wuhan,HubeiProvince,P.R.China),anditscompositionisillustratedinTable4.1.ME-DOD(10.19%tocopherols)waspreparedbyDODaccordingtotheprocedureshowninFigure4.2.
4.3 analytiCal Methods
TheapproximatecontentsofFFAsandglycerides,includingmonoglycerides,diglyc-erides,andtriglycerides)werecalculatedfromacidandsaponificationvalues(A.V.andS.V.,respectively)andexpressedasthecontentsofoleicacidandtriolein[18].
Analysesoftocopherols,sterols,andFAMEswereperformedwithhighperfor-manceliquidchromatography(HPLC)andgaschromatographwithflameionizationdetector(GC-FID),respectively[19–21].ThecompositionofME-DODandtocopherolconcentrate was determined with gas chromatograph-mass spectrometry (GC-MS).Additionally,theviscosityofthesamplesobtainedintheseparationexperimentweremeasuredwithanAR1000rheologymeter(TAInstrumentsCo.,Ltd,England)[21].
4.4 Correlation For experiMental data
TheSoave-Redlich-Kwong(SRK)EOS[22]withtheAdachi-Sugie(AS)mixingrule[23] was used to correlate the experimental data. The SRK EOS is the modifica-tionofthesimpleRedlich-KwongEOS,withwhichthevaporpressurecurvecanbereproducedwell.ThisprocedureofcorrelationwascompletedbyPE2000,whichwasdevelopedbyPfohl,Petkov,andBrunnerandcontainssomecommonEOSandprovedcapabilitytoobtainaconvergencesimilartothatfromASPENsoftware[24].
4.5 Binary phase equiliBria
4.5.1 aPParatUs anD ProCeDUre
Theexperimentalapparatususedinthisworkiscalled“gas-liquidalternatingcir-culationsystem,”whichisusedtosimultaneouslymeasurethecompositionsinboth
7089_C004.indd 107 10/8/07 11:50:34 AM
108 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
taB
le 4
.1C
hara
cter
isti
cs o
f do
d
a.V
.*
(mg
koh
/g)
s.V.
* (m
g ko
h/g
)FF
a*
(%)
gly
.* (
%)
toco
pher
ols
(%)
isom
er p
erce
ntag
e (%
)
ster
ols
(%)
isom
er p
erce
ntag
e (%
)
α-β-
+γ-
δ-C
ampe
ster
olst
igm
aste
rol
β-si
tost
erol
95.4
147.
148
.127
.29.
2311
.73
60.0
328
.24
9.45
33.5
23.5
43.0
*A
ccor
ding
toA
OC
Sm
etho
ds[1
8],t
hea
ppro
xim
ate
cont
ents
off
ree
fatty
aci
d(F
FA)a
ndg
lyce
ride
s(G
ly.i
nclu
ding
mon
ogly
ceri
des,
dig
lyce
ride
s,a
ndtr
igly
ceri
des)
wer
eca
lcul
ated
fro
ma
cid
and
sapo
nific
atio
nva
lues
(A
.V.a
ndS
.V.,
resp
ectiv
ely)
and
exp
ress
eda
sth
eco
nten
tso
fol
eic
acid
and
trio
lein
.So
urce
:Fa
ng,T
.,G
oto,
M.,
Wan
g,X
.,D
ing,
X.,
Gen
g,J
.,Sa
saki
,M.a
ndH
iros
e,T
.,J.
Sup
ercr
itic
al F
luid
s, 4
0,5
0, 2
007.
With
per
mis
sion
.
7089_C004.indd 108 10/8/07 11:50:35 AM
Extraction and Purification of Natural Tocopherols by Supercritical CO2 109
liquidandgasphases.AsshowninFigure4.3,theequilibriumsystemisimmersedinawaterbath,withtwostirrersforkeepingthesystem’stemperatureuniformwiththeprecisiontemperaturecontrolof+0.5K.
About70mLofpurecomponentisinitiallychargedintoequilibriumvessel6(170mL,max.pressure30MPa),andthenCO2flowsintotheapparatusfromcylinder1,passing throughvalve2andfiltratingpipe3, thecoolerandsyringepump4(Isco260D,max.pressure57.71MPa,TeledyneIscoInc.,Lincoln,NE,USA),valve5,andthenintoequilibriumvessel6.Afterthepressureandtemperaturereachtherequiredvalues,valve5isclosedandthenmagneticpump8ispoweredon,whichcankeepthefluidflowingupwardatabout4mLperminute.Byrotatingfour-wayvalve7,gas(lightphase)orliquid(heavyphase)ischosenasthecirculatingfluidinthesystem.Forexample,thecirculatingfluidinFigure4.3isthegasphase.Afterthegasphaseiskeptcirculatingfor1.5hours,six-wayvalve9isturnedandthefluidinsampler11(20mL)istakenasthegassample.Aftervalve15isopenedandadjusted,theCO2inthegasslowlypassesthroughsamplingbottle13andflowmeter17(precision0.01L),whichrecordstheamountofCO2.Thepipesbetweenvalve9and15andbottle13areallheatedbyanelectronicheater.Thecompoundsprecipitatedinsampler11arewashedintobottle13withn-hexaneandrinsedwithabout10mLn-hexanetoavoidthesampleloss,sincethesolutesmayformaerosolparticlesandthenpassthroughthecollectingbottlewithCO2fluid.Aftereveryrun,theresidualn-hexaneinthesampleisremovedfromcylinder20withCO2andthenmergedintothesamplebattle.Then-hexanesolutionisquantifiedbyelectronicbalance(precision0.1mg)andanalyzedbyGC (formethyloleate)orHPLC(forα-tocopherol).From theamountsofCO2andchromatographicdata,thecompositioninthegasphaseiscalculated.Asforthe
1, 20: CO2 Cylinder 2, 5, 15, 16: Valve 3, 19: Filtrating Pipe4: Cooler and Syringe Pump 6: Equilibrium Vessel 7: Four-way Valve8: Electromagnetic Pump 9, 10: Six-way Valve 11: Gas Sampler12: Liquid Sampler 13, 14: Sampling Bottle 17, 18: Gas Flowmeter
2
3
41
5
79
86
Thermostated Water Bath
(170 ml)
11
10
1317
15
19
20
1814
1612
(5 m
l)(2
0 m
l)
PI TIR
Figure 4.3 Schematic diagram of experimental apparatus. (From Fang, T., Goto, M.,Yun,Z.,Ding,X.andHirose,T.,J.Supercritical Fluids, 30,1,2004.Withpermission.)
7089_C004.indd 109 10/8/07 11:50:36 AM
110 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
sample from the liquidphase, four-wayvalve7 is turned to the liquidcirculation.Aftercirculatingfor1.5hours,six-wayvalve10isturnedforsampling.Byasimilarmethodtothegasphases,theliquidcompositionisalsoobtained.
Thisstructureofourapparatuspreventspressurefluctuationduringsampling.Toobtainmoreaccurateresults,alldatarepresentmeanvaluesofthreesamplingsatauniformcondition,andtherelativestandarddeviationsarewithin0.005mol%forgascomposition(Y1)and0.5mol%forliquidcomposition(X1).
4.5.2 high PressUre View Cell
Forobservingtheequilibriumsystems,aviewcell(30mL,max.pressure30MPa,AkicoCo.,Tokyo,Japan)wasemployedinourexperiment,asshowninFigure4.4.Amagnetic stirrer is coupled with the cell and the temperature is controlledby electric heaters embedded inside the cell’s wall. The Isco pump was used forpressurizingthesystem.
4.5.3 Phase eqUilibriUm ProPerties
Thebinaryphaseequilibriumdataformethyloleate+CO2andα-tocopherol+CO2weremeasuredandcorrelatedat313.15to353.15Kinthepressurerangesof5to23MPaformethyloleateand5to30MPaforα-tocopherol[19].Theisothermsof313.15,333.15,and353.15Kformethyloleate+CO2andα-tocopherol+CO2weremeasuredoverthepressurerangesof5to23MPaand5to30MPa,respectively.TheexperimentalresultsandtheircorrelateddataareshowninFigure4.5andFigure4.6,inwhichthedatameasuredbyotherresearchers[25–37]arealsoillustrated.
Asforthephaseequilibriumofmethyloleate+CO2,at313.15Kand333.15K,our experimental data in Figure4.5 agree well with the data reported by otherresearchers, except thedata reportedbyChenget al. [25].Chenget al. foundanunusualwaist-shapecurveatthevicinityofthecriticalpressureofCO2,aninteresting
Filtrating PipePI
TIR
View Cell
Magnetic StirrerIsco Syringe Pump
CO2 Tank
Figure 4.4 Schematic diagram of high-pressure cell for visual observation. (FromFang,T.,Goto,M.,Yun,Z.,Ding,X.andHirose,T.,J.Supercritical Fluids, 30,1,2004.Withpermission.)
7089_C004.indd 110 10/8/07 11:50:37 AM
Extraction and Purification of Natural Tocopherols by Supercritical CO2 111
20(a
) T =
313
.15
K
(c) T
= 3
53.1
5 K
(b) T
= 3
33.1
5 K
Cram
pon
et al
., 199
9In
omat
a et a
l., 1
989
Zou
et al
., 199
0Yu
et al
., 199
2Ch
eng
et al
., 198
9Th
is W
ork
Cram
pon
et al
., 199
9In
omat
a et a
l., 1
989
Zou
et al
., 199
0Yu
et al
., 199
2N
ilsso
n et
al., 1
991
This
Wor
k
16 12 P (MPa) P (MPa)
8 4 24 20 16 12 8 4
24 20 16 12 8 4
0.5
0.6
Mol
e Fra
ctio
n of
CO
2 (%)
Mol
e Fra
ctio
n of
CO
2 (%)
0.7
0.8
0.9
0.97
0.98
0.99
1.00
0.5
0.6
0.7
0.8
0.9
1.0
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
0.98
1.00
20 16 12 8 4 0.2
0.3
0.4
0.5
0.6
0.7
(d) Th
is W
ork
and
Corr
elat
ion
Resu
lt (S
RK-E
OS
and
AS
mix
ing
rule
)Th
is W
ork,
313
.15
KCa
lcul
ated
at 3
13.1
5 K
This
Wor
k, 3
33.1
5 K
Calc
ulat
ed at
333
.15
KTh
is W
ork,
353
.15
KCa
lcul
ated
at 3
53.1
5 K0.
80.
960.
90.
981.
00
This
Wor
k
Fig
ur
e 4.
5 B
inar
yph
ase
equi
libr
iaf
orm
ethy
lol
eate
+C
O2.
(Fr
omF
ang,
T.,
Got
o,M
.,Y
un,
Z.,
Din
g,X
.an
dH
iros
e,T
.,J.
Sup
ercr
itic
al F
luid
s, 3
0,1
,200
4.W
ith
perm
issi
on.)
7089_C004.indd 111 10/8/07 11:50:39 AM
112 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
(a) T
= 3
13.1
5 K
(c) T
= 3
53.1
5 K
(d) Th
is W
ork
and
Calc
ulat
ed D
ata
This
Wor
k, 3
13.1
5 K
Calc
ulat
ed at
313
.15
KTh
is W
ork,
333
.15
KCa
lcul
ated
at 3
33.1
5 K
This
Wor
k, 3
53.1
5 K
Calc
ulat
ed at
353
.15
K
(b) T
= 3
33.1
5 K
Joha
nnse
n et
al., 1
997
Chen
et al
., 200
0Sk
erge
t et a
l., 2
002
Mei
er et
al., 1
994
Pere
ira et
al., 1
993
Chra
stil,
198
2O
hgak
i et a
l., 1
987
This
Wor
k
Joha
nnse
n et
al., 1
997
Chen
et al
., 200
0Sk
erge
t et a
l., 2
002
Pere
ira et
al., 1
993
Chra
stil,
198
2Th
is W
ork
Joha
nnse
n et
al., 1
997
Sker
get e
t al.,
200
2M
eier
et al
., 199
4Ch
rast
il, 1
982
This
Wor
k
3236 28 24 20 16 12 8 4 0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
0.99
60.
998
1.00
00.
20.
30.
40.
50.
60.
70.
80.
90.
996
0.99
81.
000
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
0.99
60.
998
1.00
00.
50.
60.
70.
80.
90.
998
1.00
0
36 32 28 24 20 16 12 8 4 32 28 24 20 16 12 8 4
32
Mol
e Fra
ctio
n of
CO
2 (%)
P (MPa)P (MPa)
Mol
e Fra
ctio
n of
CO
2 (%)
36 28 24 20 16 12 8 4
Fig
ur
e 4.
6 B
inar
yph
ase
equi
libr
iaf
orα
-toc
ophe
rol
+C
O2.
(Fr
omF
ang,
T.,
Got
o,M
.,Y
un,
Z.,
Din
g,X
.an
dH
iros
e,T
.,J.
Sup
ercr
itic
al F
luid
s, 3
0,1
,200
4.W
ith
perm
issi
on.)
7089_C004.indd 112 10/8/07 11:50:40 AM
Extraction and Purification of Natural Tocopherols by Supercritical CO2 113
phenomenonthatwasnotverifiedbyotherresearchers’andourdata.Additionally,inthecaseof353.15K,nodataarereportedintheliterature.
Forthesystemofα-tocopherol+CO2,Figure4.6a–cillustratesthatourmeasureddatamatchwiththeliteraturedataatthreetemperatures.Theresults,especiallythecompositiondataingasphase,areremarkablydifferentfromauthortoauthor.Thepossible reasonsfor theerrorswere thought tobehighviscosityofα-tocopherol,pressurechange,andaerosolscausedbysuddendepressurization[34].Additionally,thedataforliquidphasearenotasabundantasthoseforgasphase.
Our data illustrate that both methyl oleate and α-tocopherol mole fractionsin gas phase increase as pressure increases at constant temperature. Meanwhile,the CO2 fraction in liquid phase rises with increasing pressure. The equilibriumconcentrationofmethyloleateinCO2isalwaysmuchhigherthantheequilibriumconcentrationofα-tocopherolinCO2.Moreover,theinfluenceoftemperatureongascompositioniscontrarytothatofpressure.Inaddition,formethyloleate+CO2,withtheincreaseoftemperature,theCO2fractioninliquidobviouslydecreases,whereasforα-tocopherol+CO2at313.15Kand333.15K,theliquidcompositionchangesslightlywithtemperature.Ontheotherhand,at353.15K,theCO2molefractioninliquidisslightlylargerthanthoseatothertemperatureswithpressureshigherthan12MPa.
In addition, an ideal correlation for our experimental data, as shown inFigure4.5dand4.6d,canbeobtainedbytheSRKEOSwiththeASmixingrule,wheretheaveragedeviationobtainedislowerthan0.12%forgasand7%forliquid.
4.5.4 solUbility
The solubilities of methyl oleate and α-tocopherol in supercritical CO2 werecalculatedfromthegasphasecomposition.TheresultsareshowninFigure4.7.ThesolubilitiesofthetwocompoundsarepresentedasafunctionoftheCO2density.
Two effects are observed. At constant temperature, solubility increases withincreasingdensity.ThisisprobablyduetotheincreasingsolventpowerofCO2athigherdensity.Atconstantdensity,ariseof temperatureresults inanincreaseofsolubilityduetotheincreaseinvaporpressureofthesolutes.SimilarphenomenawerereportedbyJohannsenandBrunner[35].
4.5.5 DistribUtion CoeffiCient
Equilibriumdataoftwophasescanbeusedtocalculatethedistributioncoefficient,whichwasdefinedby:
Kyxi
i
i
= (4.1)
whereyi and xiaremolefractionsofcomponentiingasandliquidphase,respectively.Calculation results are shown in Figure4.8. As density increases at constant
temperature,thedistributioncoefficientofmethyloleateincreasessignificantly.Inthecaseofmethyloleate+CO2,whenpressurewasmorethan12.5MPaat313.15K,
7089_C004.indd 113 10/8/07 11:50:41 AM
114 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
16
2.5
2.0
1.5
1.0
0.5
0.0
12 8
Met
hyl O
leat
e Den
sity o
f CO
2(g/
ml)
Den
sity o
f CO
2(g/
ml)
Solubility of Alph-Tocopherol (g/100g CO2)
Solubility of Methyl Oleate (g/100g CO2)
T =
313.
15 K
Calc
ulat
ed at
313
.15
KT
= 33
3.15
KCa
lcul
ated
at 3
33.1
5 K
T =
353.
15 K
Calc
ulat
ed at
353
.15
K
Alp
ha-T
ocop
hero
l
T =
313.
15 K
Calc
ulat
ed at
313
.15
KT
= 33
3.15
KCa
lcul
ated
at 3
33.1
5 K
T =
353.
15 K
Calc
ulat
ed at
353
.15
K
4 0 0.0
0.2
0.4
0.6
0.8
0.0
0.2
0.4
0.6
0.8
(a)
(b)
Fig
ur
e 4.
7 T
hes
olub
iliti
eso
fm
ethy
lol
eate
and
α-t
ocop
hero
lin
CO
2.(
From
Fan
g,T
.,G
oto,
M.,
Yun
,Z
.,D
ing,
X.
and
Hir
ose,
T.,
J.S
uper
crit
ical
Flu
ids,
30,
1,2
004.
With
per
mis
sion
.)
7089_C004.indd 114 10/8/07 11:50:42 AM
Extraction and Purification of Natural Tocopherols by Supercritical CO2 115
Met
hyl O
leat
e
1.0
0.8
0.01
2
0.00
8
0.00
4
0.00
0
0.6
0.4
0.2
0.0
Den
sity o
f CO
2(g/
ml)
Den
sity o
f CO
2(g/
ml)
Distribution Coefficient of Methyl Oleate
Distribution Coefficient of Methyl Oleate
T =
313.
15 K
Ca
lcul
ated
at 3
13.1
5 K
T =
333.
15 K
Ca
lcul
ated
at 3
33.1
5 K
T =
353.
15 K
Ca
lcul
ated
at 3
53.1
5 K
Alp
ha-T
ocop
hero
l T
= 31
3.15
K
Calc
ulat
ed at
313
.15
K T
= 33
3.15
K
Calc
ulat
ed at
333
.15
K T
= 35
3.15
K
Calc
ulat
ed at
353
.15
K
0.0
0.2
0.4
0.6
0.8
0.0
0.2
0.4
0.6
0.8
(a)
(b)
Fig
ur
e 4.
8 D
istr
ibut
ion
coef
ficie
nts
ofm
ethy
lol
eate
and
α-t
ocop
hero
lin
CO
2.(
From
Fan
g,T
.,G
oto,
M.,
Yun
,Z.,
Din
g,X
.and
H
iros
e,T
.,J.
Sup
ercr
itic
al F
luid
s, 3
0,1
,200
4.W
ith
perm
issi
on.)
7089_C004.indd 115 10/8/07 11:50:43 AM
116 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
18.5MPaat 333.15K, and22.5MPaat 353.15K, there is no remarkablediffer-enceinequilibriumcompositionsbetweenthetwophases.Thismeansthatintheexperimental range investigated, critical points for the binary mixture probablyexist.Ifthedistributioncoefficientequalstheunity,thetwophases’compositionsanddensitiesareentirelyidentical,asaresultthebiphasicsystemchangingintoamonophasicsystem,whichmeansmethyloleateandCO2arecompletelymiscibleatsuchconditions.Thecorrespondingpressureiscalledacritical pressure forthebinarymixtureunderacertaintemperature,andallthecriticalpointsatdifferenttemperatureslineuptoacritical curve, whichischaracteristicforthismixture[1]inaP-T-xdiagramforabinarysystem.Becauseaccuratemeasurementofcriticalpoints is relativelydifficult,weadopted an approximatemethodby extrapolatingthecorrelatedcurvesofmethyloleate+CO2,asshowninFigure4.5d.Theapproxi-materangesforcriticalpointswereestimatedtobe13to14MPaat313.15K,19to20MPaat333.15K,and23to24MPaat353.15K.
Forverifyingthisprediction,ahigh-pressurecellwithwindowswasusedforvisualobservation.Initially,about20mLmethyloleatewerechargedintothecell.At 313.15 K, CO2 was slowly compressed (0.5 mL/min) into the cell. When thepressurewas8 to12MPa, the interfacebetweengas and liquidwasvery clear,as shown in Figure4.9a. When the pressure reached about 12.6 MPa, the inter-facebetweengasandliquidbecamethickerandtheliquidlevelincreasedalittlebecausemoreCO2dissolvedin liquid,asshowninFigure4.9b.Thenthesysteminsidethecellbecamemoreobscureandturbidwithpressureincrease,asshownin Figure4.9c. Finally, the interface became completely unidentifiable at about13.7MPa,andthewholesystemrecoveredclearasauniformphaseat13.9MPa,as shown in Figure4.9d. At 333.15 K and 353.15 K, a similar phenomenon wasrespectivelyobservedat19.7to20.1MPaand23.5to23.8MPa.Actually,itshouldbenoticedthatthedisappearanceoftheinterfacedoesnotsuddenlyhappen.Thechangeisaprogressiveprocessandtheendpoint isdifficult tobedeterminedbyvisualobservation.However,byvisualobservation,approximatepressure rangesareclosetothepredictionfromourcorrelation.
Inthecaseofα-tocopherol+CO2,thedistributioncoefficientindicatesthatthetwocomponentsarepoorlydissolvedineachother.Thedistributioncoefficientsofα-tocopherolareonetotwoordersofmagnitudelowerthanthoseofmethyloleate.
The discussion above indicates that there are considerable differences inthe solubilities and distribution coefficients of methyl oleate andα-tocopherol inCO2. Therefore, supercritical CO2 extraction can be thought feasible to separateα-tocopherol frommethyloleate.According tophaseequilibriumdataona fattyacid-CO2system[27,28,31,36],thesolubilitydifferencebetweenfattyacidsandα-tocopherolissmallerthanthatofmethyloleateandα-tocopherol.Thus,inordertoseparatetocopherolsfromDOD,thepretreatmentshowninFigure4.2isofgreatsignificance. The pretreatment converts fatty acids and glycerides, which consistof70%to80%ofDOD,intoFAMEs,resultinginenlargingthesolubilitybetweencomponentsofsupercriticalCO2.Therefore,thesupercriticalCO2processseemstobemorefeasibleforseparatingtocopherolsfromesterifiedDODthandirectlyfromDOD.Inaddition,pretreatmentissimultaneouslyadvantageousforremovingmoststerols and other solid impurities (wax, long-chain hydrocarbons) from esterified
7089_C004.indd 116 10/8/07 11:50:43 AM
Extraction and Purification of Natural Tocopherols by Supercritical CO2 117
(a) T
= 3
13.1
5 K
, P =
8 M
Pa(b
) T =
313
.15
K, P
= 1
2.6
MPa
(c) T
= 3
13.1
5 K
, P =
13.
7 M
Pa(d
) T =
313
.15
K, P
= 1
3.9
MPa
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118 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
DOD[5,7–10]becausethesolubilityofsterolsisfarlowerinfattyacidmethylestersthanthatinfattyacids.
Compared with methyl oleate, the solubility and distribution coefficient ofα-tocopherolaregenerallyonetotwoordersofmagnitudelower.Suchalargediffer-encebetweenthe twocomponents indicates that it ispossible toconcentratenaturaltocopherolsfromesterifiedDODintheexperimentalrangeinvestigated.Thisconclu-sionneedstobeprovedfurtherbystudyoftheternarysystemandtherealisticsystem.
4.6 ternary phase equiliBria
Aspreviouslydescribed,thecomplexsystemofME-DOD+CO2canberegardedas a pseudo-ternary (methyl oleate + tocopherol + CO2) system. Regretfully, nopublishedinformationwasfoundonphaseequilibriumofME-DODinsupercriticalCO2,eventhoughsomeliteraturereportedonternaryandmulticomponentsystemsinvolvingα-tocopherolorDOD[38–41].Thus,wemeasuredthephasebehaviorsfortheternaryandrealisticsystemswiththeviewofprovidingfundamentalinforma-tionforfurtherseparationexperimentsandprocessdesign.
Inthispart,weinvestigatedtheinfluencesofthreefactorsonphasebehavior:pressure (from10 to29MPa), temperature (from313.15 to353.15K), and initialfeedcomposition.Six initial feedcompositions(0,10.19,32.44,50.46,71.93,and100mass%)wereinvestigated.Amongthese,0%and100%stoodforthepurecom-positionsofmethyloleateandα-tocopherol,respectively.Theircorrespondingphaseequilibriumdatawerecitedfromthebinarydatainpart4.5.3.ThefeedcompositionofME-DODwas10.19%.Otherfeedcompositionswerepreparedbymixingmethyloleateandα-tocopherolaccordingtodifferentproportions.
4.6.1 aPParatUs anD ProCeDUre
Anexperimentalapparatuswasestablishedformeasuringthecompositionsofbothliquid and gas phases. As shown in Figure4.10, the apparatus consisted of feed,equilibrium, and sampling systems.Aviewcell (30mL,max. pressure30MPa,AkicoCo.,Tokyo,Japan)coupledwithamagneticstirrerwasemployedastheequi-libriumvessel,anditstemperaturewascontrolledwithanelectricheatercapableofmaintainingthetemperaturewithin±0.1K.
Initiallytheequilibriumcellwaschargedwithabout15to20mLfeed.CO2thenflowedintotheapparatusfromtheCO2tankviathefilteringpipeandsyringepump(ISCO260D,max.pressure57.71MPa,Teledyne Isco Inc.,Lincoln,NE,USA),whichisoperatedinthemodeofconstantpressure,andintotheequilibriumcell.Afterthepressureandtemperaturereachtherequiredvalues,themagneticstirrerwasturnedonandthesysteminsidetheequilibriumcellwasstirredforatleast2hours.Byrotating thesix-portvalve, thesampleswereconverted toandfromgas (lightphase)andliquid(heavyphase).Forexample,thesituationshowninFigure4.10isasampletakenfromthegasphase.Duringsampling,byadjustingthemicroswitchof thedigitalbackpressureregulator(BPR,JASCO880-81,JASCOInternationalCo.Ltd.,Tokyo,Japan),gaseousCO2slowlypassedthroughthesamplingbottleandflowmeter,whichrecordedtheamountofCO2(definedascompound3)consumed.
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Extraction and Purification of Natural Tocopherols by Supercritical CO2 119
The pipes connected to the six-port valve, cell, and digital BPR were heated byelectronicheaters.Theirtemperatureswerecontrolledinamannersimilartothatoftheequilibriumcell.Additionally,becauseoftheeffectofadiabaticexpansion,thefluid temperaturedecreasedgreatlyandsuddenlyduringsampling.Consequently,somesolutesorCO2mayhavecondensedattheoutletoftheBPR,contributingmoreorlesstomeasurementinaccuracy.Toavoidsuchphenomena,theBPRoutletwasheatedandmaintainedatatemperatureof371K.Inaddition,about10mLn-hexanewasinitiallyloadedinthesamplebottlebecausethesolutesmayhaveformedaerosolparticlesandpassthroughthecollectingbottlewiththeCO2fluid[34].Then-hexanesolutionwasquantifiedbyelectronicbalance(precision10–4g)andanalyzedbyGC(formethyloleate,compound1)andHPLC(fortocopherol,compound2).Accord-ingtotheamountofCO2consumedandchromatographicdata,thegascomposition(y1, y2, y3)wascalculated.Byasimilarmethod,theliquidcomposition(x1, x2, x3)wasobtained.Additionally,becausesimilarsystemlineswereusedforsamplingfromtwophases,whenthesix-portvalvewasswitchedforsamplingfromanotherphase,thefluidfromthecellwaskeptflowingwithoutsamplingforabout1to2minutes,inordertoavoidcarryoverofthesamples.Anotherkeypointwastoensurethatthesamplewastakenfromanequilibriumsystembysamplingonlywhentheliquid-gasinterfacewasclearlyvisible.
Thestructureofourapparatusavoidedpressurefluctuationduringtheequilib-riumandsamplingstepsbecausetheequilibriumcellpressurewasmaintainedatthesetvalues(therequiredvalues)bysettingtheISCOpumpinthemodeofconstantpressure.Moreimportantly,duringsampling,theCO2flowrateshouldbekeptrela-tivelylowbyadjustingtheBPRmicroswitch.Inourexperiment,theCO2flowrate
Filtering PipeSix-part Valve
Digital BPR
Window
Equilibrium CellISCO Syringe
Pump
Feed System EquilibriumSystem
Magnetic StirrerCO2 Tank
SamplingSystem
Feed Pump
TIR PI
One-wayValve
Figure 4.10 Schematic diagram of the phase equilibrium apparatus. (From Fang, T.,Goto,M.,Sasaki,M.andHirose,T.,J. Chem. Eng. Data,50,390,2004.Withpermission.)
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120 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
wasmaintainedatlowerthan20mL·min–1and5mL·min–1(at0.1MPaandroomtemperature)duringsamplingfromgasandliquid,respectively.
To obtain more accurate results, all data represented mean values of threesamplingsatuniformconditionswithanuncertaintyof±0.001massfractionforgascompositionand±0.002forliquidcomposition,respectively.
Forgasphasesampling,theCO2amountwasadjustedtoabout2L(at0.1MPaandroomtemperature)atexperimentalpressureslowerthan20MPasincelowsolubilityatlowpressureswasamainreasonforexperimentalerror;however,forhigherpressures(≥20MPa),theamountofCO2wasabout1L(at0.1MPaandroomtemperature).Forliquidphase,theCO2amountwasadjustedtoabout0.01to0.02L(at0.1MPaandroomtemperature).Thecontentsofmethyloleateandtocopherolintheliquidsampleweregenerallyhighandrequireddilutionbeforechromatographicanalysis.
Thesamplesdissolvedinn-hexanewereanalyzedbyGCtodeterminetheconcen-trationofmethyloleate.AnalysisoftocopherolwasperformedbyHPLC[19,20].
4.6.2 inflUenCes of PressUre anD temPeratUre on Phase eqUilibriUm
Theisothermsat313.15,333.15,and353.15Kfortheternarysystemofmethyloleate(1)+tocopherol(2)+CO2(3)weremeasuredoverthepressurerangefrom10to29MPa.At313.15K,thecompositiondataat10,20,and29MPaweredrawninatriangulardiagram,as shown inFigure4.11.Obviously, the two-phase region,which is sur-roundedbytheequilibriumdata,shrinkswithincreasingpressure.Inotherwords,
Methyl Oleate
Tocopherol
Tocopherol
Carbon Dioxide
Carbon Dioxide
Methyl Oleate
(a) (b)
0.0 1.0
0.8
0.6
0.4
0.2
0.0
0.2
0.4
0.6
0.8
1.00.0 0.2 0.4
W3
W3
W2W1
W1W2
0.6 0.8 1.0
0.0
0.5
1.00.8 0.9 1.0
0.2
0.1
0.0
Figure 4.11 Influence of pressure on the phase equilibria of (w1 methyl oleate + w2tocopherol+ w3CO2)atT =313.15K:(a)Liquid-gasequilibria;(b)Theequilibriumcomposi-tionsingas;⦁,experimentaldataatP=10MPa;◾,experimentaldataatP=20MPa;▲,theexperimentaldataatP=29MPa;•,thecorrelatedtielinesatP=10MPa;•,thecorrelatedtielinesatP=20MPa;•,thecorrelatedtielinesatP=29MPa.(FromFang,T.,Goto,M.,Sasaki,M.andHirose,T.,J. Chem. Eng. Data,50,390,2004.Withpermission.)
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Extraction and Purification of Natural Tocopherols by Supercritical CO2 121
themutualsolubilityofthecomponentsincreased.Figure4.11ashowstheCO2massfractioninliquidriseswithpressureincrease,whiletheCO2massfractioningasis reduced, as shown in Figure4.11b, which means the solubilities of other com-ponents ingasincrease.At lowerpressures(10MPa),becausebothmethyloleateandtocopherolhavelimitedmiscibilityinCO2,theternaryphasebehaviorrevealsaphaseequilibriumofternarytypeII[1].Thus,thereisnocriticalpoint.Athigherpressures(20,29MPa),becausemethyloleateandCO2arecompletelymiscible,thetwo-phaseareaisternarytypeI,whichischaracterizedbyacriticalpointwherethetwophasesbecomeidentical.InthetypeIsystem,highersolubilityinthegascanbereachedthaninternarytypeIIsystem.
Inadditiontothemeasureddata,Figure4.11showsthetielinesconnectingwiththeequilibriumdataintheliquidandgasphases.ThetielineswerecorrelatedwiththeSRKEOSandtheASmixingrule.Characteristically,thegradientoftheequilib-riumtielinesgraduallychangesfromonesidelineofthetriangletotheother.Thismeansthatwiththeincreaseofmethyloleatemassfractioninfeed,phasebehaviortendstobeclosetothatofthebinarysystemofmethyloleate+CO2.
Figure4.12showstheinfluenceoftemperatureonphaseequilibriumat20MPa.Obviously,theinfluenceoftemperatureiscontrarytothatofpressure.Withincreas-ing temperature, the two-phase area expands, as shown in Figure4.12a. In addi-tion, the phase equilibria are of ternary type I at 313.15 and 333.15 K, and thenat353.15KthephaseequilibriumdevelopsintoternarytypeII,wherethebinarycriticalpressure forCO2+methyloleate isgreater than20MPa.Noticeably, the
Methyl Oleate
Tocopherol
Tocopherol
Carbon Dioxide
Carbon Dioxide
Methyl Oleate
0.0 1.0
0.8
0.6
0.4
0.2
0.0
0.2
0.4
0.6
0.8
1.00.0 0.2 0.4
W3
W3
W2 W1
W1W2
0.6 0.8 1.0
(b)(a)
0.0
0.5
1.00.8 0.9 1.0
0.2
0.1
0.0
Figure 4.12 Influenceof temperatureon thephaseequilibriaof (w1methyloleate+w2tocopherol+ w3CO2)atP =20MPa:(a)Liquid-gasequilibria;(b)Theequilibriumcomposi-tionsingas;⦁,theexperimentaldataatT=313.15K;◾,theexperimentaldataatT=333.15K;▲,theexperimentaldataatT=353.15K;•,thecorrelatedtielinesatT=313.15K;•,thecorrelatedtielinesatT=333.15K;•,thecorrelatedtielinesatT=353.15K.(FromFang,T.,Goto,M.,Sasaki,M.andHirose,T.,J. Chem. Eng. Data,50,390,2004.Withpermission.)
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122 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
influenceof temperatureonthegascompositionseemstobe lesssignificant thanthatoftheliquidcompositionat20MPa,asshowninFigure4.12b.
4.6.3 seParation faCtor between toCoPherol anD methyl oleate
Accordingtothemeasuredgasandliquidcompositiondata,theseparationfactor(S)betweentocopherol(compound2)andmethyloleate(compound1)wascalculatedby:
S=(y2/x2)/(y1/x1) (4.2)
whereyi and xiaremassfractionsofcomponentiingasandliquid,respectively.The separation factor represents the process selectivity for separating methyl
oleatefromtocopherol.Indetail,alowervalueindicateshigherselectivity,whereasa higher value indicates that it is more difficult to separate the two compoundsunder certain conditions. Furthermore, when the separation factor equals unity,thecompositioningasissimilartothatinliquidandthesupercriticalCO2processcannotseparatemethyloleatefromtocopherol.
Figure4.13 and Figure4.14 show the influences of pressure and temperatureon the separation factor, respectively. In Figure4.13, both the experimental dataandcorrelatedcurveillustratethat,ataconstanttemperature,theseparationfactorincreasesaspressureincreases,exceptforonepartat10MPa.However,asshowninFigure4.14,theinfluenceoftemperatureiscontrarytothatofpressure.Asmentionedabove, a lower separation factor indicates a higher selectivity; accordingly, thetendenciesshowninFigure4.13andFigure4.14indicatethatlowpressureandhightemperature lead to high selectivity, which is advantageous for separating methyloleate from tocopherol with supercritical CO2. In addition, at constant pressure
0.5
0.4
0.3
0.2
S
0.1
0.00.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8
x20
Figure 4.13 Influenceofpressureontheseparationfactor(S)atT=313.15K:x20, the
initialtocopherolmassfractioninfeed;▲,experimentaldataatP=29MPa;◾,experimentaldataatP=20MPa;⦁,experimentaldataatP=10MPa;····,correlatedatP=29MPa;---,correlatedatP=20MPa;—,correlatedatP=10MPa.(FromFang,T.,Goto,M.,Sasaki,M.andHirose,T.,J. Chem. Eng. Data,50,390,2004.Withpermission.)
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Extraction and Purification of Natural Tocopherols by Supercritical CO2 123
and temperature, the separation factor increases as the initial tocopherol content(x2
0) decreases. Moreover, this tendency is more obvious as pressure increases. Alowercontentoftocopherolmeansahighercontentofmethyloleatesincethefeedmainlyconsistedofmethyloleateandtocopherol,amongwhichtheformerismoresolubleinsupercriticalCO2.Consequently,moremethyloleateinthefeedgeneratesmoretocopherolfordistributioninsupercriticalCO2,resultinginanincreaseintheseparationfactor.Inotherwords,methyloleateactsasthecosolventfortocopherol.AsimilarphenomenonwasalsoreportedbyBambergeretal.[42],whomeasuredthesolubilitiesoffattyacids,puretriglycerides,andtriglyceridemixturesinsupercriticalCO2. They found that the solubilities of the less soluble triglycerides in mixtureslike tripalmitinwere enhancedby thepresenceofmore soluble triglycerides, liketrilaurin. In this situation, the more soluble compounds were said to be acting asthecosolvents.
4.6.4 eqUilibriUm lines
Usingtheobtainedequilibriumdata,equilibriumlinesweredrawninthepressureandtemperaturerangesinvestigated.Figure4.15andFigure4.16illustratetheequi-libriumlinesat313Kand20MPa,respectively.Thedatawererepresentedasthemassfractionofmethyloleate(CO2freebasis).
AsshowninFigure4.15andFigure4.16,eitherapressureincreaseoratempera-turedecreasemovestheequilibriumlineclosertothediagonalline.Infractionationdesigning, this tendencymeans thatan increase in theoretical stages isaccompa-niedbyadecreaseinprocessselectivity.Suchatendencyagreeswellwiththatofillustratedbytheseparationfactor.
0.4
0.3
0.2
S
0.1
0.1 0.2 0.3 0.4 0.5 0.6 0.7x2
0
Figure 4.14 Influenceoftemperatureontheseparationfactor(S)atP=20MPa:x20,the
initialtocopherolmassfractioninfeed;⦁,experimentaldataatT=313.15K;◾,experimentaldataatT=333.15K;▲,experimentaldataatT=353.15K;—,correlatedatT=313.15K;---,correlatedatT=333.15K; ····,correlatedatT=353.15K. (FromFang,T.,Goto,M.,Sasaki,M.andHirose,T.,J. Chem. Eng. Data,50,390,2004.Withpermission.)
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124 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
4.6.5 Phase behaVior of me-DoD
Duringthemeasurementofphaseequilibrium,therealisticsystemofME-DODwastakenasthe10.19%(tocopherolmassfraction)feedcomposition.Also,theME-DODsystemwastherawmaterialsforconcentratingnaturaltocopherols;thus,itsphasebehavior is discussed separately. Figure4.17 shows the influence of pressure andtemperatureontheseparationfactor.
1.0
0.8
0.6
0.4
0.2
0.00.0 0.2 0.4
x1
y 1
0.6 0.8 1.0
Figure 4.16 EquilibriumlinesatP=20MPa:x1’,massfractionofmethyloleateinliquid
(CO2freebasis);y1’,massfractionofmethyloleateingas(CO2freebasis);⦁,experimental
dataatT=353.15K;◾,experimentaldataatT=333.15K;▲,experimentaldataT=313.15K;—,correlatedatT=353.15K;---,correlatedatT=333.15K;····,correlatedatT=313.15K.(FromFang,T.,Goto,M.,Sasaki,M.andHirose,T.,J. Chem. Eng. Data,50,390,2004.Withpermission.)
1.0
0.8
0.6
0.4
0.2
0.00.0 0.2 0.4
x1
y 1
0.6 0.8 1.0
Figure 4.15 EquilibriumlinesatT=313.15K:x1’,massfractionofmethyloleateinliquid
(CO2freebasis);y1’,massfractionofmethyloleateingas(CO2freebasis);⦁,experimental
dataatP =10MPa;◾,experimentaldataatP =20MPa;▲,experimentaldataP =29MPa;—,correlatedatP =10MPa;---,correlatedatP =20MPa;····,correlatedatP =29MPa.(FromFang,T.,Goto,M.,Sasaki,M.andHirose,T.,J. Chem. Eng. Data,50,390,2004.Withpermission.)
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Extraction and Purification of Natural Tocopherols by Supercritical CO2 125
The trends illustrated in Figure4.17 are similar to those in Figure4.13 andFigure4.14.Noticeably,theseparationfactorsatpressureslowerthan20MPaarerelativelysmall.Forinstance,at313.15K,theseparationfactorremainedlowerthan0.2forallpressureslowerthan15MPa.Aspressureincreases,theseparationfactorgreatlyincreases,reaching0.35at20MPa.Theincreaseoftemperatureoffsetstheeffectofpressuretosomeextents.Onthebasisoftheproperty,aseparationstrategyseems tobe reasonable and feasible.A fractionation column is necessary for theME-DODliquidsystem.Firstofall,lowpressure(15to20MPa)wasusedincombi-nationwithatemperaturedistributioninthecolumntoseparateFAMEslikemethyloleate.Thenthepressurewasincreasedtoseparatetocopherolfromotherimpurities.Thisprocedureneedstobeverifiedbyafractionationoperationinwhichoperationparameterscanbedeterminedandoptimized.
Duringourexperiments,somephenomenaoftherealisticsystemofME-DOD+CO2wereobservedthroughthevisualequilibriumcell.Figure4.18showstheliquid-gas interfacechanges thatoccuraspressure increases.The interface increasinglychangesfromcleartoobscure,withtheinterfacefinallydisappearingathighpres-sureabout29MPa.Athighpressure,acriticalpointprobablyexiststhatcausesthewholesystemtobecomeentirelymiscible.Inthissituation,theliquidandgascom-positionsare identicalandtheseparationfactorequalsunity.Itshouldbenoticedthat the disappearance of the interface does not happen suddenly. The change isaprogressiveprocessandanaccurateendpoint isdifficult todeterminebyvisualobservation. The critical pressure at 313.15 K is approximately estimated in thepressurerangefrom27.8to29.0MPabyvisualobservation.
Figure4.19showsthechangesinthefeedsituationwhenME-DODwaschargedbypumpintotheequilibriumcellatdifferentpressuresandataconstant5mL/min.Atlowpressure(5MPa),ME-DODcouldsmoothlyflowintotheequilibriumcell,butathighpressures,thechargedME-DODresemblesdropsorfog.Moreovertherateforflowingdownwardatahighpressurewasslowerthanthatatlowpressure.
0.4
0.3
0.2
0.1
10 15 20 25p/MPa
S
Figure 4.17 Separationfactor(S)ofME-DODinsupercriticalCO2:⦁,experimentaldataatT=313.15K;◾, experimental data atT=333.15K;▲, experimental dataT=353.15K.(From Fang,T., Goto, M., Sasaki, M. and Hirose, T., J. Chem. Eng. Data, 50, 390, 2004.Withpermission.)
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126 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
WehypothesizedthatthemainreasonforthiswasthesmallerdifferenceindensitybetweenME-DODandsupercriticalCO2athighpressure;forexample,at20MPaand313.15K,thedensityofCO2is0.840g/mLandthatofME-DODis0.865g/mL.Suchasmalldifferenceindensityislikelytocausethedroporfogphenomenon,eventhoughtheclearinterfacebetweenliquidandgasremained.Thisphenomenonshouldbeconsideredwhendesigningacontinuouslycountercurrentoperation.
4.7 separation with superCritiCal Co2 FraCtionation
Asdescribedinsection4.1,theimportantstepinconcentratingnaturaltocopherolsfrom ME-DOD is to remove the FAMEs, which contribute more than 70% ofME-DOD. FAMEs are important chemical materials in biofuel, metal-cuttingoil,andcleaningagentproduction,aswellas in thesynthesisofotherfattyacidproducts [17]. In section 4.6, the fundamental research on ternary and realisticphaseequilibriahasestablishedapreliminaryseparationstrategy,whichmustbetestedthroughasupercriticalCO2fractionationoperation.
AfractionationcolumnisnecessaryfortheME-DODliquidsystem.First,lowpressure (the initialpressure) isused in combinationwitha temperaturegradientalongthecolumntoseparatetheFAMEs.Then,thepressureisincreasedtoseparatethetocopherolsfromotherimpurities.
20 MPa 25 MPa 27 MPa 29 MPa
Figure 4.18 The interface between liquid and gas at T = 313.15 K. (From Fang, T.,GotoM.,Sasaki,M.,Hirose,T.,J. Chem. Eng. Data,50,390,2004.Withpermission.)
P = 5 MPa P = 20 MPa P = 25 MPa
Figure 4.19 FeedsituationofME-DODatdifferentpressures(feedrate=5mL/minandT =313.15K).(FromFang,T.,Goto,M.,Sasaki,M.andHirose,T.,J. Chem. Eng. Data,50,390,2004.Withpermission.)
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Extraction and Purification of Natural Tocopherols by Supercritical CO2 127
4.7.1 fraCtionation aPParatUs anD ProCeDUre
A fractionation system was rebuilt from a supercritical CO2 apparatus [43]. Theexperimentalsetupconsistedofacountercurrentcontactcolumn(2.4m×20mmi.d.,750mL)andaseparator(600mL)forthetopproduct,asshowninFigure4.20.Thecolumnwaspackedwithstainlesssteel3mmDixonPacking(NaniwaSpecialWireNettingCo.,Ltd.,Tokyo)overalengthof1.8m.Theseparationexperimentwascarriedoutinsemicontinuouscountercurrentoperationwithamaximumfeedof200g.
Before each run, about 120 g ME-DOD was charged into the column at40±1 g/h so that an abundance of raw material had accumulated at the columnbottom.Consequently, thefreshCO2fluidcouldcomeintocontactwithsufficientME-DODatthestartofthefractionationoperation.Thisensuresthateachexperi-mentbeganatasteady-statecondition,whichmeansboththefeedandtopfractionflow in a continuous situation with relatively stable flow rates. Fresh CO2 waschargedintothecolumnthroughvalve14(V14), thepressuresofthecolumnandseparatorwereadjustedbyBPR1andBPR2,respectively.Thetemperaturegradientofthecolumnwasconcurrentlyadjustedbyeightproportionalintegraldifferentialcontrollers.Theseparatorconditionsweremaintainedat3.8to4.0MPaand333K.Whenpressureandtemperaturereachedtherequiredvalues,CO2wasintroducedfromthecolumnbottombyopeningV15andsimultaneouslyclosingV14,indicatingthestartingpointof thefractionationoperation.Duringcontinuousoperation, the
V1 V2
V3V4
V5
V6
BPR1V7
V8P1
V15
V10
V11
V12
V13
V14
MV1
V17
BPR2
Window
Windows
V9
Feed(Continuous)
P2
Windows
Gas Meter 1
V16 Raffinate
Dixon Packing
Extract
Extraction Column Heater
CoolerCO2CO2
Figure 4.20 Schematic diagram of fractionation apparatus. (From Fang, T., Goto, M.,Wang,X.,Ding,X.,Geng,J.,Sasaki,M.andHirose,T.,J.Supercritical Fluids, 40,50, 2007.Withpermission.)
7089_C004.indd 127 10/8/07 11:50:56 AM
128 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
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7089_C004.indd 128 10/8/07 11:50:57 AM
Extraction and Purification of Natural Tocopherols by Supercritical CO2 129
ME-DODfeedlocationwaschangedbyswitchingon/offV11,V12,andV13.Addi-tionally,toachievedifferentsolvent-to-feed(S/F)ratios,theflowrateofME-DODwasmaintainedataconstant40±1g/hwhile theflowrateofCO2wasvariedbyadjustingmicrometeringvalve1(MV1).Thetotalmaximumfeedusedforeachrunwasabout200g,includingtheinitialfeedof120gbeforefractionationoperation.Intheintervalbetweenconsecutiveruns,theresidualmaterialswereremovedoutofthecolumnbyopeningthebottomvalve(V16)andreleasingthecolumnpressure.
4.7.2 Pretreatment resUlt anD ComPosition of me-DoD
Beforethefractionationexperiment,ME-DODwaspreparedfromDODaccordingtotheprocedureshowninFigure4.2.Table4.2liststheresultofthescale-upexperi-mentwith350KgDOD.Firstofall,ourpretreatmentmethoddoesnotcauseobviousdamagetotocopherolsasthetocopherols’recoveryinME-DODwas97%andsimul-taneouslyabout74.8%ofsterolswereremoved.Noticeably,thetotalsterols’recoveryin crude sterols and ME-DOD was larger than 100% because some sterols werereleasedfromsterolestersduringpretreatment.Additionally,aftermethylesterifica-tion,mostFFAwasconvertedintoFAMEs,whiletheconversionrateofglycerideswasonly18.5%,indicatingthatmethylesterificationisnotenoughtosimplifythecomplicatedsystemofDODandmethanolysisisnecessaryforconvertingglyceridesintoFAMEs.Finally,asforthetwoproducts,ME-DODandcrudesterols,theirtotalamountswerealittlelargerthanthatofinitialfeedbecausealittlewaterwasmixedintotheME-DODafterthestepofwashing.
ThecompositionofME-DODanalyzedwithGC-MSisshowninFigure4.21andTable4.3.Atotalof18compoundswereidentified.Thecompositionwas83.16%FAMEs,3.55%squalene,11.18%tocopherols,and2.11%sterols.ThemainFAMEswere methyl palmitate (14.57%), methyl linoleate (39.23%), and methyl oleate
Retention Time (min)
Abu
ndan
ce
5.00 10.00
11.26
9.62
11.1710.40 16.21
16.93
16.06
15.87TIC : ME-DOD.D
28.54
21.17
26.21
29.4029.13
31.4730.51
27.4423.24
15.00 20.00 25.00 30.00
Figure 4.21 GC-MS TIC chromatography of ME-DOD. (From Fang, T., Goto, M.,Wang,X.,Ding,X.,Geng, J.,Sasaki,M.andHirose,T.,J.Supercritical Fluids, 40,50, 2007.Withpermission.)
7089_C004.indd 129 10/8/07 11:50:58 AM
130 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
(21.16%),andthethreecompoundsmadeup90.14%ofallFAMEs.Becausesomecompoundswithhighmolecularweight,suchasglycerides,sterolesters,pigments,andwax,couldnotbeidentifiedwiththecurrentanalyses,theareapercentagesofcompoundswerenotaccurateandcouldnotbeusedforquantification.Thus,HPLCandGC-FIDwereemployedfordeterminingthecontentsoftocopherols,sterols,andFAMEs,respectively.Table4.3alsoshowstheanalysisdataobtainedbyHPLCand
taBle 4.3Composition of Me-dod
Composition determined by gC-Ms
peak no.rt
(min)area (%) Compounds trivial name of FaMe
1 9.62 1.08 Dodecanoicacid,methylester Methyllaurate(C12:0)
2 10.40 0.72 Tetradecanoicacid,methylester Methylmyristate(C14:0)
3 11.18 0.40 Unidentified /
4 11.26 14.23 Hexadecanoicacid,methylester Methylpalmate(C16:0)
5 15.87 39.23 9,12-octadecadienoicacid,methylester Methyllinoleate(C18:2)
6 16.06 21.16 9-octadecenoicacid,methylester Methyloleate(C18:1)
7 16.21 1.67 7-octadecenoicacid,methylester Methyloleate(C18:1)
8 16.93 3.73 Octadecanoicacid,methylester Methylstearic(C18:0)
9 21.17 0.37 13-docosenoicacid,methylester Methylbrassidate(C22:1)
10 23.25 0.57 Docosanoicacid,methylester Methylbehenate(C22:0)
11 26.21 3.55 Squalene
12 27.44 2.28 δ-tocopherol
13 28.54 6.98 γ-tocopherol
14 29.13 0.74 β-tocopherol
15 29.40 1.18 α-tocopherol
16 30.51 0.69 Campesterol
17 30.82 0.33 Stigmasterol
18 31.47 1.09 β-sitosterol
tocopherols (%) determined by hplC and isomers’ percentage
Tocopherols(%) α-Tocopherol β and γ-Tocopherols δ-Tocopherol
10.19 12.05 61.57 26.38
sterols (%) determined by gC-Fid and isomers’ percentage
Sterols(%) Campesterol Stigmasterol β-Sitosterol
2.71 32.11 23.18 44.71
FaMes (%) determined by gC-Fid and Main FaMes’ percentage
FAMEs C16:0 C18:2 C18:1
71.28 17.66 45.52 28.28
Source: Fang,T.,Goto,M.,Wang,X.,Ding,X.,Geng,J.,Sasaki,M.andHirose,T.,J.Supercritical Fluids, 40,50, 2007.Withpermission.
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Extraction and Purification of Natural Tocopherols by Supercritical CO2 131
GC-FID, inwhich thecontentsof tocopherols,FAMEs,andsterolswere10.19%,71.28%, and 2.71%, respectively. Noticeably, most FFA and glycerides were con-vertedintoFAMEsandmoststerolsinDODwereremovedthroughthepretreatmentprocess.Asaresult,tocopherolswerepartlyconcentratedinME-DOD,similartotheresultsreportedbyLeeetal.[10].
Fromtheaboveanalysis,ME-DODcontainsFAMEs(about70%),whichmustberemovedinthefirststepattheinitialpressure.
4.7.3 effeCt of the initial PressUre
For the greatest degree of tocopherol enrichment inside the column, the initialpressure was investigated so that most of the FAMEs were extracted with littletocopherolcontent.Inthefirst2hours,about80gME-DODwaschargedintothecolumn and the operation was in continuous countercurrent fractionation mode,whereCO2wasthecontinuousphaseandthefeedoilwasthedispersedphase.Afterfeeding,theoperationwaschangedtobatchfractionationmode.Theextractedfrac-tionswerecollectedintheseparator.Every30minutesor1hour,thefractionswereremoved from the separator and weighed until the total yield from the separatorreachedabout70wt.%(140g)ofthetotalfeed(200g).Atthatpoint,theexperimentwasterminated.
Thephaseequilibriumdatainsection4.6.4indicatedthattheseparationfactorbetween FAMEs and tocopherols change markedly from 15 to 20 MPa. Conse-quently,pressuresof14,16,and18MPawereinvestigatedfor theseexperiments,whileotheroperationparameterswerekeptatsimilarvaluesthroughout.Thecol-umntemperaturegradientwassetinalineardistributionfrom313Katthebottomto348Katthetop,theS/Fratiowasadjustedto75(theflowratesofCO2andME-DODwere3±0.05Kg/hand40±1g/h,respectively),andthefeedlocationwasV13.
Figure4.22showsthattheextractionyieldofFAMEswasgreatlyinfluencedbytheinitialpressures,ashigherpressureledtohighersolubilityandfasterextraction.Forinstance,theextractionyieldat18MPareachedmorethan70%in2.5hourswithabout7.5KgCO2,whileat14MPa,ittookfarmoretime(10hours)andmoreCO2(30Kg)fortheextractionyieldtoreachthesamelevel.Ontheotherhand,higherpressureresultedinmoretocopherolsextractedtogetherwithFAMEs,withthehigh-esttocopherolcontentbeing3.2%at18MPa,whichwasaboutthreetimesofthatat14MPa.Thistrendagreedwiththecommonrulethatsolubilitygenerallyincreaseswithpressure.However,highpressurealsoresulted indecreaseof theselectivity,whichisadisadvantagefortheseparation[6,10].
Fordetaileddiscussion,averagetocopherols’content(ATC)ofallfractionsandthe averageoil loading (AOL)wereused for evaluating the separation efficiency,withtheformerstandingforthepurityoftheFAMEproductandthelatterrepre-sentingtheprocessvelocity.AOLisdefinedastheratiobetweenthetotaloilmasscollectedandtheCO2consumedoveragiventime.Practically,AOLcanbecalcu-latedfromtheslopesoftheyieldcurvesshowninFigure4.22,andtheresultsarelistedinTable4.4.Aspressureincreased,AOLincreasedfrom0.48g/100gCO2at14MPato1.97g/100gCO2at18MPa.Similarly,ATCalsoincreased,indicatingthatmoretocopherolsweresolvatedwiththeFAMEsinthesupercriticalCO2.This
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132 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
influencedthequalityoftheFAMEproductandledtoalossoftocopherolsenrichedinthefollowingprocess.Anotherphenomenonobservedwasthattheproportionoftocopherolisomersinthefractionswasgreatlyinfluencedbytheinitialpressure.Thetocopherolsatlowpressures(14and16MPa)weremainlyα-tocopherol,whereasthosepresentat18MPawerecomposedofthefourisomersinproportionssimilartothatoftherawmaterial.Hence,16MPawasselectedastheinitialpressureforseparatingFAMEs.
4.7.4 effeCt of the final PressUre
Whenthetotalyieldreachedabout70%,itmeantthatmostFAMEswereseparatedfromnaturaltocopherols,whichenrichedinsidethecolumn.Thenthesecondstepwascommenced,whereintocopherolswereconcentratedbyincreasingthecolumn
4 8 12 16 20 24 28 32 36
0
15
30
45
60
75
Consumption of CO2 (kg)
Extr
actio
n Yi
eld
of F
AM
Es (%
)
0
5
10
15
20
25
Toco
pher
ols’
Cont
ent (
%)
Figure 4.22 EffectoftheinitialpressureontheseparationofFAMEs.ExtrationyieldofFAMEs:⦁,18MPa;◾,16MPa;▲,14MPa;Tocopherols%:•,18MPa;◽,16MPa; ,14MPa.(Otherparameters:thecolumntemperaturegradientof313to348K,S/Fratio=75,thefeedlocationatV13). (FromFang,T.,Goto,M.,Wang,X.,Ding,X.,Geng, J.,Sasaki,M.andHirose,T.,J.Supercritical Fluids, 40,50, 2007.Withpermission.)
taBle 4.4effect of the initial pressure on the separation of FaMes
P (Mpa)
total Feed (g)
total yield (%)
aol (g/100 g Co2) atC (%)
proportion of tocopherol isomers
α β+γ δ
14 200 71.97 0.48 0.19 97.98 2.02 N.D.*
16 200 73.34 1.22 0.21 94.11 5.89 N.D.
18 200 73.82 1.97 2.11 16.61 71.56 11.83*: N.D.meansnotdetermined.Source: Fang,T.,Goto,M.,Wang,X.,Ding,X.,Geng,J.,Sasaki,M.andHirose,T.,J.Supercritical
Fluids, 40,50, 2007.Withpermission.
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Extraction and Purification of Natural Tocopherols by Supercritical CO2 133
pressuretoahighervalue(thefinalpressure).Theexperimentwasterminatedwhenthetotalyieldreachedabout85%to90%feed,becauseitwasfoundtobedifficulttoobtainmorethan90%yieldoffeedintheexperimentalrangeandtheextractionvelocitywaspracticallyveryslow(<5g/30min).Thetotalandtocopherolyieldswerecalculatedfromthemassflowandtocopherolcontentinthefractions.Wemainlyinvestigatedthe influenceof thefinalpressureonATCandtocopherols’recovery(TR).Figure4.23showsthefractionchangeasafunctionoftimeatdifferentfinalpressures,andFigure4.24illustratestheeffectofpressuremodesontotalyieldandTR.Inthefirst4hours,therewasnodistinctchangeinthefirststepofpressureto16MPa.Themassflowwasabout15to20g/30min(Figure4.23)and,after4hours,theyieldreachedabout70wt.%ofthefeed(Figure4.24).Evidently,themassflowdecreasedtolessthan10g/30min.Inaddition,tocopherolyieldat16MPawasonly3%to5%,whichisthatfavoredconcentrationoftocopherolsinthesubsequentstepathigherpressure. In thesecondstep,athigherpressure, themassflowandtotalyieldincreasedwithanincreaseinthefinalpressure,butthechangeintocopherolcontentat22MPawasnotasprecipitousasthatat20or18MPa(Figure4.23).Thisindicatedthat,whileahigherfinalpressureleadstoanincreaseinthetotalsolubil-ity,itsimultaneouslyresultsinadecreaseinselectivity.TheATCofallfractionsat22MPawasonly44.1%,althoughthecorrespondingTRwas82.8%.Both18MPaand20MParesulted inATCsgreater than50%,but theTRat18MPawasonly46.8%andlowerthan81.3%at20MPa(Figure4.24).Moreover,18MParesultedinalongerfractionationprocessbecauseoflowsolubility;forinstance,theextractedfractionwasalwayslessthan5g/30minafter7hours,andtheexperimentat18MPawasterminatedat9hoursbecauseoftooslowextraction.Therefore,20MPawas
1 2 3 4 5 6 7 8 90
10
20
30
40
50
Time (hr)
Mas
s Flo
w (g
)
0
20
40
60
80
Toco
pher
ols (
%)
Figure 4.23 Effect of the final pressure on mass flow and tocopherols’ content. Massflow:▲,18MPa;⦁,20MPa;◾,22MPa;Tocopherols%: ,18MPa;•,20MPa;◽,22MPa.(Other parameters: the initial pressure = 16 MPa for 4 hours,S/F ratio = 75, the columntemperaturegradientof313to348K,thefeedlocationatV12).(FromFang,T.,Goto,M.,Wang,X.,Ding,X.,Geng,J.,Sasaki,M.andHirose,T.,J.Supercritical Fluids, 40,50, 2007.Withpermission.)
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134 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
selectedastheoptimalfinalpressureespecially,at5.5hours,afractionwithpurityof80.5%wasobtained,whichwasthehighestcontentobtainedinourstudy.
4.7.5 ComPosition of toCoPherol ConCentrate
Accordingtothefractionationoperation,itprovedtobefeasibleforconcentratingnaturaltocopherolsfromME-DODwithsupercriticalCO2fractionation.Withtheoptimizedpressureparameters,about29.1ghaving57.1%tocopherolconcentrate(allfractionsat20MPa)wereobtainedfrom200gME-DOD.GC-MSwasusedtoanalyzethecompositionintheconcentrate.TheresultisshowninFigure4.25.
The area percentage of tocopherols was 68.8%, higher than the 57.1% deter-minedbyHPLC.Themainreasonwasthatthereweresomeimpuritieswithhighmolecularweight that couldnotbe identifiedwith the currentGC-MScondition.Additionally, among the determinable compounds, FAMEs (C16:0, C18:2, C18:1,C18:0),squalene,andsterolswerethemainimpurities,withtheareapercentagesof15.4%,5.8%,and10%,respectively.
4.7.6 VisCosity ComParison
Inthiswork,DODandME-DODwereusedasrawmaterialandobtaineddiffer-entfractionsbysupercriticalCO2fractionation.Besidesthecompositiondifference,thesematerials aredifferent inphysicalproperties. Inparticular, theirviscositiesattractedourinterestsinceviscosityisaveryimportantphysicalpropertycommonlyused in engineering design.For example,when designing the feeding system forME-DOD,itsviscositydatahelpdeterminewhetherpreheatingisnecessary.
1 2 3 4 5 6 7 8 9
0
20
40
60
80
Time (hr)
Tota
l Yie
ld (%
)
0
20
40
60
80
100
Toco
pher
ols’
Reco
very
%
Figure 4.24 Effectof thefinalpressureon totalyieldandtocopherols’recovery.Totalyield:▲, 18MPa;⦁, 20MPa;◾, 22MPa;Tocopherols’ recovery: , 18MPa;•, 20MPa;◽,22MPa.(Otherparameters:theinitialpressure=16MPafor4hours,S/Fratio=75,thecolumn temperature gradient of 313 to 348K, the feed location atV12). (FromFang,T.,Goto,M.,Wang,X.,Ding,X.,Geng,J.,Sasaki,M.andHirose,T.,J.Supercritical Fluids, 40,50, 2007.Withpermission.)
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Extraction and Purification of Natural Tocopherols by Supercritical CO2 135
Figure4.26 illustrates theviscositychangesofDOD,ME-DOD,andwaterasa functionof temperature.When the temperaturewasvaried from293 to303K,theviscositiesof the three samplesdecreasedquicklyand then thedecrease ten-dencybecamerelativelystableandslowathighertemperatures.Suchcharacteris-ticsindicatedthatthethreesamplesweretypicallypseudoplasticfluids.Inaddition,theviscosityofME-DOD is far smaller than thatofDODand similar to thatofwater.Thus,thepretreatmentprocessshowninFigure4.2leadstotwoadvantageousresults for continuous fractionation process. One is that the converted ME-DODhaslargersolubilityinsupercriticalCO2thanDOD;theotheristhattheviscosity
Retention Time (min)
TIC: TOCOD.D
11.24
15.8316.03
16.93 21.1723.24
23.47
26.21
27.46
28.60
29.4131.49
33.1630.53
30.8529.14
28.4028.20
5.00 10.00 15.00 20.00 25.00 30.00
Abu
ndan
ce
Figure 4.25 GC-MS TIC chromatography of 57.1% tocopherols. (From Fang, T.,Goto,M.,Wang,X.,Ding,X.,Geng,J.,Sasaki,M.andHirose,T.,J.Supercritical Fluids, 40,50, 2007.Withpermission.)
290 295 300 305 310 315 320 325 330 3350.000
0.004
0.008
0.012
0.1
0.2
0.3
0.4
0.5
Temperature (K)
Visc
osity
(Pa.s
)
Figure 4.26 ComparisonbetweentheviscositiesofDOD,ME-DOD,andH2O.◾,DOD;▲,ME-DOD;•,H2O.(FromFang,T.,Goto,M.,Wang,X.,Ding,X.,Geng,J.,Sasaki,M.andHirose,T.,J.Supercritical Fluids, 40,50, 2007.Withpermission.)
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136 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
isgreatlyreducedafterconvertingmoreviscouscompounds(FFAandglycerides)intolessviscousFAMEs.Particularlyafterpretreatment,theviscosityofME-DODisfurtherdecreasedbyremovingmostofthesterols,whichpracticallyactasakindofthickeningmaterial.
Figure4.27 shows a comparison of viscosities among the fractions obtained.Theviscosityorderwasasraffinate>57.1%tocopherols>FAMEs.Here,raffinatewastheresidualmaterialatthebottomofthecolumnafterthefractionationopera-tionanditappearedasaverystickyliquid.
4.7.7 aPPliCation in CommerCial ProDUCtion
Onthebasisofthewholeresearch,anindustrialapplicationwascarriedoutinpastyears.Scale-upexperimentswithan18Lcolumnweredoneandtheresultswerereportedinliterature[44].Inaddition,aworkshop(Figure4.28)andacommercial-scale fractionation system of 350 L × 2 (Figure4.29) were established in KaidiFineChemical IndustrialCo.Ltd. (Wuhan,HubeiProvince,P.R.China)and thetechnologywasindustrializedwithanannualprocesscapacityof750tME-DODintheyearof2000.Accordingtoourwork,threepatentswereappliedandfinallyreleasedforpublication[45–47].
4.8 ConClusions
As the first step of the whole research, binary phase equilibrium data of methyloleate + CO2 and α-tocopherol + CO2 were measured and correlated with theSoave-Redlich-Kwong EOS and Adachi-Sugie mixing rule. According to theobtaineddata,thesolubilityanddistributioncoefficientwerecalculated.Compared
290 295 300 305 310 315 320 325 330 3350.000
0.004
0.5
1.0
1.5
Temperature (K)
Visc
osity
(Pa.s
)
Figure 4.27 Comparisonbetweentheviscositiesofthefractionsobtainedatdifferentpressures.◾,Raffinateat20MPa;▲,57.1%tocopherols(fractionat20MPa);•,FAMEs(fraction16MPa). (FromFang,T.,Goto,M.,Wang,X.,Ding,X.,Geng,J.,Sasaki,M.andHirose,T.,J.Supercritical Fluids, 40,50, 2007.Withpermission.)
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Extraction and Purification of Natural Tocopherols by Supercritical CO2 137
Figure 4.28 Tocopherol concentration workshop established in Wuhan (Kaidi FineChemicalIndustrialCo.,Ltd.,Hubei,P.R.China).
Figure 4.29 Supercritical CO2 fractionation system (350 L × 2) for concentratingnaturaltocopherols.
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138 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
withmethyloleate,thesolubilityanddistributioncoefficientofα-tocopherolweregenerallyonetotwoordersofmagnitudelower.SuchalargedifferencebetweenthetwocomponentsindicatesthatitispossibletoconcentratenaturaltocopherolsfromME-DODintheexperimentalrangeinvestigated.
Second,ternaryphaseequilibriumdataofmethyloleate+tocopherol+CO2weremeasuredandcorrelated.Onthebasisoftheexperimentaldataandcorrelationresults,the separation factor and equilibrium line were investigated. The discussion indi-catedthatlowerpressuresandhighertemperaturesleadtoahigherselectivity.Also,highercontentofmethyloleateinthefeedimprovesthedistributionoftocopherolsingas,resultingindecreasedselectivity.Morenoticeably,theexperimentaldataontherealisticsystemofME-DOD+CO2ledtotheformationofaseparationstrategy.
Finally,supercriticalCO2fractionationwasemployedtoconcentratetocopherolsfromME-DOD.TheinitialpressurewasinvestigatedforseparatingFAMEs.Forthefollowingtocopherolconcentrationstep,afinalpressureof20MParesultedinrela-tivelyhighaveragetocopherolcontent(>50%)andtocopherolrecovery(about80%).On the basis of the fundamental and separation research, an application in com-mercialproductionwasalsoconductedinthepastyears.Accordingtotheobtainedresults,itcanbeconcludedthatsupercriticalCO2fractionationistechnicallyfeasibleforconcentratingnaturaltocopherolsfrommethylesterifiedDOD.
reFerenCes
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3. Martins,P.F.,Batistella,C.B.,MacielFilho,R. andWolf-Macie,M.R.,Comparisonof two different strategies for tocopherols enrichment using amolecular distillationprocess,Ind. & Eng. Chem. Res.,45,753,2006.
4. Sumner,C.E.,Jr.,Barnicki,S.D.andDolfi,M.D.,Processfortheproductionofsterolandtocopherolconcentrates,U.S.PatentUS5424457,A199506131995,1995.
5. Mau,J.andTsen,H.,Investigationontheconditionsforthepreparationofhigh-purityvitaminEconcentratefromsoybeanoildeodorizerdistillate,J. Chin. Agri. Chem. Soc. (Taipei),33,686,1995.
6. Lucas,A.,Martinez,E.O.,RincónJ.,Blanco,M.A.andGracia,I.,Supercriticalfluidextractionoftocopherolconcentratesfromolivetreeleaves,J. Supercrit. Fluids,22,221,2002.
7. Zhao,Y.,Sheng,G.andWang,D.,Pilot-scaleisolationoftocopherolsandphytosterolsfromsoybeansludgeinapackedcolumnusingsupercriticalcarbondioxide,inProc. 5th Int. Symp. on Supercritical Fluids,Atlanta,Georgia,USA,April8–12,2000.
8. Zhou,Q.,Sheng,G., Jiang,H. andWu,M.,Concentrationof tocopherolsby super-criticalcarbondioxidewithcosolvents,Eur. Food Res. Tech.,219,398,2004.
9. Nagesha, G.K., Manohar, B. and Udaya Sankar, K., Enrichment of tocopherols inmodifiedsoydeodorizerdistillateusingsupercriticalcarbondioxideextraction,Eur. Food Res. Tech.,217,427,2003.
10. Lee,H.,Chung,B.H.andPark,Y.H.,Concentrationoftocopherolsfromsoybeansludgebysupercriticalcarbondioxide,J. Am. Oil Chem. Soc.,68,571,1991.
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Extraction and Purification of Natural Tocopherols by Supercritical CO2 139
11. Shishikura, A., Fujimoto, K., Kaneda, T., Arai, K. and Saito, S., Concentration oftocopherolsfromsoybeansludgebysupercriticalfluidextraction, J. Jpn. Oil Chem. Soc.,37,8,1988.
12. Liu,Y.,Fang,T.andDing,X.,PhaseequilibriumforsupercriticalCO2andthemethylesterified product form soybean oil deodorizer distillate, J. Food Lipids, 13, 390,2006.
13. Mendes,M.F.,Pessoa,F.L.P.andUller,A.M.C.,AneconomicevaluationbasedonanexperimentalstudyofthevitaminEconcentrationpresentindeodorizerdistillateofsoybeanoilusingsupercriticalCO2,J. Supercrit. Fluids,23,257,2002.
14. King,J.W.,Favati,F.andTaylor,S.L.,Productionoftocopherolconcentratesbysuper-criticalfluidextractionandchromatography, Sep. Sci. Tech.,31,1843,1996.
15. Chang,C.J.,Chang,Y.F.,Lee,H.,Lin,J.andYang,P.W.,Supercriticalcarbondioxideextraction of high-value substances from soybean oil deodorizer distillate, in Proc. 5th Int. Symp. on Supercritical Fluids,Atlanta,Georgia,USA,April8–12,2000.
16. Brunner, G., Malchow, T., Stürken, K. and Gottschau, T., Separation of tocopher-ols from deodorizer condensates by countercurrent extraction with carbon dioxide,J. Supercrit. Fluids,4,72,1991.
17. Swern,S.,Bailey’s Industrial Oils and Fats, JohnWiley&Sons,NewYork,1986. 18. AmericanOilChemists’Society,OfficialMethodsandRecommendedPracticesofthe
AmericanOilChemists’Society,17thed.,2000,A.O.C.S.,Washington,DC. 19. Fang,T.,Goto,M.,Yun,Z.,Ding,X.andHirose,T.,Phaseequilibriaforbinarysystems
ofmethyloleate-supercriticalCO2 andα-tocopherol-supercriticalCO2,J. Supercrit. Fluids,30,1,2004.
20. Fang,T.,Goto,M.,Sasaki,M.andHirose,T.,Phaseequilibriafortheternarysystemmethyloleate+tocopherol+supercriticalCO2,J. Chem. Eng. Data,50,390,2004.
21. Fang,T.,Goto,M.,Wang,X.,Ding,X.,Geng,J.,Sasaki,M.andHirose,T.,Separationofnaturaltocopherolsfromsoybeanoilbyproductwithsupercriticalcarbondioxide,J. Supercrit. Fluids, 40,50, 2007.
22. Soave,G.,Equilibriumconstants fromamodifiedRedlich-Kwongequationofstate,Chem. Eng. Sci.,27,1197,1972.
23. Adachi,Y.andSugie,H.,Anewmixingrule-modifiedconventionalmixingrule,Fluid Phase Equilibrium,28,103,1986.
24. Weber,W.,Petkov,S.andBrunner,G.,Vapour-liquidequilibriaandcalculationsusingtheRedlich–Kwong-Aspenequationofstatefortristearin,tripalmitin,andtrioleininCO2andpropane, Fluid Phase Equilibrium, 158–160,695,1999.
25. Cheng,H.,Zollweg,J.A.andStreett,W.,Experimentalmeasurementofsupercriticalfluid–liquidphaseequilibrium,InSupercritical Fluid Science and Technology, ACS Symposium Series 406,Johnston,K.P.andPenninger,J.M.L.,Eds.,AmericanChemicalSociety,Washington,DC,1989,86.
26. Inomata,H.,Kondo,T.,Hirohama,S.,Arai,K.,Suzuki,Y.andKonno,M.,Vapour-liquidequilibria forbinarymixturesofcarbondioxideandfattyacidmethylesters,Fluid Phase Equilibrium,46,41,1989.
27. Zou,M.,Yu,Z.R.,Kashulines,P.,Rizvi,S.S.H.andZollweg,L.A.,Fluid-liquidphaseequilibriaof fattyacidsandfattyacidmethylesters insupercriticalcarbondioxide, J. Supercrit. Fluids,3,23,1990.
28. Nilsson, W.B., Seaborn, G.T. and Hudson, J.K., Partition coefficients for fatty acidestersinsupercriticalfluidCO2withandwithoutethanol,J. Am. Oil Chem. Soc.,69,305,1992.
29. Yu,Z-R.,Rizvi,S.S.H.andZollweg,J.A.,Phaseequilibriaofoleicacid,methyloleate,andanhydrousmilk fat in supercritical carbondioxide,J. Supercrit. Fluids, 5, 114,1992.
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140 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
30. Crampon,C.,Charbit,G.andNeau,E.,High-pressureapparatusforphaseequilibriastudies:SolubilitiesoffattyacidestersinsupercriticalCO2, J. Supercrit. Fluids,16,11,1999.
31. Chrastil,J.,Solubilityofsolidsandliquidsinsupercriticalgases,J. Phys. Chem.,86,3016,1982.
32. Oghaki,K.,Tsukahara,I.,Semba,K.andKatayama,T.,Afundamentalstudyoftheextraction with a supercritical fluid. Solubilities ofα-tocopherol, palmitic acid, andtripalmitin incompressedcarbondioxideat25°Cand40°C,Int. J. Chem. Eng.,29,303,1989.
33. Pereira,P.J.,Goncalves,M.,Coto,B.,deAzevedo,E.G.anddaPonte,M.N.,PhaseequilibriaofCO2+DL-α-tocopherolattemperaturesfrom292to333Kandpressuresupto26MPa,Fluid Phase Equilibrium,91,133,1993.
34. Meier,U.,Gross,F. andTrepp,C.,Highpressurephase equilibrium studies for thecarbondioxide/α-tocopherol (vitaminE) system,Fluid Phase Equilibrium,92,289,1994.
35. Johannsen,M.andBrunner,G.,Solubilitiesoffat-solublevitaminsA,D,EandKinsupercriticalcarbondioxide,J. Chem. Eng. Data,42,106,1997.
36. Chen,C.C.,Chang,C.M.J.andYang,P.W.,Vapor–liquidequilibriaofcarbondioxidewith linoleic acid, α-tocopherol, and triolein at elevated pressures, Fluid Phase Equilibrium,175,107,2000.
37. Skerget, M., Kotnik, P. and Knez, Z., Phase equilibria in systems containingα-tocopherolanddensegas,J. Supercrit. Fluids,26,181,2003.
38. Brunner,G.,Malchow,T.,Stuerken,K.andGottschau,T.,Separationoftocopherolsfrom deodorizer condensates by countercurrent extraction with carbon dioxide,J. Supercrit. Fluids,4,72,1991.
39. Stoldt, J.,Saure,C.andBrunner,G.,Phaseequilibriaof fatcompoundswithsuper-criticalcarbondioxide,Fluid Phase Equilibrium,116,399,1996.
40. Stoldt,J.andBrunner,G.,Phaseequilibriummeasurementsincomplexsystemsoffats,fatcompounds,andsupercriticalcarbondioxide,Fluid Phase Equilibrium,146,269,1998.
41. Araujo, M. E., Machado, N. T. and Meireles, M.A., Modeling the phase equilib-rium of soybean oil deodorizer distillates + supercritical carbon dioxide using thePeng-RobinsonEOS,Ind. Eng. Chem. Res.,40,1239,2001.
42. Bamberger,T.,Erickson,J.C.,Cooney,C.L.andKumar,S.K.,Measurementandmodelpredictionofsolubilitiesofpurefattyacids,puretriglycerides,andmixturesoftriglycer-idesinsupercriticalcarbondioxide.J. Chem. Eng. Data,33,327,1988.
43. Sato,M.,Kondo,M.,Goto,M.,Kodama, A. andHirose,T.,Fractionationof citrusoilbysupercriticalcountercurrentextractorwithside-streamwithdrawal, J. Supercrit. Fluids,13,311,1998.
44. Fang,T.,Goto,M.,Liu,Q.,Ding,X.andHirose,T.,Countercurrentextractionforthefractionation of natural tocopherols with supercritical CO2, in Proc. of the 6th Int. Symp. on Supercritical Fluids,Tome1,425,Versailles,France,April28–30,2003.
45. Wang,X.,Geng,J.,Liu,C.,Ding,X.andFang,T.,ProcessforfractionallyextractingnaturalvitaminEbysupercriticalCO2fluid,ChinaPatentCN1369487A,2002.
46. Wang,X.,Fang,T.,He,F.,Liu,C.,Liu,S.,Cao,Y.andWu,X.,ProcessforextractingvitaminEfromplant-oildeodorizerdistillates,ChinaPatentCN1418877A,2003.
47. Wang,X.,Geng,J.,Liu,C.,Ding,X.,andFang,T.,Anovelcolumnusedforthecon-tinuously countercurrent operation of supercritical CO2 fractionation, China PatentCN2561484Y,2003.
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141
5 Processing of Fish Oils by Supercritical Fluids
Wayne Eltringham and Owen Catchpole
Contents
5.1 Introduction................................................................................................. 1415.2 FishOilComponents:Sources,Properties,andCommercialUses............ 142
5.2.1 Omega-3FattyAcids:EicosapentaenoicAcidandDocosahexaenoicAcid..................................................................... 142
5.2.2 SqualeneandDiacylGlycerylEthers.............................................. 1445.2.3 VitaminA(Retinol)......................................................................... 1465.2.4 WaxEsters....................................................................................... 146
5.3 Separation/FractionationTechnologies....................................................... 1475.3.1 TraditionalProcessingMethods...................................................... 148
5.3.1.1 Distillation.......................................................................... 1495.3.1.2 Low-TemperatureCrystallization....................................... 1515.3.1.3 UreaCrystallization........................................................... 1525.3.1.4 ChromatographicMethods................................................. 1565.3.1.5 EnzymaticTransformation................................................. 156
5.3.2 SupercriticalFluidProcessingofFishOils..................................... 1585.3.2.1 PhaseEquilibria:SupercriticalCO2andFishOil
Components........................................................................ 1585.3.2.2 PolyunsaturatedFattyAcidProcessing.............................. 1685.3.2.3 SqualeneandDAGEProcessing........................................ 1765.3.2.4 VitaminAProcessing........................................................ 1785.3.2.5 ProcessingofOtherMarineOilComponents.................... 181
5.4 Summary..................................................................................................... 181References.............................................................................................................. 181
5.1 IntroduCtIon
Extractionandfractionationoffishoilshasbecomeamajorareaofresearchoverthelast30yearsbecauseofthepotentialapplicationoftheseextractsandfractionsin thepharmaceutical,nutraceutical,andcosmetic industries.Themajorconstitu-entsoffishoilsaretriacylglycerides(TAGs).Minorcomponentsincludefreefattyacids, squalene, tocopherols, cholesterol, wax esters, sterol esters, phospholipids,diacylglycerides,diacylglycerolethers,pigments,andvitaminsA,D,andE.Oilsofmarineoriginaresignificantlymorecomplexthanthosefromplantsandterrestrialanimals.ThefattyacidsthatconstituteTAGsinfishoilsvaryconsiderablyaccording
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142 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
to degree of unsaturation, variety of chain length, and number of isomeric com-pounds.TAGsaretypicallymadeupofstraight-chainfattyacidscontainingfrom12to24ormorecarbons,withthedegreeofunsaturationvaryingfromzerotosixdoublebonds.Fishoilsoftencontainmorethan60differentfattyacids,includingiso-mers,whichdifferaccordingtothepositionofunsaturationwithinthecarbonchain.Thecomplexcompositionoffishoilsmakesthemdifficulttoprocesstoconcentratespecificfattyacids.Highlevelsofunsaturationmaketheuseofhigh-temperatureprocessingmethodsproblematicbecauseofthesusceptibilityofthesecompoundstooxidativeandthermaldegradation.Thevariability infattyacidcompositionoffishoilishighlydependentonfishspecies,season,feedinghabits,partofthefishused(e.g.,liverorflesh),andcatchlocation.SomefattyacidprofilesofselectedfishoilsareshowninTable5.1[1],whichshowsthatliveroilcompositionisusuallylesssaturatedthanthatoftheflesh.
This chapter discusses several important fish oil constituents, includingtheir physical properties and applications in thenutritional, pharmaceutical, andcosmeticindustries.Moreover,thechapterisintendedtogiveanoverviewofthevariouslaboratory-to-productionscaletechniquesthatcanbeusedtoextractandfractionatevariousfishoil components.Acomparisonof various extraction andfractionationtechniquesisdiscussed,includinghowsomeoftheproblemsassoci-ated with “traditional” processing methods can be overcome using supercriticalfluid(SCF)technologies.
5.2 FIsh oIl Components: sourCes, propertIes, and CommerCIal uses
5.2.1 Omega-3 Fatty acids: eicOsapentaenOic acid and dOcOsahexaenOic acid
Productionofomega-3(ω3)fattyacidconcentratescontinuestobeatopicofinter-estforboththepharmaceuticalandhealthfoodindustries.Sincetheearlystudieson long-chainω3-polyunsaturated fatty acids (PUFAs) by Burr and Burr [2], thehealthbenefitsofthesecompoundshavebeenstudiedextensively.Withthegrowingpublicawarenessofthehealthbenefitsofω3-PUFAs,themarketforsuchproductsis expected to grow, increasing the demand for efficient production and isolationmethods.Themostwidelystudiedω3fattyacidsareeicosapentaenoicacid(EPA)anddocosahexaenoicacid(DHA)(Figure5.1).Extensiveclinicalfindingsontheireffectsonhumanphysiologyandtheiruseinthepreventionandtreatmentofdiseasessuchasarteriosclerosis[3,4],thrombosis[5],arthritis[6],andseveraltypesofcancer[7,8]havebeenreported.
Omega-3fattyacidsofmarineorigincanreduceserumTAGlevels[9].ThisisofparticularimportancebecauseraisedlevelsofserumTAGsareconsideredtobeariskfactorforcoronaryheartdisease[10].Urakazeetal.[5]showedthatintravenousinjectionsofanEPA-containingemulsioncanincreasetheEPAcontentinplasmaandplateletphospholipids.TheirstudyshowedthatplateletaggregationissignificantlydepressedandthatEPA-containingemulsionsmaybeusefulforpatientsrequiringpreventativecareofthrombosis.Othermedicalstudiesreportusingω3fattyacids
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Processing of Fish Oils by Supercritical Fluids 143
for the reductionofbloodpressure [11,12].AstudybyKremerandcoworkers [6]reported theeffectofEPA intake inpatients suffering from rheumatoidarthritis.Aftera12-weekdiethigh inpolyunsaturatedfat, lowinsaturatedfat,andwithadailysupplementofEPA(1.8g),patientsnotedadecreaseinmorningstiffnessandjointpain.Otherworkershavealsoreportedanti-inflammatoryactivityofω3fattyacids [13].Researchers at thePaterson Institute (Manchester,UK)have identifiedamechanismbywhichω3fattyacidsmayprevent thedevelopmentofmetastatic
table 5.1Fatty acid profiles of selected Fish oils
salt Water oils
Freshwater oils
atl
anti
c C
od
atl
anti
c C
od l
iver
spin
y d
ogfi
sh
spin
y d
ogfi
sh l
iver
paci
fic
hal
ibut
paci
fic
her
ring
mac
kere
l
men
hade
n
stri
ped
mul
let
pink
sal
mon
lake
her
ring
rai
nbow
tro
ut
Fatty acid Weight (%) of total Fatty acids
14:0 1.8 2.8 2.0 1.6 2.8 7.6 4.9 8.0 4.6 3.4 5.5 2.1
15:0 0.5 0.4 0.5 0.3 0.3 0.4 0.5 0.5 6.3 1.0 0.4 0.8
16:0 33.4 10.7 21.2 13.2 15.1 18.3 28.2 28.9 17.3 10.2 17.7 11.9
16:1 2.4 6.9 6.0 5.7 8.9 8.3 5.3 7.9 11.0 5.0 7.1 8.2
16:2 0.6 1.0 0.9 1.0 0.8 1.0 0.7 0.8 3.8 1.7 0.7 1.2
17:0 0.9 1.2 1.2 1.0 0.7 0.5 1.0 1.0 0.8 1.6 0.6 1.5
18:0 4.0 3.7 2.7 4.3 3.4 2.2 3.9 4.0 5.0 4.4 3.0 4.1
18:1 11.9 23.9 27.5 28.5 25.7 16.9 19.3 13.4 8.4 17.6 18.1 19.8
18:2ω6 1.2 1.5 1.3 0.7 0.9 1.6 1.1 1.1 3.2 1.6 4.3 4.6
18:3 0.8 0.9 0.6 0.6 0.3 0.6 1.3 0.9 .4 1.1 3.4 5.2
18:4ω3 1.2 2.6 0.7 0.8 3.6 2.8 3.4 1.9 3.0 2.9 1.8 1.5
19:0 0.6 0.7 0.9 1.5 1.6 0.8 0.9
20:1 1.6 8.8 5.8 1.5 8.0 9.4 3.1 0.9 0.7 4.0 1.2 3.0
20:4ω6 3.2 1.0 2.5 0.8 2.5 0.4 3.9 1.2 2.6 0.7 3.4 2.2
20:5ω3(EPA) 12.4 8.0 7.9 3.7 10.1 8.6 7.1 10.2 7.5 13.5 5.9 5.0
22:1 0.7 5.3 4.1 10.3 5.1 11.6 2.8 1.7 0.7 3.5 2.8 1.3
22:6ω3(DHA) 21.9 14.3 10.4 6.5 7.9 7.6 10.8 12.8 13.4 18.9 13.3 19.0
Totalsaturated 40.6 19.4 27.6 21.1 22.3 29.0 38.5 43.3 35.5 22.2 28.0 21.3
Totalmonounsaturated 16.6 44.9 43.4 46.0 47.7 46.2 30.5 23.9 20.8 30.1 29.2 32.3
Totalpolyunsaturated 41.3 29.3 24.3 14.1 26.1 22.6 28.3 28.9 33.9 40.4 32.8 38.7
Totalω3 34.3 22.3 18.3 10.2 18.0 16.2 17.9 23.0 20.9 32.4 19.2 24.0
Source: AdaptedwithpermissionfromJournal of the American Oil Chemists’ Society,41,662.©1964AmericanOilChemists’Society.
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144 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
prostate cancer [14]. They showed that theω6 polyunsaturated fatty acid arachi-donicacid(20:4ω6)isapotentstimulatorofmalignantepithelialcellularinvasion,which can increase the risk of prostate cancer development. They stated that theobservedcellularinvasionisinhibitedbyEPAandDHAintheratiosofω31:2ω6.SeveralotherstudieshavealsofocusedontheanticarcinogenicpropertiesofEPA[15–17].Omega-3fattyacids,especiallyEPA,alsoshowpromiseinthetreatmentofneuropsychiatricdisorders,suchasdepression[18],schizophrenia[19],andanorexianervosa[20].
BothEPAandDHAoccurnaturallyinthebody,wheretheyhavebeenshowntobeimportantinmembranestructureandfunction[21].Theyarefoundinespeciallylargeamountsinbraincells,eyes,nerves,andadrenalglands.Inparticular,DHAisoneofthemostabundantconstituentsofbrainstructurallipids,whereithasimpor-tanteffectsonmembraneorder(fluidity),theactivityofmembrane-boundenzymes,andsignaltransduction.DHAisconsideredtobeessentialforthevisualandneuro-logicaldevelopmentofinfants.Thelipidfractionofhumanmother’smilkcontainsDHA-to-EPAratiosof4:1,withDHAcontentbeing30timesmorethantheamountofDHAobservedincows’milklipid.IntheU.S.,80%ofinfantformulascontainDHAsothatchildrenreceivethisnutrientduringtheimportantphasesofbrainandnervoussystemgrowthanddevelopment.
5.2.2 squalene and diacyl glyceryl ethers
Thenaturaloccurrenceofsqualenewasfirstreportedin1906byMitsumaruTsujimoto,anindustrialengineerwhopioneeredthechemistryoffatsandoilsinJapan.Thirtyyearslater,NobellaureatePaulKarrerdescribedthechemicalstructureofsqualeneforthefirsttime.Squaleneisa30-carbonisoprenoid(Figure5.2)thatisusedcom-mercially as an additive in pharmaceutical preparations, cosmetics, sunscreens,dyes,lubricants,andhealthfoods[22,23].Duringthe1950s,squalenewasfoundtooccurnaturallyinthehumanbody[24].Humansebum,anaturalproductexpressedby sebaceous glands, contains around 12% squalene. Sebum helps keep the skinsuppleandhelpstomaintainskinmoisturelevels.Squalenewaslaterfoundtobe
OH
O
OH
OEicosapentaenoic Acid (EPA), 20:5 ω-3
Docosahexaenoic Acid (DHA), 22:6 ω-3
FIgure 5.1 ThechemicalstructuresofEPAandDHA.
FIgure 5.2 Thechemicalstructureofsqualene(C30H50).
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Processing of Fish Oils by Supercritical Fluids 145
theprecursortocholesterol[25],whichinturnistheprecursortoarangeofsteroidsandvitaminD.
GloorandKarenfeld[26]foundthatthebody’sconsumptionofsqualeneincreaseswhenskinisexposedtoultraviolet(UV)radiation.Kohnoandcoworkers[27]showedthatthefirstmoleculetargetedbyUVradiationintheskinwassqualene.TheseresultsledtomoreintenseresearchontheinteractionofsqualenewithUVradiationand,in1995,Kohnoetal.[28]demonstratedthatsqualenecanpreventUV-inducedoxidationoflipidsintheskin.Radioprotectiveanddetoxifyingeffectsofsqualenehavealsobeenreported[29].Dietarysqualenemayhavethepotentialtolowerbloodcholesterollevels[30,31].Anumberofresearchpaperssuggestthatsqualeneshowspotentialasananticarcinogen[32–34].Foramorethoroughtreatiseonthepropertiesofsqualene,itsactioninmetabolicpathways,anditspossiblepreventativecapabilitiesforhumandisease,thereaderisdirectedtoaworkbyDas[35].
Thereportsonthebeneficialeffectsofsqualeneonhumanhealthhaveledtoacommercialdemandforthislong-chainunsaturatedhydrocarbon.Althoughsqualeneisnaturallyfoundinsmallquantitiesinoliveoilandby-productsfromtherefiningofoliveoil,wheatgermoil,ricebranoil,andyeast,themostabundantsourcebyfaristheliversofdeep-seasharks.Othermajorcomponentsfoundintheliversofdeep-seasharksincludediacylglycerylethers(DAGEs)andTAGs.Becausesharksdonotpossessswimbladders,thepresenceoflargequantitiesoflow-densityoils(squalenedensity0.86gcm–3;DAGEdensity0.89gcm–3)intheirliversallowsthemtoachieveandmaintainbuoyancy.Table5.2showssometypicalcompositionsofsharkliveroilforseveralspeciesofshark[36,37].DAGEsareconsideredtobeefficientinwoundhealingapplicationsandinpreventingthemultiplicationofbacteria.Someresearch-ershavealso suggested thatDAGEsmayaid in the reductionofcertain typesofcancer,promoteformationofbloodcells,andprovideprotectionagainstradiation
table 5.2shark liver oil Compositions for some selected species of shark
shark species Common name squalene dages tags otherd ref.
Carcharhinus plumbeusa Sandbarshark 0.0 0.0 83.0 17.0 36
Centrophorus scalpratusb Endeavourshark 81.6 9.9 8.5 0.0 37
Centrophorus squamosusc Leafscalegulpershark 70 11 18 1.0 36
Centroscymnus crepidaterb Long-nosevelvetshark 73.0 20.0 5.0 2.0 37
Centroscymnus plunketib Plunketshark 0.9 76.6 22.5 0.0 37
Dalatias lichac Kitefinshark 79 18 2 1.0 36
Deania calceab Platypusshark 69.6 1.6 10.8 18.0 37
Etmopterus granulosusb Lanternshark 50.3 32.1 9.3 8.3 37
Hexanchus griseusa Bluntnosesixgillshark 1.0 70.0 29.0 0.0 36
Somniosus pacificusb Pacificsleepershark — 49.5 49.1 1.4 37
Squalus acanthiasc Pikeddogfish 0.0 12.0 87.0 1.0 36a Hawaiianwaters,bSouthernAustralianwaters, cChathamRise,NewZealand, dIncludingfreefatty
acids(FFA),phospholipids,sterols,pristane,waxesters,andsterolesters.
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146 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
injury[38].SharkliveroilswithdefinedlevelsofDAGEsandsqualenearecurrentlysoldasnutraceuticals.
5.2.3 Vitamin a (retinOl)
Vitamin A, a fat-soluble compound, is derived in the bodies of animals fromβ-carotene(alsoknownasprovitamin A).VitaminA,carriedinthebloodbyretinol-bindingprotein,hasbeenidentifiedasanessentialnutrientnecessaryforgrowth,reproduction,andvision.Inthebody,itisoxidizedtoretinal,akeycomponentofthevisualsystem,andtoretinoicacid,whicheffectsgeneexpressionviaspecificnuclearreceptors. The discovery of an anticarcinogenic action of vitamin A by Saffiottoetal.[39]ledtorapidgrowthinvitaminAresearch.Inanimalexperiments,vitaminAanditsmetabolite,retinoicacid,wereshowntohaveanticancerproperties.Retinoicacidisnowaningredientusedinanti-ageingcosmetics.VitaminA–richcreamsareusedforseverecasesofacne[40].Forreviewsdescribingthediscovery,structureelucidation,androlesofvitaminAinthehumandiet,thereaderisdirectedtothepublicationsofWolf[41]andUnderwood[42].
TheimportanceofvitaminAinhumanhealthhasleadtoarequirementfordietarysupplementsintheformofcapsulesandfoodfortification.Theliversofcartilaginousfish,suchassharksandrays,areapotentiallyvaluablesourceofvitaminA.VitaminAisfoundinfishliveroilsalmostentirely(96–100%)asesters[43],suchasvitaminApalmitate(Figure5.3).
5.2.4 Wax esters
Oilsofcertaindeep-seaspeciesoffisharecomposedalmostentirelyofwaxesters,which are esters of long-chain fatty acids and fatty alcohols. Fish species in theSouthPacificwhoseoilsarecomposedofwaxestersincludeorangeroughy,blackoreo,andsmall-spinedoreo (Table5.3) [44].The fattyacidportionsof theestershavecarbonchainlengthsofC14–C24andareeithersaturatedormonounsaturated.ThefattyalcoholshavecarbonchainlengthsofC16–C24andaremostlysaturated(Table5.4)[44].Waxesterswithunsaturationneartheesterbondaremorevolatilethanwaxesterswithunsaturationnear the centerof the aliphatic chainbuthavesimilaroxidativestability[45].Inteleostfish(thosewithabonyskeletalstructure),theoccurrenceofwaxestersinmuscletissuecorrelatesbetterwithseadepthandverticalmigrationpatternsthanwithtaxonomy[46].
Infish,waxestersappeartofunctionassourcesofenergy,insulation,andaidstobuoyancyandbiosonar[47].Inindustry,theyhaveapplicationsasspeciallubri-cants,cosmeceuticals,andchemicalrawmaterialsforthemanufactureofsoapsand
O
O CH2(CH2)13CH3
FIgure 5.3 ThechemicalstructureofvitaminApalmitate.
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Processing of Fish Oils by Supercritical Fluids 147
detergents[48].Oilfromtheheadof thespermwhale,whichcontainsmorethan65% wax esters (Table5.3), is particularly suitable for these applications. Owingtothepresentstatusofthespermwhaleasanendangeredspecies,however,theoilisno longeran itemofcommerceandalternativesourceshavebeen investigated.Astudyofthechemicalandphysicalpropertiesoforangeroughyoilshowedthatitcouldreadilyreplacespermwhaleoil[44].TheproductionoforangeroughyoilhasbeencommercializedinNewZealand,withseveralplantsproducingthousandsoftonnesperyear[49].
5.3 separatIon/FraCtIonatIon teChnologIes
Therearemanymethodsforthefractionationoffishoils,butonlyafewaresuitableforlarge-scaleproduction.Thesuitablemethods,whichusuallyrequireconversionofTAGstofattyacidsorethylesters,includeadsorptionchromatography,fractionalormoleculardistillation,enzymaticsplitting,low-temperaturecrystallization,ureacomplexation and SCF extraction/fractionation techniques. Standard oil refiningtechnologiesarenotconsideredhere.
table 5.3total lipid Composition and Composition of the Wax ester Fraction of orange roughy, black oreo, small spined oreo, and sperm Whale
orange roughy black oreosmall spined
oreo sperm Whale
Component Weight (%) of oil
Waxesters 94.9 91.5 95.6 65.8
TAGs 3.1 4.8 2.5 30.1
Cholesterol/alcohols 1.0 2.7 1.5 4.0
Phospholipids 1.0 1.0 0.4 0.1
total Carbon number Weight (%) of Wax ester Component
C26 4.7
C28 14.0
C30 0.2 0.5 0.5 21.1
C32 2.1 3.5 2.9 23.2
C34 11.4 11.6 9.3 19.9
C36 16.7 21.8 18.3 11.7
C38 24.8 21.3 26.2 4.4
C40 23.4 19.8 25.4
C42 14.8 10.8 12.8
C44 5.5 6.1 4.3
C46 1.1 4.6 0.3
Source: Adapted with permission from Journal of the American Oil Chemists’ Society, 59, 390.©1982AmericanOilChemists’Society.
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148 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
5.3.1 traditiOnal prOcessing methOds
Separationoffishoilfattyacidsandestersusingtraditionalmethodsiscomplicatedbyseveralfactors.First,methodsrelyingondifferencesinmolecularweight,suchasdistillation,arehinderedbytherelativelysmallmolecularweightdifferencesinthese compounds, especially when attempting to separate saturated and unsatu-ratedfattyacidsofthesamechainlength.Second,PUFAsarereadilysusceptibletodegradation,oxidation,polymerization,andstereomutation,evenatmoderatelyelevated temperatures. Berdeaux et al. [50] reported the thermal degradation ofPUFAs during the deodorization of fish oils. The high processing temperatures
table 5.4percentages of Fatty acids and Fatty alcohols of the Whole Fish Wax esters of orange roughy, black oreo, small spined oreo, and sperm Whale
orange roughy
% Fatty
black oreo
% Fatty
small spined oreo
% Fatty
sperm Whale
% Fatty
Component acid alcohol acid alcohol acid alcohol acid alcohol
saturated
<14:0 21.6
14:0 1.2 4.1 1.9 6.8 9.4 8.0
15:0 <0.1 0.8 <0.1 0.7 0.9 1.4
16:0 1.0 7.3 15.5 20.8 8.1 9.4 5.1 39.5
17:0 0.7 1.1 0.8 3.8 0.4 1.1
18:0 0.3 8.1 3.2 2.3 3.7 0.9 1.5 7.7
19:0 0.6 0.2
20:0 <0.1 0.2 <0.1 <0.1
22:0 <0.1 <0.1 <0.1 <0.1
24:0 <0.1 <0.1 <0.1 <0.1
Unsaturated
14:1 0.5 0.3 0.4 19.6
15:1 <0.1 0.7 0.1 0.2 0.2
16:1 11.8 7.9 10.9 15.6 4.1
17:1 1.0 0.8 0.8 3.7 1.3 1.0
18:1 56.0 34.6 26.9 19.0 32.8 23.3 17.8 35.4
18:2 1.9 1.0 0.9 0.5
20:1 17.8 30.6 15.8 29.6 16.5 33.7 3.9 1.4
22:1 7.8 13.8 11.6 20.3 9.5 31.6 1.4
23:1 <0.1 1.8 2.1 0.8
24:1 <0.1 5.4 8.3 3.9 <0.1 1.1 <0.1
Source: AdaptedwithpermissionfromJournal of the American Oil Chemists’ Society,59,390.©1982AmericanOilChemists’Society.
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Processing of Fish Oils by Supercritical Fluids 149
associatedwithvacuumdistillationcanbeproblematicbecauseω3fattyacidsaresusceptibletooxidativedeteriorationduetotheirhighdegreeofunsaturation.Theprimaryoxidationproducts, lipidhydroperoxides,areespeciallyunstableandcandegrade to yield volatile secondary oxidation products. The secondary oxidationproductscanimpartundesirable,unpleasantfishyodorsandflavorstoendproducts.ThemixtureofTAGsinfishoilsistoocomplexforefficientisolationofindividualcomponentsandoftenonlymodestenrichmentscanbeexpectedfromfractionation.Therefore,mosteffortshavebeendirectedtowardthefractionationofacidsandtheirmethyl/ethylesters.Dealingwiththesesingle-chaincompoundsallowsdifferencesinchainlengthordegreeofunsaturationtobeeffectivelyaddressed.TheacidsandestershavegreatervolatilitythanTAGs,allowingtheuseoftemperature-controlledseparationmethods,suchasmoleculardistillation.Afterseparation,specificTAGsorfreeacidscanbereconstituted.Mostseparationmethods,whenusedalone,canonlyseparatefishoilacidsintogroupfractions.Therefore,twoormoreproceduresareoftenrequiredtoproduceindividualcomponentsinhighpurity.
5.3.1.1 distillation
Distillation relies on differences in mixture component vapor pressures, whicharestronglyrelatedtomolecularweightsforahomologousfamilyofcompounds.Enrichment is achieved by exploiting the differences in vapor pressure throughcountercurrentcontactingofvaporandliquidphasesinstagesusingplatesorcontin-uouslyusingrandomorstructuredpacking.Ifweassumeabinary(orpseudobinary)mixturecontainingcomponentsAandB,thenthedegreeofattainableenrichmentisdependentontheratioof theindividualcomponentvaporpressures,pAandpB.Assuming A is the more volatile component, the separation factor,α = pA/pB, isgreaterthanunityandsomedegreeofseparationcanbeachieved.
Figure5.4showsanidealizedvapor-liquidcompositiondiagramforamixtureofAandBthatisbeingseparatedinastage-wisedistillationcolumn.Atthefeedpointofthedistillationprocess,theconcentrationofAintheliquidphaseisX0andthatinthevaporphaseisY0.Thevapor-liquidequilibriumcurvepredictsthattheinitialconcentrationofAinthevaporphaseisgreaterthanthatintheliquidphasebyvirtueofitsgreatervolatility.Ifthevaporiscondensed(condensationrepresentedbythehorizontallinesdrawnbetweenthevapor-liquidequilibriumcurveandtheauxiliarylineinthestrippingorenrichmentsectionofthecolumn),theresultingliquidwillhaveaconcentrationofAequaltoY0/KA =X1,whereKAisthepartitioncoefficient(K-value)ofcomponentA.TheK-valueofanycomponentiisdefinedas:
K
y
xii
i
= (5.1)
whereyiistheconcentrationofcomponentiinthevaporphaseandxiistheconcen-trationofcomponentiintheliquidphase.
Similarly,thevaporinequilibriumwiththeliquidphaseofconcentrationX1hasaconcentrationofAequaltoY1.Again,theconcentrationofAinthevaporphaseisenhancedduetoitsgreatervolatility.Ifthisvaporisthencondensed,theresulting
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150 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
liquidhasamolepercentconcentrationofAaccordingtothesamerelationship—thatis,Y1/KA = X2.Similarrelationshipsholdintherectificationsection,wherethe“heavy”componentBisconcentrated.Whenthesestepsarecarriedoutcontinuouslyinamultistageorcontinuouslypackeddevice,itistheoreticallypossibletoobtainhighlypureAas theextractandhighlypureBas theraffinate.Alternatively, theevaporationandsubsequentcondensationstepscanbecarriedoutasabatch-wisefractionaldistillationprocessuntilthedesireddegreeofenrichmentwithrespecttoAhasbeenachieved.
Stoutetal. [51]highlighted thepracticaldifficultyofobtainingω3-PUFAsinhighconcentrations in thenaturalTAGform.Moleculardistillationofmenhadenoil in itsnaturalTAGformincreased theEPAcontent in theresiduefrom16.0%to19.5%and theDHAcontent from8.4% to17.3%.Carryingout thedistillationusingthemenhadenoilethylesters,however,almostdoubledtheEPAconcentrationfrom15.9%to28.4%,whereastheconcentrationofDHAshowedanalmostfivefoldincrease,from9%to43.9%.
Fractionaldistillationoffishoils ispreferentiallycarriedoutusing fattyacidestersunder reducedpressure (0.1 to10 torr) since these,unlike free fatty acids,approximateideality.Also,thegreatervolatilityoftheestersallowsseparationtobecarriedoutat lower temperatures,which is importantconsidering the thermalinstabilityoftheω3components.Boilingpointsofunsaturatedestersaremarginallylowerthanthoseofsaturatedestersofthesamechainlength.Therefore,foragivenchainlength,unsaturatedestersareenrichedduringtheearlystagesoffractionation
Raffinate
X0 X1 X2
Y2
Y1
Y0
Feed
0 20 40 60 80 100
100
80
60
40
20
0
Extract
Rectificat
ion Section
Stripping S
ection
FIgure 5.4 SeparationofahypotheticalmixtureofcomponentsAandBbydistillation.
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Processing of Fish Oils by Supercritical Fluids 151
andsaturatedonesareenrichedintheendfractions.Duringthetransitionfromonechainlengthtothenext,thedistillatecontainsamixtureofsaturatedlowerchainlengthandunsaturatedhigherchainlengthcomponents.
Althoughthegreatervolatilityof thefattyacidestersallowstheuseof lowerprocess temperatures (compared with temperatures required for free fatty acids),temperatures are still moderately high (typically 423 to 473 K), and exposure todistillation conditions over a prolonged period of time can be detrimental to thepolyunsaturatedconstituents,causinghydrolysis,polymerization,isomerization,andthermaloxidation[52].Privettandcoworkers[53,54]foundconsiderabledecompo-sitionofarachidonateduringslowdistillationinaspinningbandcolumn.Fraction-ationofmarineoilesterscontainingchainlengthsofC20ormoreisdifficultbecauseseparationfactorsdecreasewithincreasingmolecularweight[55].
5.3.1.2 low-temperature Crystallization
Crystallization separationsarebasedon thedifferences incompositionof equili-brated liquid and solid phases. The process can be carried out using the crudeliquidoilor inasolventsolution.Anoperationwith thecrude liquidoil requiresslowcoolingandslowagitation.Thisproducesaslurryofsolidandliquidcompo-nents, the latterbeingenrichedwithPUFAs.Whenusinga solution, theequilib-rium isdependentoncomponent solubilities.Commonsolventsofchoice includemethanol,acetone,petroleumether,acetonitrile,nitromethane,andliquidpropane.Theformationofsolidcrystalsinevitablyresultsinentrapmentofsomeliquid,andsoseparationfactorsarenothigh.Thesolubilityoffatsinorganicsolventsincreaseswithincreasingunsaturationanddecreaseswithincreasingmolecularweight[56].Singleton[57]andStoutetal.[51]havemeasuredthesolubilityofseveralfattyacidsinavarietyofsolvents.Theirfindingsledtothefollowingrules:
Long-chainsaturatedfattyacidsarelesssolublethanshort-chainsaturatedfattyacids.Saturatedacidsare less soluble thanmonounsaturatedanddiunsaturatedacidsofthesamechainlength.Transisomersarelesssolublethancisisomers.Straight-chainacidsarelesssolublethanbranched-chainones.Freefattyacidsarealwayslesssolublethantheirmethylestercounterparts.
Themeltingpointsof fattyacidsvaryconsiderablyaccording to theirdegreeofunsaturation.Thiswidevariationinmeltingpointcanbeexploitedtoenabletheseparationofsaturatedandunsaturatedfattyacidcompounds[58].Atlowtempera-tures,long-chainsaturatedfattyacids(havinghighermeltingpoints)crystallizeout,leavingthePUFAsinsolution.Thefreeacidsarepronetoassociationinsolution.Although this can be overcome by processing the methyl ester analogues, thepracticaladvantageislimitedbecauseofthegreatersolubilityoftheesters.Also,theformationofeutecticspreventsachievementofthedegreeoffractionationonewouldpredictfromsolubilitydataalone.Thechoiceofsolventandprocessingtemperaturemustbeconsideredcarefullybecausethesefactorscanhaveapronouncedeffecton
•
•
•••
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152 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
theconcentrationofPUFAsobtained.Thereisalsothepossibilityofseparatingfattyacidsonthebasisofdegreeofunsaturation[59].DHA(orsaltsthereof)crystallizesat a lower temperature than EPA in solvents such as acetone. However, the verylowtemperaturesrequiredmaketheprocesssomewhatimpractical[60]. Until theintroductionofSCFfractionation techniques, low-temperaturecrystallizationwasconsideredtobethemethodleastdetrimentaltopolyunsaturatedfattyacids.
5.3.1.3 urea Crystallization
Ureacomplexationwithstraight-chaincompoundswasfirstreportedbyBengen[61].Whilepureureacrystallizesinatightlypackedtetragonalstructure(Figure5.5a),in the presence of long straight-chain molecules, it crystallizes into a hexagonalstructuretoformaninclusioncomplex(Figure5.5b)[62].Theureacomplexiscon-structedofaspiralarrangementofureamolecules,andthestraight-chainmoleculesare held in the hexagonal channels by van der Waals forces, London dispersionforces, or induced electrostatic interactions [63]. The hexagonal channel is wideenough to accept molecules with a diameter of around 5 Å, but molecules withgreaterdiametersarenoteasilyaccommodated.Theformationandstabilityofureacomplexesisthereforegovernedbyshape,size,andgeometry.
Theuseofureacrystallizationseparatesfattyacidsmainlyaccordingtotheirdegreeofunsaturation.Whenureacrystallizesfromasolutioncontainingamixtureof fatty acids, the saturated and monounsaturated fatty acids are preferentiallyincluded in thecomplexwhile thepolyunsaturatedfattyacidsremain insolution.Thisisbecausethepresenceofdoublebondsinfattyacidspreventsthechainfromorientingintothe“ideal”geometryforcomplexformation.Thiscausesanimbalanceof theoptimum intermolecular distances,whichdisrupts thenet attractive forcesanddestabilizestheinclusioncomplex.Thestabilityofureainclusioncomplexesislessenedbyshorterchainlengthsandahighernumberofdoublebonds[64].Transisomersformmorestablecomplexesthanthecorrespondingcisisomers,andcom-pounds with conjugated double bonds form more stable adducts than those withmethylene interrupted or isolated double bonds. The most influential processingvariablesaffectingtheextentofcrystallizationaretheurea–fattyacidratiosandtheprocesstemperature.Theurea–fattyacidratiocanbeusedtofractionatefattyacidsaccording to differing degrees of unsaturation. When low concentrations of urea
(b) Palmitic Acid (C16:0)/Urea Inclusion Complex(a) Pure Urea
FIgure 5.5 Crystalstructuresforpureureaandurea–fattyacidcomplexes.
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Processing of Fish Oils by Supercritical Fluids 153
areused,thedifferentfattyacidscompeteforcomplexformationaccordingtotheabilitytoformthemoststableinclusioncompound.
RoblesMedinaetal.[65]studiedtheeffectofurea–fattyacidratioonthefattyacid composition of urea filtrates (noncomplexed fatty acids) from cod liver oil.Table5.5showstheirresultsfromureacrystallizationat277KandFigure5.6showstheeffectoftheseratiosonvariousfattyacidgroupsofinterest.Withaurea–fattyacid ratio of 1:1, the saturated fatty acids were partially removed from solution,while the concentration of monounsaturates remained constant. Increasing theratioto2:1facilitatedmaximumremovalofthesaturatedacidsalongwithpartialremovalofthemonounsaturatedacids.Ataratioof3:1,theremovalofsaturatedand
table 5.5the effect of urea/Fatty acid ratio on the noncomplexed Fatty acid Composition resulting from urea Fractionation of Cod liver oil at 277 K
Fatty acid Cod liver oil
urea/Fatty acid ratio
1:1 2:1 3:1 4:1
14:0 4.2 2.7 0.7 0.5 0.7
16:0 10.6 2.0 0.2 0.5 0.0
16:1ω7 7.8 9.6 6.9 2.5 3.2
18:0 2.6 0.9 0.1 0.0 0.0
18:1ω9 17.0 17.6 3.2 2.9 0.7
18:1ω7 4.6 5.9 1.4 1.0 0.0
18:2ω6 1.5 2.0 1.6 0.7 0.7
18:3ω6 0.2 0.2 0.4 0.5 0.5
18:3ω3 0.8 1.1 1.0 0.6 0.6
18:4ω3(SA)a 2.4 3.3 6.3 8.0 8.5
20:0 0.2 0.2 0.2 0.0 0.1
20:1ω9 10.8 9.0 1.3 0.6 0.8
20:3ω6 0.1 0.1 0.1 0.2 0.2
20:4ω6 0.5 0.8 1.0 0.9 1.0
20:5ω3(EPA) 9.4 13.0 22.6 24.8 25.6
22:0 0.1 0.0 0.0 0.0 0.0
22:1ω11 8.3 3.8 0.4 0.0 0.0
22:1ω9 0.1 0.0 0.0 0.0 0.0
22:4ω6 0.5 0.7 1.5 1.7 1.8
22:5ω3 1.2 1.6 2.4 1.4 1.6
22:6ω3(DHA) 11.0 15.8 45.4 58.2 59.9
24:0 0.0 0.0 0.0 0.0 0.0
Source: Reprinted with permission from Journal of the American Oil Chemists’ Society, 72, 575.©1995AmericanOilChemists’Society.
a Stearidonicacid.
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154 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
monounsaturatedfattyacidswereatamaximum.Increasingtheratioto4:1didnotgreatlyincreasetheremovalofthesaturatedandmonounsaturatedcomponentsfromthesolution.Increasingtheurea–fattyacidratiosalsoincreasedtheconcentrationofstearidonicacid(SA),EPA,andDHAinthefiltrate.ThemaximumenrichmentofPUFAswasalsoobservedatanoptimumurea–fattyacidratioof3:1,aphenom-enonnotedbyotherresearchgroups[66–68].Inthesamestudy,RoblesMedinaandcoworkers[65]investigatedtheeffectoftemperatureonthefattyacidcompositionofureaconcentratesfromcodliveroilusingaurea–fattyacidratioof4:1;Figure5.7iscompiledfromtheirdata.ThefiltrateconcentrationsofSAandDHAwerefoundtobegreatestat261K,thetotalω3fattyacidconcentrationswerefoundtobegreatestat 277 K, and the concentration of EPA in the filtrate was maximized at around293K.Althoughdifferenttemperaturevalueswerereported,thistrendontheinflu-enceoftemperaturechangeissimilartothatobservedbyWillesandcoworkers[66],whousedureafractionationandhigh-performanceliquidchromatography(HPLC)toproducefractionsrichinPUFAsfromfishoils.Duringureacrystallization,theyreportedthattheconcentrationsofSAandDHAinthefiltrateweregreatestat268K,theconcentrationoftotalω3inthefiltratewasgreatestat283K,andthatofEPAwasmaximizedat288K.
WanasundaraandShahidi[69]optimizedtheproductionofω3fattyacidconcen-tratesfromsealblubberoilusingureacomplexation.Theyinvestigatedtheeffectsof the urea–fatty acid ratio, crystallization time, and crystallization temperature.
4:11:1CodLiverOil
Urea/Fatty Acid Ratio
SA
EPA
DHA
Total SA, EPA, DHA
Total Saturated+ Monounsaturated
Monounsaturated
Saturated
Fatty
Aci
d Co
ncen
trat
ion/
% w
/w
2:1 3:1
100
90
80
70
60
50
40
30
20
10
0
FIgure 5.6 Theeffectofurea–fattyacidratioonthenoncomplexedfattyacidcompositionresultingfromureafractionationofcodliveroilat277K.
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Processing of Fish Oils by Supercritical Fluids 155
The study was carriedout using free fatty acids.Under optimumconditions, themaximumamountof totalω3 fatty acids (88.2%;70.1%ofwhichwasDHAand9.36%ofwhichwasEPA)fromsealblubberoilwasobtainedataurea–fattyacidratioof4.5:1,acrystallizationtimeof24hours,andacrystallizationtemperatureof263K.
Thereareseveraladvantagestousingureacomplexationforthefractionationoffattyacids[70]:
Largequantitiesofmaterialcanbehandledusingsimpleequipment.Theycanbecarriedoutundermildconditions(e.g.,roomtemperature).Biocompatiblesolventssuchasethanolcanbeused.Separationsareoftenrelativelyefficientwhencomparedwithmethodssuchassolventextractionandfractionalcrystallization.Itisarelativelyinexpensiveprocess.
Ureainclusioncomplexationisusuallycarriedoutinmethanolorethanol.Oneshouldbecautiouswhenusingmethanolbecauseitmaycausemethylationofsomefattyacidsduringcomplexformation,producingamixtureoffattyacidsandmethylesters[71].UreaconcentrationshouldbenearsaturationsincetheconcentrationofrecoveredPUFAsfromthefiltratedecreaseswithdecreasingureaconcentration[70].Usingacetone,orhigherhydrocarbons,assolventsforureacomplexationshouldbeavoidedbecauseitcancompetewiththefattyacidsforinclusion[72].Ratherthanbeingusedasastand-aloneprocess,ureacrystallizationisoftenusedtopreconcen-tratefishoil fattyacidmixturesprior to furtherprocessing.Foramore thorough
••••
•
Temperature/K
SA
EPA
DHA
Total ω3
Fatty
Aci
d Co
ncen
trat
ion/
% w
/w
244
110
100
90
80
70
60
50
40
30
20
10
0252 260 268 276 284 292 300 308
FIgure 5.7 Theeffectoftemperatureonthenoncomplexedfattyacidcompositionresultingfromureafractionationofcodliveroilusingaurea–fattyacidratioof4:1.
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156 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
treatiseofthetheoryandpracticeoffractionationwithurea,thereaderisdirectedtotheworksofSwern[63],Schlenk[72],andSmith[73].
5.3.1.4 Chromatographic methods
Chromatographicseparationstakeadvantageofthedifferentratesofmigrationofmixturecomponentsthroughacolumninatwo-phasesystem,comprisingamobilephase (the phase containing the components of interest) and a stationary phase(animmobilephase,whichisinsolubleinthemobilephase).Themobilephasecanbealiquid,gas,orSCFandthestationaryphaseisusuallyasolid.Thephasesarechosensuchthatthemixturecomponentshavedifferingstrengthsofinteractionwiththestationaryphase.Componentsthathaveagreateraffinityforthestationaryphasehavelongerelutiontimesthancomponentswithaloweraffinity,whichenablessepa-rationtotakeplace.
Carefulselectionofthestationaryphase(adsorbent)canpermittheseparationoffattyacidsaccordingtocarbonchainlengthordegreeofunsaturation.Silverionsformaweakπ-bondwithsitesofunsaturation,andsosilverion–impregnatedcolumnscanbe used to fractionate polyunsaturates. High performance liquid chromatography[74]andsilverresinchromatography[75]havebeenusedforthepreparationofω3concentrates.Teshimaandcoworkers [76]haveuseda silvernitrate–impregnatedsilicacolumntoisolateEPAandDHAmethylestersfromsquidliveroil.Puritiesof85%to96%forEPAand95%to98%forDHAwerereportedwithyieldsof39%and48%,respectively.AdlofandEmiken[75]enrichedtheω3contentofcommercialω3-PUFAconcentrates from76.5%to99.8%using isocraticelutionfromasilverresincolumn.Guil-GuerreroandBelarbi[77]purifiedEPAandDHAfromcodliveroil using a silver nitrate–impregnated silica column. The oil was saponified andtreatedwithurea,andthenoncomplexedfattyacidswerethenconvertedtomethylestersbeforechromatographicprocessing.Thecolumnwaselutedwithasequenceofsolvents.Theymanagedtoobtaina64%yieldofDHAwith100%purity.TherecoveryofEPAwas29.6%,withafinalpurityof90.6%.
The purity of eluted fractions also depends on the choice of eluting solvent.Perrut[78]usedmethanol/water(90:10v/v)toseparatefishoilethylesters.Puritiesof96%EPAand85%DHAwereachieved.Willeandcolleagues[66]usedthesamemethanol/ethanol(90:10v/v)solventsystemforthefractionationoffishoilmethylestersandproducedEPA-richandDHA-rich fractionswith86%and83%purity,respectively. Despite the developments in chromatographic techniques to refineand concentrate fish oil components, the use of very large volumes of solvents,potential product solvent residues, loss of column resolution after repeated use,andthepresenceofpotentiallytoxicsilverresiduesarelikelytohinderscale-uptoproductionscalevolumes.
5.3.1.5 enzymatic transformation
The hydrolysis or esterification of fatty acids can be catalyzed by lipases. Thesereactions can be carriedout at low temperatures, which is beneficial consideringthedetrimental effectshigh temperaturescanhaveonPUFAs.Thedirectionandefficiencyofthereactiondependsontheconditionsemployed.Watercontentinthe
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Processing of Fish Oils by Supercritical Fluids 157
reactionmediumisacrucialfactorinfluencingthedirectionofthereaction.Highwatercontentshiftsthechemicalequilibriumtowardhydrolysis,whereaslowerwatercontent shifts the equilibrium toward esterification. For esterification, thewatercontentshouldbekepttoaminimumtopreventpartialhydrolysisofproducts,butatthesametime,thewatercontentshouldbesufficientlyhighinordertopreventenzymedeactivation[79].
The TAG form of PUFA is considered to be nutritionally more favorablethanfattyacidestersbecausestudieshaveshownthat theintestinalabsorptionofω3-PUFAestersisimpaired[80,81].Also,TAGsareoftenpromotedasbeingmore“natural”thanfattyacidesters,leadingtoacommercialdemandofPUFAsintheTAGform.HeandShahidi[82]studiedtheglycerolysisofω3-PUFAsobtainedfromseal blubber oil using Chromobacterium viscosum lipase. Up to 94% conversionwasachievedwith13.8%,43.1%,and37.4%ofmonoglycerides,diglycerides,andtriglycerides,respectively,intheproduct.Bottinoandcolleagues[83]reportedtheresistanceofcertainlong-chainPUFAsofmarineoilstolipase-catalyzedhydrolysis.Theciscarbon-carbondoublebondspresentinsomefattyacidsresultinbendingof the carbon chain, causing the fatty acid terminal methyl group to lie close totheester linkage.Themethylgroup in this instanceproducesastearichindrancetolipase-catalyzedhydrolysis.Anincreaseinthenumberofdoublebondsfurtherincreases thestearichindrance.However, therearesomereports inwhichlipasesfrom Chromobacterium viscosum and Pseudomonas sp. released EPA and DHAfromtriglycerides[84–86].
Gámez-Mezaetal.[87]studiedtheconcentrationofEPAandDHAfromsardineoilbyenzymatichydrolysis.TheyinvestigatedfivecommerciallipasesfromPseudomonas(threeimmobilizedandtwosoluble).Theyfoundthattheimmobilizedlipaseprepa-rationPS-CI(alipasefromPseudomonas sp.immobilizedonachemicallymodifiedceramic)providedthegreatestdegreeofhydrolysisforEPAandDHA(81.5%and72.3% from their initial content in the sardine oil after 24 hours) and attributedthisobservationtothehigherproteincontentofthislipase.Atthestartofhydro-lysis (3hours), theynoticed that the lipasesdisplayed a significantpreference forsaturatedfattyacidscontaining14to16carbonatoms.However,theresistancetoreleaseEPAandDHAdecreasedasthehydrolysisreactionprogressed.SubsequentureacrystallizationofthePS-CIhydrolyzedoilenrichedEPAfrom14.5%to46.2%andDHAfrom12.6%to40.3%,witha78.0%yield.
Schmitt-Rozieresetal.[88]studiedtherecoveryofEPAandDHAfromeffluentsofthesardinecanningindustry.Theoilyeffluentcomponentcontainedaround10%eachofEPAandDHA.Aftertheremovalofsolidparticles,proteins,andpeptidesfromthecrudeeffluent,theresultantoilwashydrolyzedandEPAandDHAwereenrichedbyselectiveenzymaticesterification.UsingLipozyme™,DHAwasenrichedup to 80% but no enrichment was observed for EPA. By immobilizing Candida rugosa lipaseonanAmberliteIRC50cation-exchangeresin,a30%enrichmentofEPAwasachieved.
Zuyi and Ward [89] studied the lipase-catalyzed alcoholysis of cod liver oilto concentrateω3-PUFAs. They studied the effect of water content on reaction,indicating that thewater content is influencedby thehydrophobicityof the reac-tionmedium; themorehydrophobic thealcohol, the lower thewater content that
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158 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
is required.Using isopropanol, theyobserved that thealcoholysisof triglyceridesincreasedwithincreasingwatercontentintherange0%to7.5%v/v.Athigherwaterconcentrations(>10%)severalnegativeeffects, includingenzymedestabilization,purificationcomplications,andpromotionofundesirablehydrolysisreactions,wereobserved.Carryingouttheisopropanolysiswith5%water(v/v)at383Kyieldedahighconcentrationofmonoglyceridecontaining40%ω3-PUFAs.
Lipase-catalyzed transesterification for the concentration ofPUFAs fromfishoilshasbeenshowntobeausefulalternativetotraditionalesterificationanddistil-lationmethods.Withaconversionof52%,Breivikandcoworkers [90]wereableproduceaconcentratecontaining46%EPA+DHA(theinitialsardineoilcontained24.7%EPA+DHA)usingPseudomonas sp.lipaseandastoichiometricamountofethanolwithoutsolvent,atroomtemperature.TheresultingTAGswereisolatedandconverted to ethyl esters using either conventional chemical means or enzymaticconversion by immobilized Candida antarctica lipase. Urea fractionation of theresulting product increased the EPA + DHA content to around 85%. The use oforganic solvents as reaction media for enzymatic conversions can be disadvanta-geousbecausethiscanleadtoenvironmentalandresidualsolventissuesassociatedwithproduct purification.SCFshavebeen investigated as alternative solvents forthisprocess.
5.3.2 supercritical Fluid prOcessing OF Fish Oils
AlthoughvariousSCFshavebeenusedtoextract/fractionatefattyacidsandtheiresters,carbondioxide(CO2)isbyfarthemostcommonlyusedsolventbecauseofitsavailability,lowcost,andnonreactivity.UsingCO2limitstheoxidation,decom-position,andpolymerizationofthePUFAspresentinfishoilsbecauseseparationsoccurunderaninertatmosphereandprocessescanbecarriedoutatmoderatelylowtemperatures.Inaddition,CO2isnontoxic,isnonflammable,andproducessolvent-residue-freeextracts,whichisparticularlyimportantifthedesiredmaterialsareforhumanconsumption.Theability tomodifysolventpropertiesbymanipulationoftemperatureandpressureorbytheadditionofacosolventgivessupercriticalCO2processesauniqueadvantage.Solventpropertiescanbetunedforspecificseparationproblemsofferinggreaterversatilityandflexibilityovermoreconventionalfraction-ationprocesses.Moreover,theincreasingsocialawarenessofthehealthbenefitsofcertainfishoilcomponentsinthediethasincreasedcommercialdemandfortheseproductsinthefoodandnutraceuticalindustries.TheincreaseddemandhasledtoareevaluationofprocessingmethodsandsupercriticalCO2methodologiesprovidesolvent-free,“natural”products,whichhavewideconsumerappeal.TheremainderofthischapterfocusesonthephaseequilibriaofCO2withvariousfishoilcompo-nentsaswellasthevarioussupercriticaltechniquesemployedtoextract/fractionateandisolatetheseproducts.Phaseequilibriadataareoffundamentalimportanceforoptimaldesignofextractionandfractionationoperations.
5.3.2.1 phase equilibria: supercritical Co2 and Fish oil Components
ThissectionprovidesabriefsummaryofsolubilitymeasurementsandmodellingofsolubilityandphaseequilibriaforfishoilcomponentsinsupercriticalCO2.Fish
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Processing of Fish Oils by Supercritical Fluids 159
oilsareacomplexmixtureoflipidcomponentsbelongingtoseverallipidclasses,includingacylglycerols,fattyacids,fattyacidesters,sterols,tocopherols,andhydro-carbons.Successful isolationusing supercritical processes requires reliable infor-mationonthesolubilitybehaviorofthesolutesofinterestasaffectedbyoperatingconditionsandsolute/solventproperties.Becausecompletepredictivemodellingofmulticomponentphasebehaviorinsupercriticalsystemsisnotyetrealized,experi-mentaldatastillplayanessentialroleinprocessdesignandthedevelopmentofbothsimpleandrigorousthermodynamicmodels.
SimpleempiricalmodelsbasedontheChrastilmodel[91]havebeenwidelyusedforpredictionofsolubilityinCO2atagiventemperatureanddensity.TheChrastilcorrelationcanonlybeusedforthevapor-phaseconcentrationofsolutesandgivesnoinformationontheliquid-phasecomposition.Equationofstatemodels,suchasthePeng-Robinson[92,93],Soave-Redlich-Kwong[94],excessfunction(gE)[95],groupcontribution[96],andlatticemodelequationsofstate(EOS)[97],havebeenshowntoprovidethemostrigorousmethodforpredictingphaseequilibriumbehavior.
TheChrastil correlation [91] for estimating lipid solubilities inSCFs takestheform:
ln ln /c k a T b= + +ρ (5.2)
wherecisthesolutesolubility,kistheassociationnumberrepresentingthenumberofmoleculesinthesolute-solventcomplex,ρisthepuresolventdensity,andaandbareempiricalconstants.Parameteraisdependentonthetotalheatofreaction(heatofsolvation+heatofvaporization),andbisdependentontheassociationconstantandsoluteandsolventmolecularweights.Parameterkreflectsthedensitydependenceofsolubilityatconstanttemperature,andparameterareflectsthetemperaturedepen-denceatconstantdensity.Table5.6providessomeChrastilcorrelationparametersforthesolubilityofseveralfishoillipidcomponentsinCO2[98–101].
Although, binary lipid/CO2 systems have been studied extensively, multi-componentdata are relatively scarce. In suchmulticomponentmixtures, complexintermolecularinteractionsmayleadtosignificantdeviationsfrompurecomponentsolubilities.However,purecomponentsolubilityinformationisstillimportant,asitcanbeusedtogiveaguidetothedegreeofseparationpossiblebetweentwoormoreclassesoflipidatagiventemperatureandpressure.GeneralsolubilitytrendsinbinarysystemscontributetoourbasicunderstandingoftheprincipalsoflipidsolubilityinSCFs.Thisinformationprovidesasoundbasisonwhichtoevaluatethesolubilitybehaviorofmore complexmulticomponent systems.However, researchers shouldexercisecautionwhenmakingsolubilitymeasurements, interpretingthedataand,ultimately,designingseparationprocessesbasedon this information.TemelliandGüçlü-Üstündag[102]reportseveraldiscrepanciesbetweenreportedsolubilitydataforlipid/CO2systemsfromdifferentlaboratories.Anexampleofsuchdiscrepanciesis given inFigure5.8 for some selected fatty acids inCO2[103–107]. Impurities,sampledegradation,isomericpurity,andlimitationstoexperimentalmethodsareallcontributingfactorstothereporteddatavariations.
Solutevaporpressuresandsolute-solventandsolute-soluteintermolecularinter-actions govern solubility behavior. In binary systems of a particular homologous
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160 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
table 5.6Chrastil parameters for some selected Fish oil Components (all solubilities and densities are in units of g·l–1 unless otherwise stated)
lipid Componentk ± standard
errora ± standard
errorb ± standard
errorrange t/K;
p/mpa ref.
Fatty acids
Myristicacid,C14:0 6.42±0.33 –9300±1727 –10.2±5.9 98
Palmiticacid,C16:0 7.00±0.39 –12029±1043 –7.0±4.1 98
Stearicacid,C18:0 5.81±0.54 –15890±741 12.0±3.7 98
Oleicacid,C18:1 7.92±0.37 –3982±691 –38.1±2.3 98
Linoleicacid,C18:2 9.71±0.90 –5211±1626 –46.3±5.3 98
triglycerides
Triolein 10.28±0.66 –2057±480 –61.5±4.6 98
ethyl esters
Stearicacid 5.80±0.50 –2446±857 –26.7±3.9 98
Oleicacid 7.78±0.34 –1947±503 –40.9±2.7 98
Linoleicacid 7.17±0.63 –2193±896 –36.2±4.4 98
EPA 8.62±0.17 2473±262 –45.2±1.2 98
DHA 7.76±0.32 –1784±529 –42.1±2.5 98
hydrocarbons
Squalenea 6.54±0.06 –3936.6±155 –28.24±0.7 313–333;10–30
99
minor Components
VitaminAa 5.07±0.44 –3072±339 –21.7±2.12 313–353;20–35
100b
VitaminApalmitatea 7.66 0 –49.2 333;12.5–30
101
β-carotenea 8.63±0.61 –11576±461 –23.3±3.04 313–353;20–35
100b
Fish oils
Codliveroila 10.91±0.18 –4078±122 –59.2±0.98 313–333;20–30
99b
Spinydogfishliveroila 9.97 0 –65.4 333;20–30 101
Orangeroughyoila 7.79 0 –50.6 333;20–30 101a Solubilitiesareing·kg-1anddensitiesareinkg·m–3;bDerivedfromdatapresentedinthisreference.
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Processing of Fish Oils by Supercritical Fluids 161
series,whereintermolecularinteractionsaresimilar,molecularweightandvaporpres-suresdeterminecomponentsolubilities.Forexample,inFigure5.8,thesolubilityoffattyacidsincreaseswithdecreasingmolecularweight(chainlength).Ofthesystemsreportedintheliterature,fattyacidestershavethehighestvaporpressures,followedby fattyacids.Vaporpressuresof theglyceride lipidclass follow the trendmono-glycerides>diglycerides>triglycerides[98].EsterificationwithaC1orC2alcoholsubstantiallyincreasesthesolubilityoffattyacidsinCO2becausethepolaracidgroupisconvertedtoalesspolarestergroup[108].Forfattyacidsofthesamechainlength,meltingpointsdecreasewithincreasingdegreeofunsaturation(Figure5.8).Inthisinstance,solubilityisaffectedbythephysicalstateofthecompound.
JohannsenandBrunner[100]measuredthesolubilitiesoffat-solublevitaminsinsupercriticalCO2in the temperaturerangeof313to353Kandpressurerangeof20 to35MPa.The solubility for bothβ-carotene (provitaminA) andvitaminAincreasedwithincreasingpressure.Overthetemperaturerangestudied,vitaminA shows retrograde condensation behavior (solubility decreases with increasingtemperature)uptoaround30MPa(Figure5.9).Athigherpressures,thesolubilitycurvesofvitaminAexhibitacrossoverpointandthesystemexhibitsnonretrogradebehavior.ThesolubilityofvitaminApalmitatehasbeenmeasuredbyCatchpoleetal.[99]at333Kand12.5to30.0MPa.Thesolubilityiscomparedtothatofthevitamin A free acid in Figure5.9. The decrease in polarity of the ester over thefreeacidiscounterbalancedbythelargeincreaseinmolecularweight,leadingtoamodestdecreaseinsolubility.
Mollerupetal.[109–111]carriedoutaseriesofphaseequilibriameasurementsforfishoilfattyacidethylesters(FAEEs)ofthesandeelwithCO2.MeasurementswereobtainedusingthecrudefishoilFAEEs(283to343K,2to22MPa),urea-fractionated fish oil FAEEs (313 to 343 K, 1.6 to 25 MPa), andω3-rich fish oilFAEEs(313to343K,8to26MPa).TheinitialFAEEoilcompositionsaregiven
Pressure/MPa
C16:0; 318 K; ref. 104C16:0; 318 K; ref. 103
C18:0; 318 K; ref. 103C18:0; 318 K; ref. 104
C18:1; 323 K; ref. 105C18:1; 323 K; ref. 106C18:2; 313 K; ref. 107C18:2; 313 K; ref. 103
Solu
bilit
y/g
kg–1
of C
O2
5 10 15 20
50
40
30
20
10
025 30 35 40 45
FIgure 5.8 ComparisonoffattyacidsolubilitiesinCO2fromvariousliteraturesources.(Lineshavebeendrawntoaidtheeye.)
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162 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
inTable5.7.TheK-values,onaCO2 freebasis, for someselectedω3-FAEEsaregiven inFigure5.10 at 313Kand343 Kas a functionof pressure.TheK-valuesdependstronglyontemperature,pressure,andmixturecomposition.Highselectivi-ties(higherrelativedifferencesbetweenK-values)andlowsolubilitieswereobservedatlowpressures,whereasathighpressurestheselectivitywaslowbutthesolubilitywashigh.ThecrudeFAEEmixturewasmoresolublethantheurea-fractionatedandω3-richmixtures (whichhadsimilar solubilities)because thecrudemixturecon-tainedalargeamountofsaturatedandmonounsaturatedFAEEsofmediumchainlength(C14–C18).Forthecrudefishoilesters,theK-valueswerefoundtovarywithchainlengthbutnotspecificallythedegreeofunsaturationandpositionofdoublebonds.Theauthorsreportedthatsolubilitiesincreasewithincreasingtemperature,and Figure5.10 shows that the selectivities at 343 K are greater than those at313K. The selectivities are largest in theω3-rich system because the number ofcomponentsandtheamountofmedium-chain-lengthmaterialhasdecreased.Usingurea-fractionationasapreconcentrationstepundertheconditionsinvestigated,theauthorsdeterminedthatoptimumseparationconditionsintermsofselectivityandsolubilitywereintherangeof16to18MPaat343K(correspondingtoCO2densitiesof550to615kgm–3).
Catchpole and von Kamp [92] studied the phase equilibria of the systemsqualene/CO2andsharkliveroil/CO2overtherange313to333Kand10to25MPa.Thesharkliveroilconsistedofaround50%squaleneand50%ofamixtureofTAGsandDAGEs.Therewasalso0.5%bymasspristane.TheTAG/DAGEmixturehasbeenassumedtobeasinglepseudocomponent,withahypotheticalcarbonnumberof54.Thephaseequilibriadataforsqualene/CO2at313Kand333KareshowninFigure5.11.Theamountofoildissolvedinthevaporphaseincreaseswithincreasingpressureanddecreasingtemperature,asdoestheamountofCO2dissolvedintheoilphase.Theexperimentaldataforthebinarysystemssqualene/CO2andC54-TAG/CO2weremodelledusingthePeng-RobinsonEOSwiththeusualmixingrules[92].ThePeng-Robinson EOS was also used to predict phase equilibrium and separation
Pressure/MPa
Vitamin A: 313 KVitamin A: 333 KVitamin A: 353 KVitamin A palmitate; 333 K
Solu
bilit
y/g
kg–1
of C
O2
15
30
25
20
15
10
5
020 25 30 35
FIgure 5.9 SolubilityofvitaminAandvitaminApalmitateinCO2.
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Processing of Fish Oils by Supercritical Fluids 163
table 5.7the Initial Faee Fish oil Compositions used in the studies of mollerup et al. [109–111]
Crude Fish oil urea-fractionated ω3-rich
Component Weight (%) of Faee
10:0 0.4
12:0 0.2
14:0 7.5 0.8
14:1ω5 0.5 0.2
15:0 0.5
15:1ω5 0.2 0.1
16:0 18.5
16:1ω7 12.4 0.5
16:2 1.4 2.3
16:3ω3 0.6 1.6 0.3
16:4ω3 0.8 2.9 0.2
18:0 2.2 0.03 0.5
18:1ω9 10.2 0.6
18:1ω7 2.3
18:2ω6 2.9 1.2
18:3ω6 0.4 0.8
18:3ω3 1.3 0.9
18:4ω3 3.8 12.7 2.3
20:0 0.2
20:1ω9 4.2 0.6
20:2ω6 0.3 0.5
20:3ω3 0.2 0.1
20:4ω3 0.7 1.0 2.9
20:5ω3 10.0 35.9 52.5
22:1ω11 6.5 0.3
22:1ω9 1.0 0.1
21:5ω3 0.4 1.5
22:5ω3 0.5 1.1 2.5
22:6ω3 9.6 33.2 36.1
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164 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
factors for the ternary system C54-TAG/squalene/CO2. The predicted vapor andliquidmolefractionsofsqualeneintheternarysystemareshownforselectedtem-peraturesandpressuresinFigure5.12.TheliquidmolefractionsequatetodiscretemassfractionsofsqualeneonaCO2freebasisrangingfrom0to1.Itisinterestingtonotethattheequilibriumrelationshipisalmostlinear,asshownbytheregressionlines in Figure5.12. The mass fraction of CO2 dissolved in the liquid phase at agiventemperatureandpressurestaysalmostconstantevenwhenthesqualenemassfractionvariesfrom0to1(onaCO2-freebasis).Thepredictedvaporandliquid-phasemassfractionsofsqualeneforthesamesystemaregivenonaCO2-freebasisin Figure5.13. The K-values for squalene are also shown. The selectivity towardsqualeneisbestatlowpressureandlowmassfractionofsqualene.ThesolubilityofTAGsdecreasesmoresharplywithdecreasingpressurethansqualene,andsotheincreaseinselectivityistobeexpected.TheK-valuedecreasesasthetemperatureandvapor-phasedensityincrease,althoughitisstillsufficientlyhighat333Kand25.0MPatoenableseparationofsqualeneandC54-TAG.
Ruivoetal. [112]measuredthephaseequilibriaof the ternarysystemmethyloleate/squalene/CO2overtherange313to343Kand11to21MPa.Fourdifferent
Original Fish Oil
OriginalFish Oil
Pressure/MPa Pressure/MPa Pressure/MPa
Pressure/MPaPressure/MPaPressure/MPa
K-va
lues
at 3
43 K
(CO
2 fre
e bas
is)K-
valu
es at
313
K(C
O2 f
ree b
asis)
12 14 16 18 20 22 10
1088 9 10 11 12 13 14 15 10 12 14 16 18 11 12 13 14 15 16
12 14 16 18 20 2212
0.00.20.40.60.81.01.21.41.61.82.02.2
3.5
4.0
3.0
2.5
2.0
1.5
1.0
0.5
0.014 16 18 20 22
Urea-fractionated Fish Oil
Urea-fractionated Fish Oil ω3-Rich Fish Oil
ω3-Rich Fish Oil
C16:3 ω3C16:4 ω3C18:4 ω3C20:4 ω3C20:5 ω3 (EPA)C22:5 ω3C22:6 ω3 (DHA)
FIgure 5.10 Partitioncoefficients(K-values)onaCO2-freebasisforwholesandeeloil,urea-fractionated sand eel oil, andω3-rich sand eel oil [109–111]. (Reprinted from Fluid Phase Equilibria,161,169,©1999.WithpermissionfromElsevier.)
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Processing of Fish Oils by Supercritical Fluids 165
Liquid
0.725
26
24
22
20
18
16
14
12
10
80.750 0.775 0.800 0.825 0.995 0.996 0.997 0.998 0.999 1.000
Vapour
313 K333 K
Mole Fraction of CO2
Pres
sure
/MPa
FIgure 5.11 Liquidandvapormolefractionsforthesqualene–CO2system[92].
Liquid Phase Mole Fraction Squalene0.00 0.05 0.10 0.15 0.20 0.25
0.0040
0.0035
0.0030
0.0025
0.0020
0.0015
0.0010
0.0005
0.0000
Vapo
r Pha
se M
ole F
ract
ion
Squa
lene
250 Bar, 333 K200 Bar, 313 K200 Bar, 333 K125 Bar, 313 K
FIgure 5.12 Liquidandvaporphasemolefractionofsqualeneatselectedtemperaturesandpressures[92].Points:Peng-Robinsonequationofstatepredictions;lines:linearregressions.(ReprintedwithpermissionfromIndustrial and Engineering Chemistry Research,36,3762.©1997AmericanChemicalSociety.)
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166 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
feedcompositionswereusedcontaining0.1079,0.3350,0.6447, and0.8779molefractionsof squalene.The selectivityof afluid canbequantified in termsof theseparationfactor,α,whichinthisexampleisgivenby:
α = ⋅
⋅y x
x yM S
M S
(5.3)
whereyisthemolefractionconcentrationinthevaporphase,xisthemolefractionintheliquidphase,andthesubscriptsMandSpertaintomethyloleateandsqualene,respectively.TheselectivityofCO2towardmethyloleateoverarangeofpressuresisdemonstratedinFigure5.14aasafunctionoftemperature(initialsqualenefeedmolefractionof0.6447)andFigure5.14basafunctionofsqualenefeedconcentrationat313K.Figure5.14showsthatCO2ishighlyselectiveformethyloleate,withsepa-ration factors ranging from2 to8.The separation factordecreaseswithdecreas-ing temperature and with increasing pressure, which results in a higher loading,givinggreaterthroughputattheexpenseofselectivity.Anincreaseinsolubilitywithtemperatureatfixeddensityisadvantageousforpackedcolumnfractionation,whichrequires adensitydifferencebetween the supercritical andoilphases tobe largeenoughtopreventflooding.
Additionof cosolvents, suchas ethanol, canenhance the solubilityof solutesin supercritical CO2. Cosolvents have also been shown to act as entrainers. Theentrainereffecthasbeendefinedasaphenomenoninwhichthesolventpowerofafluidisincreasedbytheadditionofcosolvents,whilsttheselectivityofthatfluidismaintainedorenhanced[113].Inmanystudies,theenhancedsolubilitieshavebeenattributedtosolute-cosolventinteractions,suchashydrogenbondingordipole-dipole
Mass Fraction Squalene in the Liquid
VLE
K Factors125 Bar, 313 K250 Bar, 333 K
125 Bar, 313 K250 Bar, 333 K
Mas
s Fra
ctio
n Sq
uale
ne in
the V
apou
r
Equi
libriu
m C
oeffi
cien
t K
0.0 0.2 0.4 0.6 0.8 1.0
8 1.0
0.8
0.6
0.4
0.2
0.0
7
6
5
4
3
2
1
0
FIgure 5.13 MassfractionintheliquidandvaporphaseandK-valuesforsqualeneonaCO2-freebasis[92].(ReprintedwithpermissionfromIndustrial and Engineering Chemistry Research,36,3762.©1997AmericanChemicalSociety.)
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Processing of Fish Oils by Supercritical Fluids 167
interactions.Specificintermolecularinteractionsbetweencosolventsandsolutescanenhancethesolubilityofthosespecificcomponents,whichcanbeparticularlyben-eficialforimprovingseparationselectivities.
Catchpoleetal.[99]measuredthesolubilityofsqualene,orangeroughyoil,codliveroil,andspinydogfishliveroilinsupercriticalCO2andCO2/ethanolmixturesat313to333Kand20to30MPa.Ethanolmassconcentrationsup to12%(onasolute-freebasis)wereused.Catchpoleandcoworkersfoundthatethanolsubstan-tially increased the solubility of all fish oil components studied (Figure5.15). At333K,theauthorscorrelatedtheincreaseinsolubilityduetotheadditionofethanolusingthefollowingequation:
Temperature 313 K 323 K 343 K
0.8779 0.6447 0.3350 0.1079
Mole Fraction ofSqualene in Feed
Pressure/MPa (a) (b)
Pressure/MPa
Sepa
ratio
n Fa
ctor
, α
Sepa
ratio
n Fa
ctor
, α
10 12 14 16 18 20 22 10 12 14 16 18 20 22
8
7
6
5
4
3
2
8
7
6
5
4
3
2
FIgure 5.14 TheseparationfactorsformethyloleateandsqualeneinCO2[112].(ReprintedfromJournal of Supercritical Fluids,29,77,©2004.WithpermissionfromElsevier.)
0
100
10
12 4
Mass % Ethanol (solute free basis)
Squalene
Solu
bilit
y (et
hano
l fre
e bas
is)/g
kg–1
of C
O2
Orange Roughy OilCod Liver OilSpiny Dogfish Liver Oil
6 8 10 12 14
FIgure 5.15 Enhancement of fish oil component solubilities as a function of ethanolcosolventconcentrationat333Kand20MPa[99].(ReprintedwithpermissionfromJournal of Chemical and Engineering Data,43,1091.©1998AmericanChemicalSociety.)
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168 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
ln( / ) ln( / )S g kg S g kg kX⋅ = ⋅ +− −10
1 (5.4)
whereSistheenhancedsolubility,S0isthesolubilityinpureCO2,kisaconstant,andXisthemasspercentofethanol.Thekconstantsforsqualene,orangeroughyoil,andspinydogfishliveroilare0.14,0.15,and0.21,respectively.
ThesolubilityenhancementofsqualeneinCO2hasalsobeeninvestigatedusingn-hexane,toluene,andethanolintherangesof303.2to313.2Kand9to10MPa[114].Thedependenceofsolubilityonentrainerconcentrationwasdescribedbyaparabolicfunction.Theauthorscharacterizedtheinitialslopeof thisfunctionforeachsolute-entrainerpairwithasinglenumber,termedtheentrainer efficiency, E.TheEvaluewasrelatedtothesimilaritiesofthemolecularstructureandpolarityof theentrainerand thesolute.TheEvalues followed the trendn-hexane (3.6)>toluene(2.4)>ethanol(1.5),indicatingtheorderofsolubilityenhancementatfixedtemperature,pressure,andentrainerconcentrations.
Nilssonetal.[115]investigatedtheeffectsofadding5%ethanolasacosolventontheK-valuesandselectivityofmenhadenoilfattyacidestersat333Kand12.5MPa.Itwasnoted thatK-values increasedwith increasingnumberofdoublebonds foragivenchainlength.Additionofethanol increasedtheK-valuesforallfattyacidesters,regardlessofchainlengthordegreeofunsaturation.TheratiooftheK-valuesforCO2-ethanolandpureCO2rangedfromaround1.5forC14estersto3.1forC22esters,demonstrating that the solubilityenhancementachievedby theadditionofethanolincreaseswithincreasingchainlength.Thefluidselectivities,definedhereastheratiooftheK-valuesfortheC14:0estertothoseoftheothermixturecompo-nents,decreasedforallfattyacidestersupontheadditionofethanol.K-valuesforthefattyacidestersinpureCO2werealsomeasuredat333Kand13.1MPa.ThemeasuredpartitioncoefficientsinpureCO2undertheseconditionsyieldedK-valuesvery similar to those obtained using CO2-ethanol at 333 K and 12.5 MPa. Theyconcluded that ethanol serves no useful purpose as a cosolvent with CO2 for theconcentrationofEPAandDHAfromfattyacidestermixtures.
Althoughtheuseofcosolventsmaybebeneficial in termsof increasingsolu-bility, the complex nature of fish oils means their application may be limited intermsofselectivityenhancement.Theiruseshouldbeconsideredcarefullybecauseitcanincreasethecomplexityofprocessdesign.Anincreaseinsolventloadingmayresult in the coextraction of undesirable components. Also, using cosolvents canaffectmass transfer,greatly increaseprocessingcosts,andcanpotentially inducedegradation of the desired extract. For a more detailed discussion of binary/CO2andmulticomponent/CO2lipidsystems,thereaderisdirectedtothecriticalandin-depthreviewsofTemelliandGüçlü-Üstündag[98,102].Severalarticlescontainingcomprehensivetabulateddataonlipid/CO2systemsstudiedarealsoavailableintheliterature[98,116–118].
5.3.2.2 polyunsaturated Fatty acid processing
ThemainfocusofstudiesinvolvingSCFprocessingoffishoilshasbeentheisola-tionofω3-PUFAs,andinparticular,theisolationofEPAandDHA.Itiswellknown
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Processing of Fish Oils by Supercritical Fluids 169
thatonlymodestenrichmentsusingatriglyceridefeedstockcanbeachievedunlesstheoilisalreadyrichinDHA.Thecomplextriglyceridestructure,containingdiffer-entchainlengthacidsanddegreeofunsaturation,makesseparationstoachievecon-centrationoflong-chainPUFAsimpractical.Forthisreason,triglyceridesareeithersaponifiedorconvertedtofattyacidalkylesterspriortofractionation.TheresultantfattyacidsorestershavegreatersolubilityinCO2byvirtueoftheirlowermolecularweightandgreatervolatility[119].Somegroupshaveinvestigatedthefractionationofthefreefattyacidsbutthereporteddegreeofseparationwaslow.
SCFextraction/fractionationprocessesinvolvingethylestersarecarriedoutincontactingdevicescomprising twofluidphases:1)amoredense,ester-richphasecontainingdissolvedCO2and2)alessdense,CO2-richphaseinwhichsomeestersare dissolved. Separation processes exploit solubility differences of various feedcomponentsinthefluid-richphase.Eisenbach[120]carriedoutthefractionationofcodliveroilfattyacidalkylestersusingCO2inabatch-continuousprocess.Here,aquantityoffeedmaterialisloadedintothebaseofthefractionationcolumn,andCO2 is continuously passed through the column until the feedstock is depleted.The low-molecular-weight componentswith short chain lengths arepreferentiallyextracted.Toenhanceseparation,atemperaturegradientalongthecolumnwasusedtogenerate internalrefluxinthecolumncausedbyareductioninsolubility.Thiscausedthehigher-molecularweightcompoundstoprecipitateandindividualchain-lengthfractionswerecollectedinaseparatorasafunctionoftime.InEisenbach’sstudy[120],therefluxwasgeneratedbythepresenceofa“hotfinger”at363Kinthetopofthefractionationcolumn.Extractionswerecarriedoutat15.0MPausingaCO2flowrateof25Lhour–1.Theextractorandcolumntemperatureswere298and323K,respectively.Theestersremaininginsolutionexitedthetopofthecolumnandwerecollectedinaseparationvesselbypressurereduction.Usingthismethod,Eisenbachwasabletosuccessfullyseparatethefattyacidestersaccordingtocarbonnumber. He reported that a fraction containing C20 esters with greater than 95%puritywasobtained,consistingofaround13%ofthefeedmaterial,whichhadaninitialEPAcontentof14.5%.TheC20fattyacidesterfractionhadanEPAcontentof48.2%.
InalaterstudybyNilssonandcoworkers[121],abatch-continuousprocessforthefractionationofmenhadenoilwascarriedoutat15.2MPausingatemperaturegradientalongtheheightofthecolumn.Temperaturesinthecolumnvariedacrossfourtemperaturezonesfrom293Katthebottomto373Katthetop.Fractionationwascarriedoutwithandwithoutureacomplexationasapreconcentrationstep.TwoofthefeedmaterialsinvestigatedareshowninTable5.8.Fractionationofthewholeesters(Feed1)demonstratedthatsuccessfulseparationaccordingtocarbonnumbercouldbeachieved.Fractionsinexcessof60%puritywithrespecttoC20and90%puritywithrespecttoC22esterswereachievedwithEPAandDHAcontentsof51.9%(oftheC20fraction)and59.5%(oftheC22fraction),respectively.ThelowpurityoftheC20fractionemphasizedthedifficultyinseparatingtheC18andC20esters.Separationusingaurea-treatedfeed(Feed2)producedfractionsinexcessof80%puritywithrespect toC20estersandgreater than95%puritywithrespect toC22esters.Morethan95%of theC20 fractioncouldbeattributed toEPAandDHAaccounted foraround90%ofthetotalC22fraction.TheenhancementoftheoverallC20puritywas
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170 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
attributedtothefactthatmostoftheC18fattyacidestersintypicalmenhadenoilaresaturatesandmonounsaturates,whichwerepreferentiallyremovedduringureacomplexation.Thesemaximumenrichmentswereachievedusingasolvent-to-feedratio(S/F)ofaround475.TheS/Fhasasignificantimpactontheeconomicviabilityofaprocess;highS/Fvaluesleadtolengthyfractionationtimesand/orhighenergycosts.AreductioninS/Fcanbeachievedbyincreasingtheoperatingpressureordecreasingtheoperatingtemperature,attheexpenseofseparationperformance.An
table 5.8Composition of Feed materials used in study by nilsson et al. [121]
Feed 1a Feed 2b
Fatty acid Weight (%) of Fatty acid esters
14:0 7.8
16:0 15.6
16:1ω7 10.9 <1.0
16:3ω4 1.1 5.3
16:4ω1 1.5 5.8
18:0 3.1
18:1ω9 7.6
18:1ω7 3.1 <1.0
18:2ω6 1.3 <1.0
18:3ω3 1.6 <1.0
18:4ω3 2.9 7.6
20:1ω9 1.2
20:4ω6 1.0 1.4
20:4ω3 1.5 <1.0
20:5ω3(EPA) 16.5 48.6
21:5ω3 <1.0 1.3
22:5ω3 2.5 <1.0
22:6ω3(DHA) 10.9 22.2
total by Carbon number
C14 9.0 <1.0
C16 32.7 13.1
C18 21.3 9.7
C20(%EPA) 21.8(75.7) 50.5(96.2)
C22(%DHA) 14.5(75.2) 25.0(88.8)
Source: AdaptedwithpermissionfromJournal of the American Oil Chemists’ Society,65,109.©1988AmericanOilChemists’Society.
a Wholeesters,bUreafractionatedesters.
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Processing of Fish Oils by Supercritical Fluids 171
appropriatebalancebetweenproductyieldandS/Fshouldbeinvestigatedduringtheoptimizationprocess.
Asanextensiontothiswork,Nilssonetal.[122]employedanincreasingpres-sureprograminconjunctionwithatemperaturegradientforfractionationofurea-crystallizedfishoilethylesters.ThefeedsamplehadacompositionverysimilartothatgivenforFeed2inTable5.8.Usingseventemperaturezonesalongtheheightofthecolumn,rangingfrom313Kinthesecondzoneto353Katthetop(thebottomzone was at ambient temperature), and pressures ranging from 13.1 to 15.7 MPa(~6.9MPaincrements), theywereabletorecover85%ofEPAand88%ofDHAfromthefeedwithapurityof90%.ThemaximumenrichmentsinthisstudywereachievedusingaS/Fof340.TheauthorsconcludedthatatagivenpressuretheS/Fisprimarilygovernedbythetemperatureoftheuppermostzoneandisinsensitivetothelowerzonetemperaturesandflowrate.Increasingthetotalnumberoftem-peraturezonesandreducingthetemperatureatthetopofthecolumncanmaintainselectivitywhileloweringtheS/F.
Batch-continuousprocessingisinappropriateforlarge-scaleproductionbecauseprocessing costs are adversely affected by operating parameters, such as highS/Fvalues.Continuousprocessesaremoreefficient forproductionof largequan-tities of PUFAs. Krukonis [123] described a continuous countercurrent processfor fractionationof fattyacidestersand large-scaleproductionofEPAandDHAconcentrates.Inthisprocess,thefeedstreamiscontinuouslysuppliedtothetopofacolumn,whereitiscontactedbytheextractingsolventthatiscontinuouslyflowingintheoppositedirection.Thedirectionofflowdependsonthedensityofthetwofluids.Generally,forCO2countercurrentextractionoffishoils,theheavierfeedoilstreamflowsdownwardandthelighterCO2phaseflowsupward.Usingthestageconcept,Krukoniscalculatedthatatotalof13stageswererequiredtoobtainEPAof90%purityinthetopproduct(extractphase)andDHAofsimilarpurityintheraffinate.TheS/Fwassignificantlydecreasedtoaround30,whichismuchlowerthantheS/Ffor thebatch-continuousprocessesdescribedabove.Thecostofcarryingout thisprocesswasestimatedtobecomparabletotheproductionofω3fattyacidconcen-tratesusingconventionalmethods.
Riha and Brunner [124] investigated the continuous countercurrent fraction-ationofsardineoilFAEEswithsupercriticalCO2inthetemperatureandpressureranges313to353Kand6.5to19.5MPa,respectively.Theexperimentsfocusedonseparating low-molecular-weight components,with carbonnumbers ranging fromC14–C18,andhigh-molecular-weightcomponents(HMCs),C20–C22.AneconomicaloperationalpointwasdeterminedandtheprocesswasdeemedtobeusefulfortheenrichmentofHMCs,whichcouldbefurtherprocessedtoobtainfractionsrichinEPAandDHA.UsingaS/Fof63at353Kand19.5MPa,ina12mcolumnwithreflux(numberoftheoreticalplates=40),theHMCfractionswereobtainedingreaterthan95%puritywithgreaterthan95%yield.
ThePUFA-richfiltrateobtainedfromaureapreconcentrationstepalsocontainsuncomplexedurea.Thefattyacidsareusuallyrecoveredfromthefiltratebysolventextractionwithamixtureofanonpolarorganicsolvent,suchashexaneorisooctane,in which the urea is insoluble, and water in which the urea is soluble. The useof nonfood-grade organic solvents such as hexane in the extraction of PUFAs is
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172 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
undesirable,particularlyiftheproductisintendedfordietarysupplementation.Lossof PUFAs may occur during the reduction of organic solvent residues to regula-tory levels.ApatentbyCatchpoleet al. [125]describesacombinedsupercriticalextraction-supercriticalantisolventprocessforextractingawiderangeoflipophiliccompoundsfromurea-containingsolutionsusingnear-criticalfluids,suchaspoly-unsaturatedfattyacidsfromfishoils.Thenear-criticalfluidiscontinuouslycontactedwiththeureafiltratesolution.Thesolventpropertiesofthenear-criticalfluidenableextractionofthefattyacid/alkylestersfromthesolution(alongwithethanol)whileantisolventpropertiesresultinurea(andwater)beingprecipitated.Thefattyacidsand ethanol can then be recovered separately by consecutive pressure-reductionsteps. Figure5.16 [126] shows the results for a single-stage urea fractionation oftunaoil(initialEPAandDHAcontentof5%and22%,respectively),andadouble-stageureafractionationofhokiliveroil(initialEPAandDHAcontentof6%and9%,respectively).ThesupercriticalantisolventprocessiscarriedoutafterthefinalstageofureaprocessingandseparatesureaandoxidationproductsfromthedesiredPUFAs. Typical antisolvent fractionation conditions are 333 K and 30 MPa. Thefinalproductscontainaround40%to50%DHA,andanω3contentof60%(tunaoil)andgreaterthan90%(hokiliveroil).
Kulas and Breivik [127] describe a process in which PUFA ethyl esters areextractedfromsolidurea/ethylestercomplexes.Thecomplexeswereobtainedfromthepriorureafractionationoffishoilethylesters.TheauthorsnotethatthePUFAsareselectivelyextractedduetothelowstabilityofthecomplex,whilestronglyboundmonounsaturatesarenotextracted.Theyalsonotethatsomeureaisextracted.Itisalsopossibletousesolidureaasaselectiveadsorbentforfattyacidsoralkylestersand CO2 or CO2 + ethanol as the mobile phase [125, 128]. The same processingconsiderationsapplyasforureasolutions:saturatesaremoststronglybound,andthestrengthofthebondingistemperaturedependent.Figure5.17[129]showsthebatch-continuousfractionationofhydrolyzedlingliveroil.Afattyacid/ethanolmix-turewascontinuouslymixedwithCO2andthenpassedthroughtwobedspackedwith finely ground solid urea. The resultant extract was around 50% DHA and
60
50
40
30
20
Feed Oil 1st Stage Feed Oil 1st Stage
Hoki Liver OilTuna Oil
% of
Fat
ty A
cid
Type
2nd Stage
10
0
SaturatesMonounsaturatesEPA, C20:5DPA, C22:5DHA, C22:6
FIgure 5.16 Fattyacidextractcompositionforsingle-stageureafractionationoftunaoilanddouble-stageureafractionationofhokiliveroil.
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Processing of Fish Oils by Supercritical Fluids 173
greaterthan90%ω3-PUFAsuntilaratioofureatofattyacidofapproximately5:1wasreached.Theprocesswasdeemednotsuitableforcommercialscaleoperationbecausethesolidureacomplexcouldnotberegeneratedin-situandbecamerockhardandverydifficulttoremovefromextractionvesselbaskets.
Supercriticalfluidchromatography(SFC)canofferthesamedegreeoffattyacidseparationas thatofferedbySCFextractionofurea-concentrated feeds,butonlyif thestationaryphasecanspecifically interactwithdoublebonds.Higashidateetal.[130]enrichedEPAandDHAfromesterifiedsardineoil(initialEPAandDHAcontentsof12%and13%,respectively)byextractingtheoilwithsupercriticalCO2anddirectlyintroducingtheresultingsolutionontoasilicagelcolumncoatedwithsilvernitrate.Extractionswerecarriedoutat313Kand8MPawithaCO2flowrateof9gmin–1.Chromatographicseparationswerecarriedoutat313Kusingpressureprogramming.Fivefractionswerecollectedsequentially.Using thismethod, theyobtainedEPA-andDHA-richfractionswithpuritiesof93%and82%,respectively.ThechromatographicoperatingconditionsandfractioncompositionsaregiveninTable5.9.Theorderof elutionwas in accordancewith the interactionof carbon-carbondoublebondswithsilver ions; theinteractionincreaseswithanincreasingdegreeofunsaturation,resultinginlongerelutiontimes.
Pettinelloetal.[131]investigatedtheproductionofEPA-enrichedmixturesfromfishoilusingSFCatboth laboratoryandpilot scale.Their startingmixturecon-tained67.6%EPAethylesters(EE)and6.1%arachidonicacid(AA;20:4ω6)-EE,whichwerefractionatedusingasilicaadsorptioncolumnandsupercriticalCO2astheelutingsolvent.Sampleswereanalyzedusingcapillaryandpackedcolumngaschromatography(GC).Theyusedandcomparedseveraltypesofsilicagelandinvesti-gateddifferentprocessoperatingconditions.Itwasemphasizedthatsampleloadingshould be optimized because this can strongly influence the yield obtained. Twomodesofoperationwerecarriedoutatthelaboratoryscale:1)constanttemperature
100
80
60
40
20
00 50 100 150
Polyunsaturated
DHA
EPA
MonounsaturatedSaturated
g Oil/kg Urea
% Co
mpo
sitio
n of
Ext
ract
200 250 300
FIgure 5.17 Extractcompositionversusfattyacid–urearatioforbatch-continuousfrac-tionationofhydrolyzedlingliveroilusingCO2andpackedbedsoffinelygroundsolidureaat290Kand30MPa.
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174 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
withstepwiseincreasesinpressure(pressureprogramming)and2)constanttemper-atureandpressure.Duringthepressureprogrammingexperiments,thepressurewasraisedduringoperationinordertochangethesolubilityandselectivityoftheFAEEinsupercriticalCO2.Thepressurewasraisedfrom18.0MPato22.0or24.0MPaat343Kduringthechromatographicrun.Thelightercomponentswereelutedduringtheearlystagesoftheprocess,andtheEPA-EEremovalwasenhancedatthehigherpressures.Thepressureprogrammodeofoperationgave largeryieldsof agivenpurity(EPA-EE+AA-EEpurityof90%,yield49.0%),eveniftheconstantpressureoperation was carried out using a lower feed loading (EPA-EE + AA-EE purityof 90%, yield 10.0%). They reported that the selectivity obtained using pressureprogrammingwasnotsatisfactoryandsotheeffectoftemperatureonselectivitywasalsoinvestigated.Thetemperaturewasraisedto353Kandanoperatingpressureof20.8MPawasusedtogivethesameCO2fluiddensityasthatat343Kand18.0MPa.AreductioninretentiontimeandasharpeningoftheGCpeakwasnoted,indicatingthatsilicagelhasaweakerinteractionwithFAEEmoleculesathighertemperatures.A95%EPApuritywasachieved,withayieldof11%.ThepurityofEPAdroppedto90%whentheyieldwasincreasedto43%.
In the pilot-scale operation, Pettinello and coworkers [131] used a system inwhichtheCO2wasrecycled.Quantitiesoffeedmaterialsoftheorderofhundredsofgramswereprocessed.Thechromatographicfractionationswerecarriedoutusingtwotypesofsilicagel(75to200µm,60Åporesizeand75to200µm,40Åporesize),and theresearchersfoundthat thebehaviorofbothwassimilar in termsofseparationperformance.Intermsofeconomics,fortheprocesstobeusefulonanindustrialscale,itisimportantthatthesilicaadsorbentcanbeusedseveraltimes.Theresearchersshowed,usingthreesuccessiverunswiththesamecolumnofsilica,thatan84%pureEPAfractioncouldbeobtainedwithyieldsof34.3%,29.8%,and28.2%forruns1,2,and3,respectively.Pettinelloetal.[131]statedthataftertheinitialdeactivationofsilicaduringthefirstrun,theadsorbentmaterialmaintainedanacceptablelevelofactivity.Theeffectoffeedmass,pressure,andpressurepro-grammingwerealsoinvestigatedduringpilot-scaleoperation.TheEPA-EEpuritywasgreatestat93%with24.6%yieldwhenusingafeedmassof275.0g,atempera-tureof343K,andapressureprogrammingmethodintherange15.0to24.0MPa.
ApatentbyPerrutetal.[132]describesusingSFCforfishoilprocessinginacontinuous mode of operation by applying simulated countercurrent moving bed
table 5.9Chromatographic operating Conditions and Fraction Compositions from the Work of higashidate et al. [130]Fraction pressure (mpa) elution time (mins) % epa % dha
1 8 0–110 0 0
2 8 110–180 57 0
3 12 180–250 93 0
4 20 250–310 46 18
5 20 310–370 0 82
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Processing of Fish Oils by Supercritical Fluids 175
(SCMB)technology.Theauthorsclaimedthata93%EPA-EE-richfractionandan85%DHA-EE-richfractionwereachievedusingthecombinedSFC-SCMBmethod.In the first step, a starting mixture containing 32.8% EPA and 20.9% DHA waspassedthroughaSFCcolumn(300×60mm)at323Kand16.0MPausingRPC-18(octadecyl silica gel, 12 to 45 µm) as a stationary phase and a CO2 flow rate of40kghour–1.Theyobtaineda73.6%EPA-EE-richfractionwithayieldof67.1%(thisEPA-richfractioncontained15.1%DHA).TheDHA-EE-richfractionwasobtainedwith56.3%puritywithyield76.1%(thisDHA-richfractioncontained35.4%EPA).In a second step, these fractions were reprocessed using eight RPC-18 columns(100×80mm)at323Kand13.0MPawithaCO2flowrateof55kghour–1.Inthesecondstep,thetotalyieldsforbothEPAandDHAwere99%andthefractionpuri-tiesincreasedto93%and85%forEPA-EEandDHA-EE,respectively.
Alkioandcoworkers[133]studiedtheeconomicfeasibilityofproducinglargequantitiesofEPAandDHAfromtunaoilusingSFC.UsingsupercriticalCO2asthe mobile phase, they carried out a systematic study to find optimum processparametersformaximumproductionrate.At338Kand14.5MPa,usingoctadecylsilane-typereversed-phasesilicaas thestationaryphase,DHAandEPAcouldbeproducedsimultaneouslyinonechromatographicstepwithpuritiesof80%to95%and50%,respectively.Aprocessforproducing1,000kgofDHAand410kgofEPAconcentrateperyearrequires160kgofstationaryphaseand2.6tonshour–1ofCO2recycle.Theystated that thestationaryphasewouldpreferablybepacked in fourparallel600mm i.d. columnsand theestimatedcostof suchaplantwasaroundU.S.$2million.Assumingthatthestationaryphasewouldhavetobereplacedonceayear,theprocessoperatingcostswerecalculatedtobeU.S.$550perkilogramofDHAandEPAconcentrate(calculationswerebasedonmaterialandoperatingcostsfortheyear2000).
Enzymaticmethodsofenrichmenthavealsobeeninvestigatedundersupercriticalconditions.Linetal.[134]studiedtheenrichmentofω3-PUFAcontentinTAGsofmenhadenoilby lipase-catalyzed transesterification in supercriticalCO2.Prior toreaction,menhadenoilwastreatedbytheureainclusionmethodtoproducean80.1%ω3-PUFAconcentrate,71.2%ofwhichwasEPA+DHA.Using the immobilized1,3-regiospecific lipase, IM60fromMucor miehei, theauthorsstudied theeffectsofseveraloperatingparametersonthereaction,includingcosolventconcentration,reactiontime,temperature,pressure,andsubstrateratio(freeω3-PUFA:TAG).Forallreactions,theenzymeconcentrationwaskeptat10%w/wofthetotalsubstrates.Bothwater and ethanolwere examined as cosolvents andbothfluids exhibited amaximumforω3-PUFAcontentinTAGsasafunctionofconcentration.Thereac-tionwasstudiedwithawatercontentrangingfrom0%to10%w/wandamaximumω3-PUFAcontentof46%wasobservedat4%watercontent.Asimilartrendwasobservedforethanol(studiedintherange0%to15%w/w)withamaximumω3-PUFAcontentof56%beingobtainedat10%w/w.Following thisobservation, allotherreactionswerecarriedoutusing10%w/wethanolasacosolvent.Foraprocesstobeeconomicallyviable,optimumresultsmustbeobtainedwithintheshortesttime-frame.At323Kandpressuresrangingfrom10.3to20.7MPa,thetotalcontentofω3-PUFAsinTAGsincreasedwithtimeupto5hours,irrespectiveofpressure.Thetemperatureeffectonreactionwasinvestigatedintherange313to333K,anditwas
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176 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
found that theω3-PUFAcontent inTAGs increasedwith increasing temperature.However,itshouldbenotedthatelevatedtemperaturesshouldbeusedwithcautionbecausemostproteindenaturationoccursat318to323K.Also,Kametetal.[135]notedthatbelow313K,CO2reactsreadilywiththefreeaminogroupontheenzymesurfacetoformacarbamate-enzymecomplex,whichreducesenzymeactivity.Thetotalω3-PUFAcontentinTAGsdecreasedwithincreasingpressure.Twopossibili-tieswereofferedtoexplainthisphenomenon:1)Thesuppressionofthethree-dimen-sionalmolecularstructureoftheactivesiteontheenzymeleadstoareductionofenzymeactivity;2)Atelevatedpressures,ahigherdissolutionofCO2intothewateronthesurfaceoftheenzymecausesadecreaseinpHandinducesthereversereac-tion.Theω3-PUFAcontentinTAGsalsoincreasedasthefreeω3-PUFA:TAGratioincreased.Underoptimumconditions(10%ethanol,5hourreactiontime,10.3MPa,323K,andasubstrateratioof4:1),theauthorswereabletoproduceTAGscontain-ing56%w/wofω3-PUFAsusingthismethod.
5.3.2.3 squalene and dage processing
Catchpole andcoworkers [101,136,137] investigated the continuousextractionofsqualenefromsharkliveroilinlaboratoryscale(5mLmin–1oilprocessingcapability)pilotscale(30mLmin–1oil)andproductionscale(1Lmin–1oil)packedcolumnplantandalaboratoryandpilotscalestaticmixerapparatususingsupercriticalCO2.Sepa-rationperformancewasdeterminedasafunctionoftemperature,pressure,oil-to-CO2flowrateratio,packedheight,staticmixerdimensions,packingtype,andrefluxratio.The initial shark liver oil contained 50% by weight squalene and 0.1% by weightpristane(C19H40),withthebalancebeingnonvolatiletriglyceridesandglycerylethers.ThepilotscalepackedcolumnapparatusisshowninFigure5.18.ThebasicapparatusconsistedofaCO2compressor,a2.5m×56mmi.d.packedcolumn,highpressurepistonpumpsforsupplyoftheliquidsharkliveroilandliquidreflux,andtwojack-eted separationvessels for the recoveryof squalene,fishodors, andpristane.CO2waspassedupwardthroughthecolumnatoperatingpressure.Thesharkliveroilwaspumpedintothetopofthefirst(withnoreflux)orsecond(withreflux)sectionofthecolumn.Therefluxliquidwaspumpedintothetopofthefirstsection.Theraffinate,whichishighlyenrichedinDAGEsandTAGsandstrippedofsqualene,wascollectedat regular timeintervals fromthebottomof thecolumn.TheCO2solutionpassedthroughapressurereductionvalveintothefirstseparationvessel,wherethebulkofthesqualenewasrecovered.Thefishodorsandpristanewererecoveredinthesecondseparationvesselbyfurtherpressurereduction,andCO2wasrecycled.Theproduc-tionscaleplant(Figure5.19)wasoperatedinasimilarmanner[101].Thelaboratoryscaleexperimentsalsousedthesamemethodology,althoughCO2wasnotrecycled.Thetotaloilloadingwasinvestigatedasafunctionofoil-to-CO2ratio(theratioofthetotalmassoftopproductfromthecolumntotheCO2massthathaspassedthroughthecolumnoveragiventime)overthetemperaturesandpressures313to333Kand12.5to25.0MPa,respectively.Investigationswerecarriedoutwithoutrefluxatfixedtemperatureandwithinternalrefluxusingatemperaturegradientovertheheightofthecolumnintherange313to333K.Theloadingfollowedthepatternofsqualenesolubility,withthehighestvaluesobtainedat333Kand25.0MPa,andthelowestat
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Processing of Fish Oils by Supercritical Fluids 177
313Kand20.0MPa.Pristanewasthemostsolubleoilcomponentandwasvirtuallycompletelyextractedfromthefeedoil,evenathighoil-to-CO2massratios.Pristaneisaskinirritantandisthusundesirableinsqualenethatisdestinedforuseincosmeticapplications.Separatorconditionswereoptimizedtomaximizetherecoveryofsqua-leneinthefirstseparator(313Kand9.0MPa),andmaximizingpristanerecoveryinthesecondseparator(313Kand6.0MPa).
Theeffectofpackingtype,scaleofoperation,andcountercurrentversuscocur-rentcontactingonmass transferefficiencywas investigated.Stainless steelwool,Raschigrings,andFenskeheliceswereinvestigatedaspackingsacrossarangeofpackedheightsatalaboratoryscale.NoreliableresultsusingFenskeheliceswereachievedduetoexcessivehold-upofliquidinthecolumn.Raschigringsgavepoorermass transfer performance than stainless steel wool. Other researchers have alsofoundthatRaschigringscomparedpoorlywithwirewooltypepackingsforlipid/near-criticalfluidpackedcolumnseparations[138,139].Theperformanceofwirewool also diminished significantly when only one packed section was used. Theauthorsconcludedthatatleast0.8mofpackingwasrequiredtoachieveahighlevelofseparationofsqualenefromtriglyceridesatalaboratoryscale,and2.5matapilotscale.Thepackedheightavailableinthedemonstrationscaleplantwasinsufficienttoachievemass transfersignificantlybetter thanastaticmixer,whichgivesonlyoneequilibriumstage(Figure5.20).Underoptimumconditionsatpilotscale,a92%
P P
P
P
P
P
H1
Air
CO2 Supply Cylinders
Cooling Water
Raffinate Squalene
Separation Vessel 1
Separation Vessel 2
RefluxPumpPiston
Pump Liquid Feed
Tank
Pristane
Hot Water System
Hot Water System
H2
H3
FIgure 5.18 ThepilotscalepackedcolumnapparatususedbyCatchpoleetal.[101,136,137]forthefractionationoffishoils.(ReprintedwithpermissionfromIndustrial and Engi-neering Chemistry Research,36,4318.©1997AmericanChemicalSociety.)
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178 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
squalenebymassproductwasobtainedinseparator1atcolumnconditionsof333Kand25.0MPa.Usingthe92%squalenefractionasarefluxfeedstock,experimentswereperformedwithafixedratiooffeedoiltoCO2usingarangeofrefluxpumprates.Increasingtherefluxtofeedoilmassflowratiocausedboththetopproductstreamloadingandconcentrationofsqualeneinthetopproducttoincrease,asshowninFigure5.21.TheoilloadingincreasedlinearlytowardtheequilibriumloadingofpuresqualeneinCO2,withincreasingreflux-to-feedratio.SincethemassflowofthefeedoilandCO2wasfixed,therewasalsoanincreaseofsqualeneconcentrationintheraffinatewithincreasingrefluxratio.Toachieveoptimumseparationperfor-manceintermsofproductpurityandlossinraffinate,itisdesirabletouserefluxofthetopproductwithaloweredfeedrateofsharkliveroilatfixedCO2flowrate.Pilot-scaleoperationunderoptimumprocessconditionswithrefluxyieldeda99%bymasssqualene-richfractioncontaininglessthan0.5%pristane,withalossoflessthan5%bymassofsqualeneintheraffinatestream.
5.3.2.4 Vitamin a processing
The processing of fish oils to recover fractions enriched in vitamin A was firstcarriedoutusingnear-criticalpropane [140].Amulticolumnprocesswasused to
FIgure 5.19 Production scaleplantused for fractionationof squalene fromshark liveroil[101].
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Processing of Fish Oils by Supercritical Fluids 179
100
80
60
40
20
00 1 2
Extract
L/GK, Ratio of Flow Rates and Equilibrium Coefficient
% Sq
uale
ne in
Ext
ract
and
Raffi
nate
Raffinate
‒ Lab Scale Static Mixer‒ Lab Scale Packed Column‒ Demo Scale Packed Column
3 4 5
FIgure 5.20 Extractandraffinateconcentrationsofsqualeneforstaticmixer,laboratoryscale,anddemonstrationscalecolumnsversusmodifiedoil-to-CO2flowrateratio.
100 28
26
24
22
20
18
16
80
60
Squa
lene
Con
cent
ratio
n, %
by M
ass
Oil
Load
ing,
g /k
g of
CO
2
40
20
00.00 0.05 0.10 0.15
Product Squalene ConcentrationRaffinate Squalene ConcentrationOil Loading
Reflux to Feed Oil Mass Ratio0.20 0.25 0.30 0.35 0.40
FIgure 5.21 Squalene concentration in the top product and oil loading in CO2 as afunctionofrefluxtofeedmassratio[136].(ReprintedwithpermissionfromIndustrial and Engineering Chemistry Research,36,4318.©1997AmericanChemicalSociety.)
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180 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
fractionatemenhadenandcodliveroilsbyutilizingdifferencesinsolubilityofoilcomponents near to, but below, the critical point. In the first column, conditionsarechosensuch that theoil ismiscibleexcept forcolorcomponentsandnonlipidcontaminants.Insubsequentcolumns,thesolubilityisprogressivelyreducedtogivefractionsenriched inpolyunsaturatesuntil thefinal column,wherein thevitaminA-rich(mostsoluble)fractionisrecovered,isreached.ThisprocessfelloutoffavorwhensyntheticvitaminAataneconomicallycompetitivepricebecameavailable.
Catchpoleetal.[101]carriedoutthecountercurrentextractionofvitaminAfrommodelfishoilmixturesusingsupercriticalCO2.ThemodelmixturesweremadetosimulatetheliveroilsofsurfacedwellingsharksthathavehighvitaminAcontents[141].VitaminApalmitateisthepredominantformofvitaminAinfishoils[142].TheremainderofthesharkliveroilisaTAGformthathassimilarfattyacidcompo-sitionstocodliveroil[143,144].Therefore,separationswerecarriedoutusingcodliveroilandvitaminApalmitatemixtures,withvitaminAconcentrationsrangingfrom1%to20%bymass.Theseparationfactorwaslowduetosimilarsolubilitiesofthevitaminesterandthenon-esterifiedoil.ExtractionswerealsocarriedoutusingmixturesofcodliveroilethylestersandvitaminAasthefreealcohol.Thesolubil-ityofthefreealcoholismuchlowerthanFAEEsatlowtomoderatepressures(9to12MPa),andvitaminAwaspreferentiallyrecoveredintheraffinate.Theresultsofthefractionationexperimentsforcodliveroil/vitaminApalmitateandcodliveroilethyl esters/vitaminAare shown inFigure5.22.TheconcentrationofvitaminApalmitate(codliveroil)intheextractwasenhancedoverthatoftheraffinateandtheenhancementwasnotstronglypressuredependent.Highlossesofthevitaminester
50
Cod Liver Esters
45
40
35
Extr
act V
itam
in C
once
ntra
tion,
% b
y Mas
s
Extr
act V
itam
in C
once
ntra
tion,
% b
y Mas
s
30
25
20
15
10
5
0 5 10 15Raffinate Vitamin Concentration, % by Mass
20 25
1.00
0.80
0.60
0.40
0.20
0.00
2 %, 313 K, 9.5 MPa2 %, 313 K, 10.0 MPa1.8 %, 333 K, 13.0 MPa4.2 %, 333 K, 13.5 MPa
Cod Liver Oil5 %, 27.5 MPa5 %, 30.0 MPa20 %, 25.0 MPa20 %, 27.5 MPa20 %, 30.0 MPa
0
FIgure 5.22 FractionationofvitaminA/codliveroilandethylestermixtures[101].Filledsymbols:⦁,◾,▲,▼,♦ExtractionofvitaminApalmitatefromcodliveroil;hollowsymbols:•,◽, , ExtractionofvitaminAfromfattyacidethylesters.(ReprintedfromJournal of Supercritical Fluids,19,25,©2000.WithpermissionfromElsevier.)
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Processing of Fish Oils by Supercritical Fluids 181
occurred in the raffinate. Increasing the extract to raffinate ratio can reduce thisloss,butthisresultsinlowerextractconcentrations.Theseparationwassubstantiallyimprovedwhenusingcodliveroilethylesters.ThemajorityoffattyacidesterswereextractedfromthefeedtoleavearaffinateenrichedinvitaminA.Theconcentrationofthevitaminintheextractdidnotexceed0.5%.
5.3.2.5 processing of other marine oil Components
Waxesteroilswereprocessedusingthepilotscalesupercriticalpilotplantdescribedearlier for theprocessingof squalene from shark liveroil [101].Thefishoilwaspartially degraded due to extended storage at room temperature and had highperoxidelevels.CO2+ethanolwasusedasthesolvent,whichwascountercurrentlycontactedwiththewaxesteroil.Ahighoil-to-solventratiocouldbeusedduetothehighsolubilityoftheoilmixtureinthesolventphase.High-molecular-weightestersand astaxanthin were recovered in the raffinate, and medium-molecular-weightesterswererecoveredinthefirstseparator,alongwithpartoftheethanol.Theover-allextracthadveryhighperoxidevalues.Theextractmixtureseparatedinto twophases,atopwaxester-richphase,andabottomethanol-richphase,whichalsocon-tainedmostoftheperoxidesandmalodorouscompoundspresentintheoriginaloil.Thefinalseparatorcontainedlargelyethanol,withvolatileodorcompounds.
Theextractionofgreen-lippedmusseloil,soldunderthenameLyprinol™,usingsupercriticalCO2hasbeencarriedoutcommerciallyforseveralyears.ThemusselsareendemictoNewZealand.Theoilhasanti-inflammatorypropertiesandhasfoundappli-cationasanantiarthriticandantiasthmaticnaturalremedy.TheextractionprocessandpropertiesoftheextractaredescribedinapatentbyMacridesandKalafatis[145].Theoilisacomplexmixtureoffreefattyacids,TAGs,sterols,andsterolesters[146].
5.4 summary
Theconventionalmethods for isolationofhigh-valuefishoil components includevacuumdistillation,ureacrystallization,hexaneextraction,andconventionalcrys-tallization. These methods have the disadvantages of requiring high processingtemperatures,resultinginthethermaldegradationordecompositionofthethermallylabile compounds or employing flammable or toxic solvents, which have adversehealtheffects.Inthisinstance,separationsemployingSCFtechnologiesoffernewopportunitiesforresolvingtheseseparationproblems.
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Processing of Fish Oils by Supercritical Fluids 187
115. Nilsson, W.B., Seaborn, G.T. and Hudson, J.K., Partition coefficients for fatty acidestersinsupercriticalfluidCO2withandwithoutethanol,J.Am.Oil Chem.Soc.,69,305,1992.
116. Staby, A. and Mollerup, J., Separation of constituents of fish oil using supercriticalfluids:Areviewofexperimentalsolubility,extraction,andchromatographicdata,Fluid Phase Equilib.,91,349,1993.
117. Brunner,G.andDohrn,R.,High-pressurefluidphaseequilibria:Experimentalmethodsandsystemsinvestigated(1988–1993),Fluid Phase Equilib.,106,213,1995.
118. Christov,M.andDohrn,R.,High-pressurefluidphaseequilibria:Experimentalmethodsandsystemsinvestigated(1994–1999),Fluid Phase Equilib.,202,153,2002.
119. Krukonis,V.J.,Supercriticalfluidfractionationoffishoils.Concentrationsofeicosapen-taenoicacid,J.Am.OilChem.Soc.,61,698,1984.
120. Eisenbach,W.,Supercriticalfluidextraction:Afilmdemonstration,Ber.Bunsenges.Phys.Chem.,88,882,1984.
121. Nilsson,W.B.etal.,FractionationofmenhadenoilethylestersusingsupercriticalfluidCO2,J.Am.Oil Chem.Soc.,65,109,1988.
122. Nilsson,W.B.,Gauglitz,E.J.,Jr.andHudson,J.K.,Supercriticalfluidfractionationoffishoil estersusing incremental pressureprogramming anda temperaturegradient,J.Am.Oil Chem.Soc.,66,1596,1989.
123. Krukonis, V.J., Processing with supercritical fluids: Overview and applications, inSupercritical Fluid Extraction and Chromatography: Techniques and Applications,ACS Symposium Series number 366, Charpentier, B.A. and Sevenants, M.R., Eds.,AmericanChemicalSociety,Chicago,1988,26.
124. Riha,V.andBrunner,G.,Separationoffishoilethylesterswithsupercriticalcarbondioxide,J.Supercrit.Fluids,17,55,2000.
125. Catchpole,O.J.,MacKenzie,A.D.andGrey,J.B.,PatentWO03/089399,US2006035350,2003.
126. Catchpole,O.J.etal.,Unpublisheddata. 127. Kulas,E.andBreivik,H.,PatentWO01/10809,US6528669,2001.128. Hiroshi,U.andHiroshi,S.,PatentJP60214757,1985.129. Catchpole,O.J.etal.,Unpublisheddata.130. Higashidate,S.,Yamauchi,Y.andSaito,M.,Enrichmentofeicosapentaenoicacidand
docosahexaenoicacidestersfromesterifiedfishoilbyprogrammedextraction-elutionwithsupercriticalcarbondioxide,J.Chromatogr.,515,295,1990.
131. Pettinello,G.etal.,ProductionofEPAenrichedmixturesbysupercriticalfluidchroma-tography:fromthelaboratoryscaletothepilotplant,J.Supercrit.Fluids,19,51,2000.
132. Perrut,M.,Nicoud,R.M.andBreivik,H.,U.S.Patent5719302,1998.133. Alkio,M.etal.,Purificationofpolyunsaturated fattyacidesters fromtunaoilwith
supercriticalfluidchromatography,J.Am.Oil Chem.Soc.,77,315,2000.134. Lin,T-J.,Chen,S-W.andChang,A-C.,Enrichmentofn-3PUFAcontentsontriglycer-
idesoffishoilbylipase-catalyzedtrans-esterificationundersupercriticalconditions,Biochem.Eng.J.,29,27,2006.
135. Kamet,S.etal.,Biocatalyticsynthesisofacrylatesinorganicsolventsandsupercriticalfluids.III.Doescarbondioxidecovalentlymodifyenzymes?,Biotechnol.Bioeng.,46,610,1995.
136. Catchpole,O.J.,vonKamp,J-C.andGrey,J.B.,Extractionofsqualenefromsharkliveroilinapackedcolumnusingsupercriticalcarbondioxide,Ind.Eng.Chem.Res.,36,4318,1997.
137. Catchpole,O.J.etal.,Fractionationoflipidsinastaticmixerandpackedcolumnusingsupercriticalcarbondioxide,Ind.Eng.Chem.Res.,39,4820,2000.
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188 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
138. Czech,B.andPeter,S.,Efficiencyofdifferentpackingsincounter-currentnear-criticalfluid extraction, in Pre-prints of High Pressure Chemical Engineering, 2nd Inter-nationalSymposium, DECHEMA-GVC,1990,419.
139. Böhm, F. et al., Design, construction, and operation of a multipurpose plant forcommercial supercritical gas extraction, in ACS Symposium Series, Vol. 406,Johnston,K.P.andPenninger,J.M.L.,Eds.,AmericanChemicalSociety,WashingtonD.C.,1989,502.
140. Passino,H.J.,Thesolexolprocess,Ind.Eng.Chem.,41,280,1949. 141. Shortland,F.B.,TheaquaticanimaloilresourcesofNewZealand,N.Z.J.Sci.Technol.,
2,30,1950.142. Winholz,M.,The Merck Index,Vol.9,Merck,NewYork,1976,1133.143. Hilditch, T.P. and Williams, P.N., The Chemical Composition of Natural Fats,
ChapmanHall,London,1964.144. Vlieg,P.andBody,D.R.,LipidcontentsandfattyacidcompositionofsomeNewZealand
freshwaterfinfishandmarinefinfish, shellfish,and roes,N.Z.J.Marine Freshwater Res.,22,151,1988.
145. Macrides,T.andKalafatis,N.,U.S.Patent6,083,536,2000.146. Wolyniak, C.J. et al., Gas chromatography-chemical ionization-mass spectrometric
fatty acid analysis of a commercial supercritical carbon dioxide lipid extract fromNewZealandgreen-lippedmussel(Perna canaliculus),Lipids,40,3552005.
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189
6 Supercritical Fluid Extraction of Active Compounds from Algae
Rui L. Mendes
Contents
6.1 Introduction................................................................................................. 1896.2 SupercriticalFluidExtractionfromAlgae................................................. 191
6.2.1 Botryococcus Braunii...................................................................... 1916.2.2 Chlorella Vulgaris........................................................................... 1936.2.3 Dunaliella........................................................................................ 1966.2.4 Haematococcus pluvialis................................................................. 1986.2.5 Hypnea charoides............................................................................ 2016.2.6 Nannochloropsis..............................................................................2026.2.7 Spirulina (Arthrospira)....................................................................205
6.2.7.1 Spirulina maxima...............................................................2056.2.7.2 Spirulina platensis.............................................................207
6.3 Conclusion...................................................................................................209References..............................................................................................................209
6.1 IntroduCtIon
Microalgaearealltheeukaryoticphotosyntheticmicroorganisms,whichpresentagreatgeneticvariety.Someauthorsincludeinthisdefinitionprokaryoticphotosyn-theticorganisms[1],suchasthecyanobacteria.Phytoplankton,whichcomprisetheautotrophicprokaryoticandeukaryoticmicroorganismssuspendednear thewatersurface,arethebaseoftheaquaticlifefoodchain.
Morethan50,000speciesofmicroalgaearesupposedtoexist,butonlyabout50havebeenwellstudiedandamuchlowernumberhasbeencultivatedinlargescale[2].Microalgaeproduceagreatvarietyofsecondarymetabolites,whicharesynthesizedattheendofgrowthphaseandatthestationaryone.Theunlimitedstructuraldiver-sityofthesecompoundscanstillbeenlargedapplyingtechniquesofcombinatorialchemistry[3]. The bioactive molecules produced by these microorganisms can bebeneficialorharmful,butevenphycotoxinsandrelatedproductsmayserveasmaterialsforusefuldrugs[4].Ontheotherhand,untilnow,microalgaehadnotbeenusedmuchfortheproductionofchemicalsandbiochemicals,althoughsome,suchassomepoly-unsaturatedfattyacids(PUFAs),arethegreatestreserveofthebiosphere[5].
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190 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
Infact,microalgaecanproduce, insignificantamounts, lipidssimilar toveg-etableoils,fuels,proteins,andessentialfattyacids,withdietaryapplications,suchaslinoleic,g-linolenic,eicosapentaenoic,docosahexaenoic,andarachidonicacids;vitamins(β-carotene,B12,andE);pigments(carotenoids,phycobiliproteins),waxes,biosurfactants, sterols, andother chemical specialties [6–8].Reviews focusingoncommercial aspects of microalgae biotechnology have recently appeared [9, 10].Figure6.1summarizesthemainapplicationsofmicroalgae[11].
Themarinemacroalgae,alsoknownasseaweed,havebeenusedashumanfoodandfertilizersandtoobtainagar,alginicacid,andcarrageenan,amongotheruses.In this field, the discovery of metabolites with biological activity has increasedsignificantly in the last threedecades [12].Thepharmaceutical industryhaspri-marilyfocuseditsattentiononthefollowingsubstances:sulphatedpolysaccharidesas antiviral substances, halogenated furanones as antifouling compounds, andkahalalideFasapossibletreatmentoflungcancer,tumors,andacquiredimmuno-deficiencysyndrome[12].
The lipid content of microalgae can reach 85% (dry weight basis), althoughvaluesbetween20%and40%aremoretypical[7].Theproductionyieldofthesecompoundsreliesontheconditionsofculture,withtheamountofnitrogenbeingoneofthemainfactorstocontrollipidcontent[5].Thefinalyieldalsodependsontheextractionmethod,degreeofcrushing,andtypeofsolventsused.
Themostimportantfeatureofalgaeistheirphotosyntheticcapacity.Therefore,thecultureofmicroalgaeisusuallymadeinopenponds,butthiscanleadtotheircontamination and to diseases, restricting this method to species that are more
Micro Algae
Food Nutraceutical, Functional
Food,Food Additives (Emulsifier,
Thickner), Sweets
Commercial Products
Hydrocarbons (Fuel) Adsorbents
Enzymes and Other Reserach Materials
Colorants Food (Ice creams, Jellies. Confectionaries, Juices)
Cosmetics (Lipsticks, Creams,
Lotions)
Pharmaceutical Products
Pharmaceutical Antibiotic, Antibacterial, Bulking Agent, Binder, Hard and Soft Capsule
Shell, Thickener Diagnostic Agents
FIgure 6.1 Microalgae applications in various fields. (Source: Adapted from Dufosséetal.,Trends in Food Science & Technology,16,389,2005.Withpermission.)
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Supercritical Fluid Extraction of Active Compounds from Algae 191
resistant, such as Dunaliella, Spirulina, and Chlorella [13]. Dunaliella salina iscultivatedinpondswithhighlevelsofsalt,whereasSpirulina canbecultivatedinhighlyalkalinewater.Ontheotherhand,thecostsassociatedwithharvestingthealgae(whichcan includemicroscreen,centrifugation,orflocculation)canpreventtheeconomicsuccessofthisprocess.
Geneticengineeringoffersthepossibilityofconvertingphotosyntheticmicro-algaeabletoproduceautotrophicallychemicalspecialtiesintoonesabletoproducethemheterotrophically[14].Theuseoffermentersandphotobioreactors(whichcanbetanksprovidedwithalightsource,polyethylenebags,plasticorglasstubes,etc.)fortheselectiveproductionofparticularcompoundsisapromisingmethodforthecultureofmicroalgae.Thefutureofthistechnologyalsopassesbytheuseofmixo-trophiccultures[15].Geneticengineeringhasalsobeenusedtocreateoverproduc-ingstrains,aconditionthathelpssomespeciesbemorecompetitive,aswellastomodifytheminordertoobtainspecificcompounds[16,17].
Supercriticalfluidextraction(SFE)ofthistypeofcompoundhassomeadvan-tages over the conventional methods. These compounds can be obtained withoutthermaldegradationandwithoutsolvents,thereforethereareneitherissuesrelatedto the toxicityof theorganic solventsusednor the legal restrictionsof theiruse.Moreover,withSFEitispossibletoobtainahighefficiencyofextraction,ashort-ened extraction time, and, in some cases, a higher yield. On the other hand, theselectivityforcertaincompoundsismuchhigherwithSFEthanwithorganicextrac-tion.Generallyallalgaehasaremainingbiomassafterlipidextractionthatconsistsmainlyofprotein(about50%)andcarbohydrates.Theorganicsolventextractioncanleadtodenaturationoftheproteins,unliketheSFE,whatwouldbedetrimentaltoitsuseinfoodorfeedapplications[18,19].
Someofthemoreinterestingearlystudiesofsupercriticalcarbondioxide(CO2)extractionofcompoundsfromalgaewerecarriedoutusingScenedesmus obliquus [20],Dilophus ligulatus(abrownmacroalga)[21],Dunaliella salina[22],Skeleto-nema costatum [46], and Ochronomas danica [46], with the objective of obtain-ing fatty acids, several secondary metabolites, eicosapentaenoic acid (EPA), andβ-carotene, respectively. Also, some reviews on this field have appeared[23–25].Table6.1showsacollectionofresearchliteratureonseveralmacroalgaeandmicro-algae (including also other microorganisms, such as fungi and yeasts), and therespectivetargetcompounds,whichhavebeentheobjectofSFE.
TheaimofthischapteristoreviewinsomedetailthestudiesofSFEtoobtainseveralactivecompoundsfromsomeofthemostimportantmicroalgaeandalsosomeseaweedspecies,focusingonthetypesofcompoundsextracted,comparisonwiththeconventionalmethodstoobtainthem,andthemostrelevantaspectsoftheirSFE.
6.2 superCrItICal FluId extraCtIon From algae
6.2.1 Botryococcus Braunii
Botryococcus braunii is a species of green microalgae that live in large naturalcolonies, either in salt water or freshwater, in temperate and tropical regions. Itpresents a unique particularity among the known photosynthetic microorganisms
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192 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
becauseitscontentinhydrocarbonscanreach85%ofthedrybiomass.Thesealgaehavealreadybeenproposedasafuturerenewablesourceoffuel[51].Thelocaliza-tionofthehydrocarbonsisextracellular.Eachcellissurroundedbyseveralexternalwalls, where most of the hydrocarbons accumulate in globular formations. Theremaininghydrocarbons(about5%)arelocatedinthecytoplasm[52].
Race A of these algae produces and accumulates linear alkadienes, C25–C31,withanoddnumberofcarbonatomsandthetrieneC29H54[53].Thesecompounds,afterhydrogenationorfunctionalization,canbeusedassubstitutesforparaffinicandnaturalwaxes,withcosmeticandpharmaceuticalapplications[54].
SupercriticalCO2studieswerecarriedoutat40ºCandpressuresof125,200,and300bar[33].Figure6.2showsthecumulativecurvesofthehydrocarbonextrac-tionyieldversusthehexaneextraction.Themaximumextractionyieldsofhydro-carbonsobtainedwere76g/kg(dryalgaebasis)and72g/kgforhexaneandSFEextractions,respectively.
About95%oftheextracellularhydrocarbonswererapidlyextractedat300bar.Moreover,thesupercriticalextractswerelimpidandgoldenduetothenonextractionofchlorophyll,andtheycontainedabout60%hydrocarbons,unlikewhathappenedwithhexaneextracts,whichcontainedonly37%ofthesecompounds.Whenhydro-carbonsweredepleted from thealgae, theextractsbecamemoreviscous.On the
table 6.1typical Compounds extracted from algae and other related organisms
organism target Compounds refs.
Arthrospira (Spirulina) Lipids,γ-linolenicacid,carotenoids,phycocyanin 26,27,28,29,30,31,32
Botryococcus braunii(raceA) Linearalkadienes(C25,C27,C29,C31),triene(C29) 33
Chlorella vulgaris Astaxanthin,canthaxanthin,lipids 34,35
Dilophus ligulatus Lipids,secondarymetabolites 21
Dunaliella bardawill Trans-β-carotene,cis-β-carotene 36
Dunaliella salina Trans-β-carotene,cis-β-carotene 37,38
Isochrisis galbana Lipids,EPA,PUFAs 39
Haematococus pluvialis Astaxanthin,astaxanthinesthers 30,40,41
Hypnea charoides ω3fattyacids(EPA,DHA) 42
Mortierella (fungus) Lipids,GLA 43
Nannochloropsis gaditana Chlorophylla,β-carotene,vaucheriaxanthine 44
Nannochloropsis sp. Lipids,EPA 45
Ochronomas danica Lipids,EPA,PUFAs 46
Phaffia rodozyma(yeast) Astaxanthin 47
Pilayella littoralis Nonhumiccompounds 48
Saprolegnia parasitica(fungus) Lipids,EPA 49
Scenedesmus obliquus Lipids,proteins 20
Skeletonema costatum Lipids,EPA,PUFAs 46
Torulaspora delbrueckii(yeast) Squalene 50
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Supercritical Fluid Extraction of Active Compounds from Algae 193
otherhand,C27andC29intheextractsdecreasedproportionatelyduringtheextrac-tionprogress,whileC31alkadieneproportionincreased[35].Hydrocarbonfractionincreasedwithpressure: the initialconcentrationofhydrocarbons increasedmoresteeplythanthatofintracellularlipids,leadingtoextractsmorerichinhydrocarbonsathigherpressures[55].
6.2.2 chlorella Vulgaris
Chlorella vulgarismicroalgaearecarotenoidproducers (mainlyofcanthaxanthinand astaxanthin) [56]. Carotenogenesis can be induced through saline, luminous,ornutritionalstress.Ontheotherhand,thecontentincarotenoidscanbetailoredthroughthedurationoftheprocessandtheintensityoftheimposedstresses.
Carotenoids belong to a hydrocarbon class (carotenes) and their oxygenatedderivatives(xanthophylls).Theirbasicstructure,reflectingitssynthesispath,con-sists of eight isoprenoidunits,which are assembled in suchway that twomethylgroupsnearthemoleculecenterareinposition1,6,whiletheothermethylgroupsstayinposition1,5[57].Thesetofconjugateddoublebonds(eleventothirteen)con-stitutesthechromophoreresponsibleforthecolorofthesecompounds.Thesecolorsrangefromyellowtoredandareinfluencedbythepresenceofmoredoublebonds,functionalgroups,andthetypeofmolecularconformation.
Canthaxanthin(β-β-carotene-4,4’-dione),C40H52O2,aredpigment,togetherwithastaxanthin,isoneofthemostimportantketocarotenoids[13].Itisusedascoloranttoimprovethecolorofpoultrymeatsandeggyolksaswellasinaquaculturetogiveapinktonalitytosalmonandtroutflesh.Althoughlackingpro-vitaminAactivity,canthaxanthinhasanticarcinogeniccapacity[58].
0
20
40
60
80
0 20 40 60 80 100CO2/Dry Alga (kg/kg)
Hyd
roca
rbon
s/D
ry A
lga (
g/kg
)
FIgure 6.2 Hydrocarbons supercritical extraction yield, as a function of solvent-algaeratio,fromthemicroalgaeBotryococcus brauniiinsupercriticalCO2at313.1K.♦12.5MPa,⦁20.0MPa,◾30.0MPa,×hexaneextraction.(Source:Mendesetal.,Inorganica Chimica Acta,356,328,2003.Withpermission.)
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194 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
ThesemicroalgaeweresubmittedtosupercriticalCO2atpressuresbetween10.0and35.0MPaandtemperaturesof40ºCand55ºC.Theextractionswerecarriedouton5goffreeze-driedChlorellaatseveralphysicalconditionsofthemicroalgae:notcrushed(whole),partiallycrushed,andtotallycrushedcells[34,35].
The initialconcentrationof lipids(asdeterminedby theslopeof thecumula-tivecurveoftheextractionatorigin)insupercriticalfluidincreasedwithpressure,for both temperatures, either with whole or crushed cells, but with the latter theincrease wasmore significant (Figure6.3).Above 15.0 MPa, therewas an initialconcentrationincreasewithtemperatureforwholecells,butwiththecrushedalgae,thatvaluewasaround25.0MPa.Forcrushedcells,thehighestvalueforthiscon-centrationwasobtainedat35.0MPa/55ºC(19mg/LCO2)andthelowest(3mg/L)at20.0MPa/55ºC.Thispressurewasthelowestusedforcrushedcells.Forwholecells,thehighestconcentrationobtainedwas5mg/LCO2at35.0MPa/55ºC.ThisbehaviorcanberelatedtothedifferentamountandtypeoflipidsavailableinthesupercriticalCO2,accordingtothephysicalconditionofthealgae.
Fortheconditionsofpressureandtemperaturestudied,usingwholecells,theglobalyieldoflipidsobtainedincreasedeitherwiththepressureatconstanttemperatureorwithtemperatureatconstantpressure(20.0and35.0MPa)[34].Withcrushedcellsat20.0MPa,theyielddecreasedwithtemperature,whereasat35.0MPa,itincreased.
The highest yield of lipids obtained by SFE was 13.3% (dry weight, partialcrushedcells)at35.0MPa/55ºC;thisvaluedroppedto5%atthesameconditionsusingthewholealgae.Theyieldoforganicsolventextractionforcrushedcellsusingacetoneandhexanewere16.8%and18.5%,respectively.
Theextractionofcarotenoidsshowedasimilarbehaviortothatoflipidsforpres-sureandtemperaturevariations.However,theyieldofcarotenoidswashigherwithsupercriticalCO2at35.0MPa(50mg/100gdryweightalgae) than thatobtained
Pressure (MPa)
Conc
entr
atio
n (m
g/L)
205
5
10
15
20
035 50
FIgure 6.3 Initial concentration of lipids in CO2 at standard temperature and pres-sure(STP),asafunctionofpressure.Wholecells,◾40°C,⦁55ºC.Crushedcells,♦40°C,▲55°C.(Source:Mendes,R.L.andPalavra,A.F., inChemistry, Energy, and the Environment,Sequeira,C.A.C.andMoffat,J.B.,Eds.,RoyalSocietyofChemistry,Cambridge,51,1998.Withpermission.)
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Supercritical Fluid Extraction of Active Compounds from Algae 195
either with acetone (40 mg/100 g dry weight algae) or hexane (30 mg/100 g dryweight algae). When the degree of crushing increased, the yields of lipids andcarotenoidsalsoincreased[35].
The fraction of carotenoids in the extracted oils increased with the pressure.Forwhole cells, this fraction ranged from18mg/100goil (at 20.0MPa/55ºC) to171mg/100gofoil(at35.0MPa/55ºC)when100gofCO2on5galgaewereused[34]. For crushed cells, the fractions obtained were higher: 100 mg/100 g oil at20.0MPa/55ºCand275mg/100gofoilat35.0MPa/55ºC[59].
When a completely crushed Chlorella is used, with a carotenoids content of3mg/gofdryalgae,theyieldofcarotenoidsextractionincreasedsteeplywhenabout20%ofthesecompoundswereextracted(Figure6.4).Thisincreasecanbeattributedtoahigheraccessibilityofthesupercriticalfluidtothecarotenoidsboundtothecellfragmentsafterthelipids’outsideparticlesareextractedortoacompetitiveeffectbetweenthelipidsandcarotenoidsforthesupercriticalsolvent[35].
Astaxanthin and canthaxanthin account for about two-thirds of the carot-enoidsinC. vulgaris.Theratioofastaxanthintocanthaxanthininthesupercriticalextracts(0.8)isthesameasthatfoundinacetoneextractswhencompletelycrushedcellsareused.However,whenpartiallycrushedorwholecellsareused,theratioof astaxanthin to canthaxanthin in the supercritical extracts drops consider-ably,from0.6(withacetone)to0.2.Possibleexplanationsforthisbehaviorareastrongerbondbetweenastaxanthinandthecellularmatrixoradifferentlocaliza-tionofthetwocarotenoids.
An unsteady model to describe the SFE of lipids from the microalgaeC. vulgariswasbasedonthemodelusedtodescribetheextractionoflipidsfromfungi (Saprolegnia parasitica) using supercritical CO2 and supercritical CO2 +cosolvent(10%ethanol)[49].Themodelassumesthattheaxialandradialdisper-sionsarenegligible,thepropertiesofthefluidandalgalbedremainconstant,andthecomplexmixtureoflipidsisconsideredasasinglecomponent[60].Themass
0
10
20
30
40
50
0 10 20 30 40 50CO2/Dry Alga (kg/kg)
%Car
oten
oids
FIgure 6.4 Recovery of carotenoids from Chlorella vulgaris, as a function of solvent-algaeratio,at35.0MPaand328.1K.▲Wholecells,⦁Slightlycrushedcells,◾Wellcrushedcells.(Source:Mendesetal.,Inorganica Chimica Acta,356,328,2003.Withpermission.)
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196 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
balanceinfluidandsolidphasecanthereforebedescribedbythefollowingsetofdifferentialequations:
ερ ρ∂∂
= − ∂∂
+ −( )y
tu
y
hApK y y* (6.1)
1−( ) ∂∂
= − −( )ε ρs
x
tApK y y* (6.2)
whereεistheporosityofthealgaebed,ρisthesupercriticalfluiddensity,ApKistheoverallmasstransfercoefficient(basedonvolume),uisthesuperficialvelocityofthefluid,xisthemassoflipidspermassoflipid-freealgae,ρsisthealgaedensity,his theaxialdistancefromthebottomof thealgaebed,y is the lipidconcentra-tioninsupercriticalfluid,tisthetime,andy*isthelipidsolubility.Theinitialandboundaryconditionsarex=xoatt=0foranyh,andy=0ath=0andt≥ 0.
Tohaveacompletedescriptionoftheextractioncurve,theoverallmasstransfercoefficient(whichaggregatestheexternalandtheintraparticleresistances)mustvarythroughoutthewholeextraction,accordingtoanempiricalexpression(3)[49,60],foundbyatrial-and-errormethod:
ApK ApKo x x x xo o= ( ) −( ) −( ) exp ln .0 01 shift (6.3)
where xshift is the concentration of lipids (in the algae) at which the diffusion-controlledregimestartsandxo is theinitialconcentrationof lipids.Masstransfercoefficientsweredeterminedusinga least squares regressionof theexperimentalextractioncurves.
ThemodelwasappliedtodifferentphysicalconditionsofC. vulgaris atapres-sure of 35.0 MPa and 55ºC, using a CO2 flow rate of 0.8 g/min. For algae withwholecells,theshiftforthediffusion-controlledregimeoccurredwhen4%ofthelipidswereobtained,inthecaseofaC. vulgariswithathickercellularwall,and10%whenanalgawithathinnercellularwallwasused.ThethicknessofthiswallseemsrelatedtothecarotenoidcontentofC. vulgaris[61].Withapartialcrushing,theshiftoccurredwhenabout25%ofthelipidswereextracted,whereaswithatotalcrushing(almostallthecellsdisrupted),theshiftoccurredwhen55%ofthelipidswereextracted.ThelastvalueissimilartothoseobtainedwithSFEoflipidsfromfungiandofoilfromrapeseed,althoughinthiscase,theshiftoccurredbecausetheremainingoilwasinotherformofbindingtothematrix[62].Thecalculatedmasstransfercoefficients(ApKo)rangedbetween0.171kg/m3sand0.531kg/m3sfortheextractionusingwholecellsand0.7kg/m3sand2.2kg/m3s usingcrushedcells[60,61].
6.2.3 Dunaliella
ThemicroalgaeDunaliella salinacanproduceβ-caroteneup to14%(dryweightbasis) [2],with this compound being mainly a mixture of twogeometric isomers(all-trans and 9-cis). The synthetic compound is the crystalline all-trans isomer,
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Supercritical Fluid Extraction of Active Compounds from Algae 197
whichisnotreallyfat-soluble;however,naturalβ-carotene(fromalgae)isnotcrys-tallineandishighlyfat-soluble.
Separating the two isomers is advantageous because the cis is more easilyabsorbedinhumantissues[63]andhasmoreantioxidantactivity[63].Theconven-tionaltechniquesofisomerseparationinvolvelargeamountsoforganicsolventsandareverytime-consuming.Ontheotherhand,formedicalandfoodapplications,itisimportantthatthisseparationisdonewithouttheuseoftoxicsolvents.
TheseparationofisomerscanbecarriedoutthroughSFEifthereisasignifi-cantdifferenceinsolubilityofthecompounds.Thecisisomerisaboutthreetimesmoresolublethantheall-trans,andbothcarotenespresenthighersolubilitythanthesyntheticall-transisomer.
The concentration in supercritical CO2 of the cis-β-carotene and all-trans-β-carotene from a solid mixture of carotenoids from Dunaliella salina is shown inFigure6.5.ThismixturewasobtainedbyacetoneextractionfromwetDunaliella[37].Theorganicextractwassaponified inorder to remove the lipidsandchlorophyll,and the separation of phases was carried out with diethyl ether. The carotenoidscontainedin theetherphasewererecoveredbyevaporationof thissolventwithastreamofnitrogen.
Gamlieli-Bonshteinetal. [36]usedadifferentapproach tocompare thesolu-bilityoftheβ-caroteneisomers.Duetononavailabilityofthepure9-cisisomertocarryoutsolubilitymeasurements, thesolubilityof thecompoundwascalculatedindirectly.SupercriticalCO2extractionofβ-carotenefromDunaliella bardawil wasperformedat448barand40ºCfromaconcentrateextractobtainedwithamixtureethanol/hexane/water.Theseconditionsofpressureandtemperaturewerepreviouslyfoundtobetheoptimalonesforthesupercriticalextractionofβ-carotene[36].Enter-ingwiththeratiooftheinitialratesoftheextraction(fromtheconcentrate)ofthetwoisomersandknowingthesolubilityofthetrans isomer,whichwaspreviouslymeasured,theauthorscalculatedthesolubilityofthe9-cisβ-caroteneasbeing,at448bar/40ºC,7,64×10–5gisomer/gCO2,nearlyfourtimesthevalueoftheall-trans
0 10 15 20 25 30 35Pressure (MPa)
10–3
10–1
10–2
10–4
10–5
100
Solu
bilit
y (g/
dm3)
5
FIgure 6.5 Solubility of β-carotene isomers in supercritical CO2 (40°C), ⦁ all-trans-β-carotene (mixture), ◾ cis-β-carotene (mixture), • synthetic trans-β-carotene. (Source:AdaptedfromMendesetal.,Inorganica Chimica Acta,356,328,2003.Withpermission.)
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198 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
isomer. Supercritical CO2 extraction was also performed on raw algae under thesameconditionsofpressure and temperature.At the initial stages, the extractionrateof thecis isomerwashigher thanthatof the trans isomer,but thedifferencebetweentherateswasnotaslargeasinthecaseoftheconcentrate.Thissuggestedtotheauthorsthat,inthefreeze-driedalgaepowder,theeffectofinternaldiffusionlimitationsonβ-caroteneextractionrateissignificant.Therefore,theresultsindicatethatdecreasingthealgaeparticlesizeshouldincreasetherecoveryandselectivityofisomerseparation.
SupercriticalCO2extractionofcompoundsfromfreeze-driedDunaliella salina wasalsocarriedoutbyMendesetal. [38]atpressuresof200and300barandatemperatureof40ºC,inasemicontinuousapparatusattwoflowrates,of18.9g/minand10.8g/min,on27gofalgaehavingacontentof0.5%(dryweight)ofβ-caroteneand10%(dryweight)lipids.Theyieldoftheextractionoflipidsincreasedwiththepressure,butnoincreaseintheextractionyieldofβ-carotenewasobservedwiththepressure.Whenthesupercriticalextractionoflipidsandβ-carotenewerecomparedforthetwodifferentflowratesat300bar,itwasverifiedthattheextractionyieldwashigherforthelowerflowrate.Recoveriesof25%and80%werereachedforlipidsandβ-carotene,respectively,attheflowrateof10.8g/min.Itwasalsoverifiedthatitwaspossibletoobtainvaluesofthecis-transratiowellaboveoftheinitialoneinthealgae(1.3).Forinstance,whentheextractionwascarriedoutat300barandataflowrateofabout18.9gCO2/min,thecis-transratioreachedthehighestvalue(3.6)atitsbeginninganddecreasedexponentiallyalongtheextraction.ForalowerflowrateofCO2(10.8g/min),theratiodecreasedto2.2.Theseresultsshowedthatthecis-β-carotenewasquicklydepletedatthesurfaceofthealgaeparticles.ThisbehaviorcanbeexplainedbythehighersolubilityofthecisisomerinCO2andbythepresenceofahigheramountofthiscompoundintheouterpartofthealgaeparticles(wherethetransisomerwasmoreeasilyisomerized),asthecapacityofDunaliella toproducethecis isomer is related to lightexposition [65].Thevaluesof thecis-trans ratiowere higher (about 4) whenβ-carotene concentrates of Dunaliella salina [37] orDunaliella bardawil[36]wereused.
Inanattempttoincreasetheselectivityoftheseparationofβ-caroteneisomers,Nobreetal.usedacombinationofsupercriticalCO2andsilicagel[66].Theexperi-ments were carried out at a pressure of 200 bar and a temperature of 40ºC in aflowapparatus.Theextractorwasa32-cm3vesselfilledwith20gofglassbeads,inwhichabout100mgβ-carotene(obtainedfromDunaliella)hadbeenpreviouslyprecipitated.Asecond5-cm3vesselinseriescontainedthesilicagel.Severalloadsofsilicawereused:1.5g,0.5g,and0.15g.Thecis-transratioincreasedwhentheamountofsilicageldecreased,beingalwayshigherthantheoneobtainedbysimpleSFE,reachingavalueof7.4forthelowestamountofsilicagelused.ThisvalueoftheratiowasaboutthreetimestheoneobtainedbySFEatthesameconditionsofpressureandtemperature.
6.2.4 haematococcus pluVialis
Haematococcus pluvialis is a freshwater flagellate that accumulates astaxan-thininitsaplanospores.Itcanproduceastaxanthininrelativelyhighyields(upto
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45mg/gdryweight algae) in laboratory conditions, but due toheavy contamina-tion, theuseofopenpondsisnotsatisfactorytoobtainthehighyieldsnecessaryforalgalastaxanthintocompetewiththesyntheticone.OthermethodsforgrowingH. pluvialis havebeentriedtoovercomethatdrawbackandalsotoincreaseyields[13].Olaizola[67]exploredthestrategyusedbyapharmaceuticalcompanytomakeHaematococcus astaxanthin more acceptable to consumers and to make it morecompetitive.ThisstrategyincludestheuseofSFE.
Astaxanthin (3,3´-dihydroxy-β,β-carotene-4,4´-dione), C40H52O2, is one ofthe most important xanthophylls either from a commercial or a biotechnologicalpoint of view, being the most abundant carotenoid and pigment found in certainaquatic animals, such as salmon, trout, shrimp, and lobster. Several isomers arefound inastaxanthinpresent innature,with3S,3S’being themainonefound inH. pluvialis[68].ThepresenceofthehydroxylandketogroupsintheiononeringexplainswhymostoftheastaxanthinappearsinHaematococcus inesterifiedforms(monoanddiester)andalsowhyitisverypronetooxidation.Theactivityofthecom-poundisseveraltimeshigherthanthatofβ-caroteneandvitaminE.Guerinetal.[68]reviewedthemainapplicationsforhumanhealthandnutritionofHaematococcus astaxanthin,namelyitsusesagainstultraviolet-lightphotooxidation,inflammation,cancer,andagingandage-relateddiseasesandinthepromotionofimmuneresponse,liverfunction,andheart,eye,andprostatehealth.Ontheotherhand,asinglehighdose(100mg)wasadministeredtohumansinastudyofbioavailability,anditwasverifiedthatastaxanthinwasreadilyabsorbedandincorporatedinhumanplasmalipoproteinataconvenabledegree[69].
Valderramaetal.[30]andMachmudahetal.[41]carriedoutsupercriticalCO2extraction of astaxanthin from H. pluvialis.Another study of the same type thatfocusedonastaxanthin,aswellasontheothercarotenoidspresentinthesemicro-algae,wascarriedoutbyNobreetal.[40].Pressuredfluidextraction(PFE),whichusesconventionalsolventsatcontrolledtemperaturesandpressures,hasalsobeenperformedtoextractcarotenoidsfromH. pluvialis[70].
InthestudiesofValderramaetal.[30],thesemicontinuoussupercriticalappa-ratuswasprovidedwithanextractionvesselof450ml,andtheexperimentswerecarriedoutat300barand60ºC.Threetypesofrunswereperformed:run1,inwhichthedriedsamplesofmicroalgaewerecrushedbycuttingmillspriortoextraction;run2,inwhichthesampleswerecrushedlikeinrun1,followedbymanualgrindingusingdryice(solidCO2);andrun3,inwhichthesamplesweretreatedasinrun2,but theextractionwasperformedusingsupercriticalCO2containingethanolasacosolvent(9.4%weight).
Theastaxanthinyielddependedstronglyonsamplepreparationandontheuseofethanolasacosolvent.Amoreefficientgrindingledtohigheryield.WithpureCO2,astaxanthinisrecoveredonlypartially.WithH. pluvialis wellcrushedandusingthecosolvent,anastaxanthinrecoveryof97%wasreached.Thisrecoverywasdeter-minedfromtheinitialandfinalcontent(residueoftheextraction)ofthecompoundinthemicroalgae.Valderramaetal.[30]didnotprovidethemethodbywhichthosecontentsweredeterminedandnomentionofesterifiedastaxanthin,theusualformof astaxanthin in thesemicroalgae,wasmade.The supercritical extraction (yield
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200 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
versussolvent-feedratio)wasmodeledwiththesameempiricalmodelusedinthesupercriticalextractionoflipidsfromSpirulina(seesection6.2.7.1).
Nobreetal.[40]carriedoutamoredetailedstudyofsupercriticalCO2extrac-tionofcarotenoidsfromH. pluvialisusingfreeze-driedsamplesgroundwithaballmill.Theeffectsofpressure(200and300bar),temperature(40and60ºC),degreeofcrushing(relatedtomillingtime),andtheuseofethanol(10%)ascosolventontheextractionyieldweredetermined.Theextractionwithacetonewascarriedoutusingglassbeadsmixedwiththealgaeuntiltotalabsenceofcolorwaspresentinthebiomass.
Besidesfreeastaxanthin(whichrepresentsabout2%ofthecarotenoids),theyieldofesterifiedastaxanthin(73%ofthecarotenoidspresentinH. pluvialis),β-carotene(7%of thecarotenoids),canthaxanthin,andluteinwereassessedandtherecover-iesobtainedbySFEwerecomparedwiththetotalcontentofcarotenoidsfromthemicroalgae.Figure6.6showsthetotalastaxanthinrecoveryasafunctionofsolvent-feed ratio, showing the beneficial effect of ethanol as a cosolvent as well as thedegreeofcrushingofthemicroalgaecells.Arecoveryofabout90%wasobtained.TheimprovementinyieldduetoethanolwasascribedtotheincreaseofastaxanthinsolubilityinsupercriticalCO2duetoitspolarcharacterand,ontheotherhand,totheswellingofthemicroalgaeparticlepores,whicheasedthereleaseofthecompounds.Therecoveryimprovementduetothecrushingofthecellscanbeattributedtotheincreaseinthenumberofdisruptedcellsandthedegreeofdisruption, increasingtheamountofextractablecarotenoids.Inthiswork,theresearchersalsoverifiedanincreaseintherecoveriesofcarotenoidswhenthepressureincreasedfrom200to300bar,butonlyaslightimprovementwasobtainedwhenthetemperature,atthesamepressures,increasedfrom40ºCto60ºC.
All the carotenoids identified in H. pluvialis (esterified and free astaxanthin,β-carotene,lutein,canthaxanthin)wererecoveredwithvaluesnearorhigherthan90%atapressureof300barandatemperatureof60ºC,usingethanolasacosolvent.
0
20
40
60
80
100
0 100 200 300CO2/Dry Alga (g/g)
Tota
l Ast
axan
thin
Rec
over
y (%)
FIgure 6.6 Recoveryoftotalastaxanthin(freeplusesters)asafunctionofCO2amountwith(◾)andwithout(▲)ethanolasacosolventusingslightlycrushedalgae,andCO2withethanolusingwell-crushedalgae(♦),at60°Cand300bar.(Source:Nobreetal.,Eur. Food Res. Tecnol., 223,787,2006.Withpermission.)
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Supercritical Fluid Extraction of Active Compounds from Algae 201
Machmudahetal. [41]studiedthesupercriticalCO2extractionofastaxanthinfromH. pluvialis usingaflowapparatusprovidedwitha50-cm3extractionvesselcontaining7gofalgaeineachexperiment.Thecontentofastaxanthinwas3.33%(drybasis)asdeterminedbySoxhletusingdichloromethane.Theconditionstestedwere temperature (313 to 353 K), pressure (20 to 55 MPa), CO2 flow rate (2to4cm3/min),andethanolconcentration(ethanol-solvent=1.67–7.5%).Thealgaewerenot crushed, unlike in the aforementioned studies.Without ethanol entrainer, theamountoftotalextract,theamountofastaxanthinextract,andtheastaxanthincon-tent in the extract increased with an increase in temperature. This behavior wasattributedtotheincreaseofvaporpressureofastaxanthinandalsotothedecom-positionofthecellwallwiththetemperature,whichcontributedtotheincreaseoftheextractablecompounds.Theincreaseofpressurealsoledtoanincreaseofthoseyields.Theamountofthetotalextractandtheamountofastaxanthinextract,butnottheastaxanthincontentintheextract,slightlyincreasedwithCO2flowrate.
The highest astaxanthin extracted (recovery) and astaxanthin content (in thetotalextract)were77.9%and12.3%,respectively,whichwereobtainedat55MPa,at343K,andataCO2flowrateof3cm3/min.
Usingethanolasanentrainer,ahigheramountofastaxanthin(80.6%)couldbeextractedatamoremoderatepressure(40MPa).Ontheotherhand,theincreaseinentrainerconcentration,upto5%(v/v)ethanol,increasedtheamountofastaxanthin.With theuseofentrainer, theCO2flowrateconsiderablyaffected theastaxanthinextractedandahigheramountofastaxanthinwasextractedasCO2flowratedecreased.Thislastpointsuggestedtotheauthorstheimportanceoftheinternalmassdiffusionin the extraction of astaxanthin fromH. pluvialis. This is in accordance with thepreviousstudiesofValderrama[30]andMendes[40],whichdemonstratedthatthecrushingofthealgaecontributedhighlytothesuccessoftheextraction.
InstudiesofPFEusingacetoneassolvent,higherorequalamountsofcarotenoidswereextractedthanwereextractedwiththetraditionalorganicsolventmethod[70].Thetimeofextractionwas20minuteswithPFE,whereas90minuteswereneces-sarytoperformthetraditionalacetoneextraction.
6.2.5 hypnea charoiDes
Hypnea charoidesisasubtropicalredseaweed.Amongtheseaweeds,theredonesareknownfortheirhighomega3(w3)fattyacidcontents[71].Amongthefattyacidconstituentsofalgallipids,themostimportantarethepolyunsaturatedfattyacids(PUFAs).TheyrangefromC12(twelvecarbonatoms)toC22andcancontainuptosixallylicbondsseparatedbyacarbonatom.Withinthisgroup,theessentialfattyacids(EFAs)forhumansarelinoleicacid,g-linolenicacid(GLA),dihomo-g-linoleicacid,arachidonicacid,andEPA.TherearetwotypesofPUFAs:w3andomega6(w6),knownofficiallybyn-3orn-6fattyacids,respectively[72].Thesenumberscorrespond to the position of the last double bond counted from the last methylgroup.Formosteukaryoticalgae,whichcontainpredominantlymonoandsaturatedfatty acids, the triglycerides constitute up to 80% of the lipid fraction [73]. In ageneralway,themarinealgaearerichinw3fattyacids,namelyEPA(C20:5,w3)anddocosahexaenoicacid(DHA,C22:6,w3).Themainsourceforhumanconsumption
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202 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
of thesePUFAs ismarinefish,whose feedingbasis isconstitutedbymicroalgae.Withthedepletionoffishstocks,microalgaeandmacroalgae(seaweeds)couldbealternativesourcesofw3fattyacids[42].
Alargenumberofepidemiological,animal,andclinicalstudieshaveshownthatintakeofw3fattyacidsisbeneficialforpreventingacertainnumberofdiseases(e.g.,cardiovasculardiseases,someformsofcancer,anddiseaseswithimmunoinflamma-torycomponents)andcanalsoplayaroleinbrainandnervedevelopmentofgrowingfetusesandinfants[72].Thetherapeuticeffectsofthesefattyacidshavealsobeenshown[74].
Cheung[42]carriedoutsupercriticalCO2extractionofn-3fattyacidsfromthesemacroalgae,withemphasisontheeffectsoftemperature,pressureontheyield,andcompositionoftheextracts.Theexperimentswereperformedon2gofdriedandground(1mmsieve)algae,attemperaturesof40ºCand50ºCandpressuresof24.1,31.0,and37.9MPaataCO2flowrateof2mg/min.Theextractioncellwasprovidedwitha10-cm3stainlesscartridge.
Cheungfoundthatlipidextractionincreasedwiththepressureatconstanttem-perature.Athigherpressures(31.0and37.9MPa),anincreaseintemperatureledtoanincreaseintheyieldandalsointherateofextraction(intheinitialperiod).However,atthelowestpressureused,theyielddecreasedwhenthetemperaturepassedfrom40ºCto50ºC.TheseresultsaresimilartopublisheddataofsupercriticalCO2extrac-tionoflipidsfromChlorella vulgaris[34].Thisbehaviorisexplainedintermsofthecombinedeffectofthepressureandtemperatureonthedensityofthesolventandvaporpressureofthesolutes.
Themaximumyieldof lipids fromH. charoides at theconditions studiedbyCheung was 67.1 mg/g (dry weight algae) at 37.9 MPa/50ºC and the lowest was33.7mg/gat24.1MPa/50ºC.Sixw3fattyacidswerefoundintheextracts,represent-ingattheconditionsofmaximumyield(37.9MPa/50ºC),24%and39%ofthelipidsandtotalfattyacids,respectively.EPA(20:5w3)representedabout60%ofthetotalw3fattyacidsandα-linolenicacid(18:3w3)16%ofthesefattyacids.DPA(22:5w3)andDHA(22:6w3)werealsofoundtorepresent8%and12%,respectively,ofthetotalw3fattyacids.
WiththeexceptionofDPAandDHA,theyieldofallthew3fattyacidshadasimilarbehaviortothatofthetotallipids,withthevariationofpressureandtempera-ture.Evenathigherpressures,theC22w3fattyacidsshowedaloweryieldathighertemperatures.ThisbehaviorcouldbeattributedtothefactthatchainlengthisamoreimportantfactorinsupercriticalCO2solubilitythanthedegreeofsaturation.
Theauthorconcludes thatalgal lipidsofH. charoides couldbeanonconven-tionalalternativetow3fattyacids.
6.2.6 nannochloropsis
Nannochloropsisspeciesaremarinemicroalgaeabletoproduceahighcontentofbothlipidsandeicosapentaenoicacid(EPA).ThiscompoundisthemajorfattyacidofthosefoundinNannochloropsis(rangingfrom29%to33%),withthecontentofn-3PUFAsbeingabout40%ofthetotalfattyacids[45].Itisalsoasourceofvaluablepigments,suchaschlorophylla,astaxanthin,zeaxanthin,andcanthaxanthin[75].
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Supercritical Fluid Extraction of Active Compounds from Algae 203
Andrichetal.[45]verifiedthatinsupercriticalextractionstudiesofalgaelittleinformation about the kinetics of the process and the influence of the operatingconditionsonthecompositionofthelipidextractswereavailable.Therefore,theyfocusedtheirstudiesatexaminingthesepoints.
Supercritical CO2 extraction of bioactive lipids from the microalgae Nanno-chloropsis sp. wascarriedoutinapilotplantapparatusatpressuresof40,55,and70MPaandtemperaturesof40ºCand55ºC.Anamountof180gmicroalgae,mixedwith100gof3-mmglassspheres,wasusedforeachrun.ThesupercriticalCO2flowratewas10kg/h.Theauthorsalsoperformedextractionbypercolationwithhexane(Soxhlet).
Thefollowingequation isused todescribe theevolutionofextractedoilovertime(t),forbothsupercriticalextractionandhexaneone:
Oe H Os e kt= −( )−* 1 (6.4)
whereOeistheamount(g)ofoilextractedatatime(t)pergramofalgalbiomass,H*isaconstantrangingfrom0to1,[Os]istheamount(g)ofoilpresentin1gofstartingmaterial,andkisthekineticconstant.TheproductH*[Os]representstheasymptoticvalueoftheextractioncurve.Inahighlyefficientprocess,H*tendsto1,meaningthattheamountofextractableoilpergramofbiomasscoincideswiththeconcentrationofoilinthestartingmaterial.
Themaximumextractionrate(Rmax)isgivenby:
R kH Osmax = + (6.5)
Theauthors interpreted thecumulativeextractioncurvesasa functionof time intermsofthekineticparameters.Intermsofoilextractable,alltheprocesses(super-criticalCO2extractionsatalltheconditionsandhexaneextraction)aresubstantiallyequivalent(about250mglipids/gdryalgae).Thevalueofkincreasesatconstanttem-peraturewiththeincreaseofpressureandalsoincreasesforagivenpressurewhenthe temperature increases, although in this case more moderately. The combinedeffectofpressure-temperature(P-T)affectskmorethanPandTalone,increasingitsvaluethreetimesfromtheextractionat40ºCand400bartotheextractionat55ºCand700bar.SFEwasclearlyfasterthantheextractionbyhexane(Rmaxwasseveraltimeshigher).
Rmaxwasexpressedintermsof theconcentrationof lipidsinthesupercriticalfluidatthebeginningoftheextraction,R*max(g/l),andrelatedtothedensity,r(g/l),andtemperature,T(K),ofthesolventthroughtheequationduetoChrastill[76]:
R ea b T Cmax* = +( )ρ (6.6)
The values of the parameters, obtained from the several extraction conditionsstudied,werea=10.92±2.57;b=3506.57±1225.65;andc=62.68±16.18.Theauthorsclaimthatthiskineticmodelseemssuitabletoevaluatetheeconomicfeasibilityoftheprocess,althoughmoreinformationisneededforitsgeneralization.
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204 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
ThepercentageofEPA in the total fatty acids ranged from29.4% to33%fortheconditionsofextractionstudied.However,thesevaluesseemlowerthanthe37%claimedforthecommercialproduct.TheauthorsattributedthistothefactthattheuseofsupercriticalCO2causedsomelossesinthemostpolarfractions,whereEPAispos-siblyprimarilylocated.Ontheotherhand,noparticulardifferenceswerefoundinthefattyacidprofilesofextracts.However,aslightincreaseinEPAandDPA(C22:5n-3)seemsperceivablewhenpassingfromthemildesttothehardestSFEconditions.
Macías-Sánchezetal.[44]carriedoutsupercriticalCO2extractionofpigmentsfrom Nannochloropsis gaditana in a microscale apparatus, which is providedwithtwoextractorsof10mleach.Inoneoftheextractorswasinsertedacartridgecontaining0.2goffreeze-driedmicroalgaeand,after15minutesofstaticextrac-tion, the runswereperformedat aflowrateof4.5mmol/min for3h.A totalof15experiments were carriedout in a random way, in order to fulfill amultilevelfactorialdesigntodeterminetheeffectoftemperatureandpressureontheextractionofthecarotenoidsandchlorophylla.
Organic solvent extraction, using methanol, was performed by sonication on0.2gofN. gaditana,with5mlof solvent foreachof the14cyclesnecessary toobtainthemethanolwithoutanycoloration.Thealgaepelletremainedgreenishafterthesolventextraction.Theyieldsobtainedwere0.8mg/mgand18.5mg/mg(dryweightbasis)forcarotenoidsandchlorophylla,respectively.
SFEwascarriedoutattemperaturesof40ºC,50ºC,and60ºCandpressuresof100,200,300,400,and500bar.Theanalysisoftheexperimentaldesignshowsthatthetemperature,pressure,andinteractionofbothvariablessignificantlyinfluencetheprocess(p-value<0.05).Theyieldincreaseswithpressureatagiventempera-turefrom100to400bar,wherethemaximumyieldisreachedat60ºC(400bar)forbothcarotenoidsandchlorophylla(0.343mg/mgand2.238mg/mg,respectively).At 100bar, no pigments were extracted and at each pressure the yield increasedwithtemperatureforpressuresabove200bar.Asusual,thisbehaviorisexplainedintermsofbalancebetweensolventdensityandvaporpressure.Ontheotherhand,at500bar,theyielddecreased.Thefactofthemaximumyieldbeobtainedatanintermediatepressure(400bar)isexplainedintermsofthedecreaseofthediffu-sioncoefficientduetotheincreaseoftheCO2density,whichreducesthepenetrationcapacityofthefluidandtheyieldatthehighestpressureused.ThisalsoalreadyhadbeensuggestedfortheextractionofcarotenoidsfromSpirulina[28].
Thefollowingempiricalcorrelationsforcarotenoids(6.7)andchlorophylla(6.8)wereobtained[44]:
R T P= − + + −0 233163 0 00492577 0 00121779 0 000. . . . 00923077
0 00002705 0 00000353013
2
2
T
PT P+ − −. . (6.7)
R P T= − − −3 43203 0 00140362 0 14499 0 00000725. . . . 9952
0 0001963 0 001144
2
2
P
PT T+ +. . (6.8)
whereRistheyieldofpigment(mg/mg),Tisthetemperature(ºC),andPispres-sure(bar).
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Supercritical Fluid Extraction of Active Compounds from Algae 205
Furthermore, the ratio of carotenoids to chlorophylls (carot/chlor) in theextracts isalwayshigher than thatobtainedwithmethanol(carot/chlor=0.043)anddecreaseswithpressure,withthehighestratioobtainedat200barand60°C(carot/chlor = 1.389). This leads the authors to conclude that supercritical CO2ismoreselectivefor theextractionofcarotenoidsinthepresenceofmorepolarpigmentslikechlorophylla.
The SC extraction of chlorophyll in this work contrasts previous work fromotherauthors,inwhichthenonextractionofthistypeofcompoundbysupercriticalCO2fromBotryoccocus braunii[33],Skeletonema costatum [46], andOchronomas danica[46]wasreported.However,inthestudyofsupercriticalCO2extractionofoilfromScenedesmus obliquus [20],theextractionofchlorophyllaismentioned.ThismatterisdiscussedbyBalabanetal.[23],whosuggestedthatentrainedparticlesofalgaemighthavecausedthediscrepancybetweenthisresultandpreviousreportsofinsolubilityofchlorophyllinsupercriticalCO2.
6.2.7 spirulina (arthrospira)
Spirulinaisoneofthemostpromisingmicroalgae.Itisrichintheessentialfattyacidall-cis-6,9,12-octadecatrienoicacid(GLA);pigments(phycocyanin,myxoxantho-phyl,zeaxanthin,andβ-carotene);proteins;andsulfolipids[77]. Likeotheralgae,suchasChlorella, Spirulina has alsobeenusedas functional food (foodderivedfromnaturalsourceswhoseconsumptionisbeneficialtohealthofthehumanbody).Inthiscase,algaeareprovidedeitherassupplementsorcompletefood[78].ResearchhasalsoshownthetherapeuticvalueofSpirulina anditsextractsinalargenumberofdiseases[79].
TheGLAfromSpirulina actuallycannotcompeteeconomicallywiththeGLAfromhigherplants(e.g.,eveningprimrose,blackcurrant,andborage),buttherearesomeadvantagestousingSpirulina microalgaeasaGLAproducer.Thiscompoundisfoundmainlyintheglycolipidfractionofthelipids,whicheasesitspurificationasapharmaceuticalcommodityand,ontheotherhand,unliketheGLAofthehigherplants,itisnotassociatedwithundesirablefattyacids[77].
GLApresentshigherEFAactivityandantithromboticandhypolipidemiceffectsthanlinoleicacid(18:2w6)[80].Italsohasbeenusedinseveralmedicalapplica-tions, such as the treatment of schizophrenia, multiple sclerosis, atopic eczema,premenstrualsyndrome,diabetes,andrheumatoidarthritis[80,81].Itcanalsoplayanimportantroleinthesynthesisofakindofprostaglandin,ahormoneinvolvedin essential tasksof thehumanbody,namely the controlof the arterial tension,cholesterol,andinflammation.
TheantioxidantactivityofextractsfromSpirulinaplatensis obtainedwithpres-surizedliquidextractionwasstudiedandattributedtothepresenceofcarotenoids,phenoliccompounds,anddegradationproductsofchlorophyll[82].
6.2.7.1 Spirulina maxima
SupercriticalCO2extractionoflipidsfromS. maxima wasstudiedbyMendesetal.[32],Canelaetal.[29],andValderramaetal.[30].ThefirstauthorsfocusedtheirworkontheextractionofGLA,thesecondonesontheextractionoffattyacidsand
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carotenoids,andthethirdontheresidueoftheextractioninordertoobtainthepig-mentphycocyanin.
TheobjectiveoftheworkofMendesetal.[32]wastocarryoutthesupercriticalCO2extractionoflipids,focusingontheGLAcontentoftheselipidsandtoassessthe influenceofpressure, temperature,and theuseofethanolasentraineron theextractionyieldsandselectivityoftheseveralfattyacidsandclassesoflipids.TheextractionoftheSpirulina lipidswasalsoperformedwithorganicsolvents(ethanol,hexane,acetone,andamixtureofwater,chloroform,andmethanol),havinginviewthecomparisonofthetwotypesofextraction(SFEandorganic).Thesupercriticalexperimentswerecarriedoutinaflowapparatuson3.6gofash-free,freeze-driedArthrospira (Spirulina) maximaataflowrateof2g/minatpressuresof250barand300barandtemperaturesof50ºCand60ºC.At250barand50ºCwithpureCO2,ayieldof0.05%(GLA/drybiomasswt%)wasobtainedwhen1.4kgCO2wasused,but above a solvent-feed ratioof300g/g, the concentrationof lipids inwasverylow.At the same conditions, using supercritical CO2plus ethanol (10mol%), theyield improvedto0.17%,buthigheryieldsarepossiblebecause theconcentrationofthelipidsinthesolventgrewsteadilyevenforhighsolvent-feedratios.Ethanolcan have an entrainment effect on the extraction of the lipids, which are mainlypolar,increasingitssolubilityand,ontheotherhand,cancounterbalancethehydro-genbondsand ionic forcesbetween themembrane-associated lipids andproteins[83],allowingthelipidstobeavailableforextractionbythesupercriticalfluid.Theincreaseoftemperatureat250barledtoanincreaseinGLAyield,buttheincreaseinpressureto350barat60ºCledtothehighestGLAyieldobtained(0.44%).TheGLAyields reachedwithethanol, acetone,andhexanewere0.68%,0.63%,and0.01%,respectively.Inanotherstudy[31],inwhichethanolwasmixedwithSpirulina,GLAyield increasedwith the amountof ethanol and, for equalmassesof ethanol andmicroalgae,theyieldwasmorethan10timesthatobtainedwithsupercriticalCO2extractionfromnon-pretreatedSpirulina.
Valderramaetal.[30]extractedtheCO2-solublematerialfromSpirulina maxima,withtheaimofconcentratingthepigmentphycocyaninintheextractionresidue.ThefirstextractionwascarriedoutusingpureCO2at300barand60ºC,whichledtoalipidyieldof1.1%.Inasecondsetofexperiments,theco-solventethanol(10%)wasused,withanincreasedyieldof3%.ThecontentinphycocyanininthesamplesofSpirulina maximaincreasedfrom6.17wt%to8.3%whenpureCO2wasusedandfrom6.91%to8.4%whenthesolventwasCO2plus10%ethanol.
An empirical model for the concentration of phycocyanin correlates well thelipidextractionyield(Y)withthesolvent-feedratio(X):
Y e X= −( )−α β1 (6.9)
Inthemodel,αandβareempiricalconstantsobtainedfromtheexperimentalyield.Y = 0 for X = 0, and the yield has as a limit the empirical maximum, which isrepresentedbytheparameterα.
Canela et al. [29] studied the supercritical CO2 extraction of fatty acids andcarotenoids from S. maxima. The experiments were conducted at temperaturesbetween20ºCand70ºCandwithpressuresupto180bar.Theseauthorspreviously
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determinedSpirulina composition(3.1%lipids,54.5%proteins,9.1%humidity,and12.2% ashes). The total amount of CO2-soluble material was determined, usinga3.5-cm3extractionat180bar (30ºCand40ºC)and150bar (30ºC,40ºC,60ºC,and70ºC).Thehighestamount,0.93%(dryweightbasisofSpirulina),wasobtainedat150barand60ºC.
KineticexperimentrunsofsupercriticalCO2extractionoflipidsfromS. maximawereperformedusingastandardextractionunitprovidedwithanextractioncellofabout 368 cm3. For each run, an amount of 175 g microalgae, mixed with equalamountofglassbeads,wasused.Thesolventflowratewasmaintainedat0.12kg/h.The effects of temperature andpressurewerequantifiedusing a factorial experi-mentaldesign.Theoptimalextractionconditionswere150barand60ºC.Buttakingintoaccount the targetcomponents(fattyacidsandcarotenoids),whicharepronetodegradationathightemperatures,atemperatureof50ºCorlowerissuggested.Canela et al. [29] reported the composition of the supercritical extracts obtainedin terms of total carotenoids. The yields are very low when compared with thecarotenoidcontentofthemicroalgaereportedbyCohen[77].Thisbehaviorcanbeattributedinparttothelowpressuresused,asthecarotenoidspresentaverylowsolubilityatthesepressures[84].
Supercritical extractions curves were modeled according to the Goto et al.
model [85], which was developed for the supercritical extraction of essential oilfrom peppermint. This model treats the solid substrate as a porous matrix. Thesoluteisextractedafteritsdesorptionfromthesolid.Diffusionoccursinsidetheparticlepores,andthereisamass-transferresistanceinthefilmsurroundingtheparticles.Themodelgaveagood representationof theexperimentaldata, andapartitioncoefficientandacombinedmass-transfercoefficientwereobtainedfromthefittingoftheexperimentalresultstothemainequationofthemodel.
6.2.7.2 Spirulina platensis
Santosetal.[26]performedexperimentsofSFEinsamplesofS. platensis usingCO2atatemperatureof40ºCandapressureof200bar.Bedsof10galgae(andsome-times20g)wereplacedinahalf-literextractionvessel.
Toevaluate theeffectof themoisturecontentof thealgae, threeexperimentswerecarriedout.Thefirstone,directlyonthemicroalgaesupplied(moisturecon-tentof3.24%),ledtoalipidyieldofabout1.1%(drybasiswt);thesecondtestwasperformed on a sample that had been dried in P2O5 desiccator for several hours;thethirdtestwasontheresiduefromthesecondone,whichhadbeendampedtogiveamoisturecontentof7%.Thedryingofthemicroalgaeproducedasubstantialfallinthelipidyield(0.3%inadryweightbasis).Furtherdampingoftheresidueenabledmorelipidicmaterialtobeextractedfromtheresidue,butstillalowyieldwasobtained(0.5%).
Inthiswork,thefattyacidsfromtheglyceridefractionobtainedintheexperi-mentswereidentified.Atypicalpercentageoftheobtainedfattyacidsissimilartothisone:palmitic(33%),palmitoleic(8%),stearic(2%),oleic(4%), linoleic(21%),andg-linolenic(32%).
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InordertomodeltheSFE,theauthorsconductedexperimentsattwosuperficialvelocities. When the amount of extracted lipids is plotted against the CO2 massused,thecurvesforthehigherflowratefallbelowthatcorrespondingtothelowerflowrate,butwhenyieldsareplottedagainsttime,theexperimentalpointsfallina common curve. This is consistent with a mass transfer mechanism within thealgaeparticles(bydiffusion),whichissoslowthattheextractantphasecomposi-tionchangesvery littlealong thebedandneverapproachesclose to theequilib-riumvalue.Theexternalfilmresistanceisnegligible.Basedontheseassumptions,Santosetal.[26]appliedthewell-knownsinglesphereextractionmodelandfoundthatthebestfitforDe/r2,whereDeistheeffectivediffusivity(ofthelipids)andtheristhealgaeparticleradius,ledtothefollowingvalue:Deπ2/r2=0.00120,forthedataresultingfromtheSFEoflipidsfromS. platensisatapressureof200barandtemperatureof40ºC.
Qiuhui[27]usedsupercriticalCO2extractionoflipidsfromthesemicroalgae,withthegoalofremovingitsbadsmell,anobstacletothemarketingandacceptanceofSpirulina,whichissoldandexportedasapowder.Infact,thisauthorpretendedtoseparateandpurifytheactivecomponentsofthealgae.TheSFEexperimentswerecarriedoutinasemicontinuousapparatus,providedwithrecyclingofCO2,ataflowrateof24kg/h;pressuresof30,35,and40MPa;extractiontimeof2,3,and4h,andtemperatureof40ºC.
Thehighestlipidyield(7.2%)wasobtainedat35MPaand4hofoperation,avaluenearthatobtainedat40MPa(7%)forthesameextractiontime.However,foranextractiontimeof2h,theyieldobtainedat40MPa(6.5%)washigherthantheoneobtainedat35MPa(5.9%).TheyieldofGLAincreasedwithpressure,havingobtained0.12%at20MPa,whileat40MPa0.29%wasreached.Bothyieldvaluesarehigherthantheone(0.05%)obtainedbyMendesetal.[32]at25MPa/50ºCfromS. maxima.
Theremovingofthedeleterioussmellwasalsoreached.Moreover,aftertreat-mentwith supercriticalCO2,theprotein contentwaspracticallynot altered,withonlyaslightreductionofabout1%inessentialaminoacidshavingbeendetected,therebypreservingthenutritivevalueofSpirulina.
Carerietal.[28]carriedoutstudiesofSFEonthestrainpacifica(acarotenoid-richdietaryproduct)ofthemicroalgaeS. platensis.Thetargetcompoundsforthestudy were the carotenoidsβ-carotene,β-cryptoxanthin, and zeaxanthin, and theauthorsproposedtofindthebestexperimentalconditionsfortheirrecoverythroughan experimental design procedure. Four parameters were investigated: pressure(150,250,and350bar), temperature(40,60,and80ºC),dynamicextraction time(40,70,and100min),andpercentageofethanol(involume)addedtotheCO2(5,10,and15%).Otherexperimentalconditionswere0.5gofdrypowderedalgae(50mmparticlesize)ina7-cm3extractor.Theextractionbyorganicsolventwasperformedusingtetrahydrofuranandpetroleumether[86].
Forallthreecarotenoids,thehighestpressure(350bar)ledtothehighestrecov-eries.Thetemperaturesthatmaximizedtherecoverieswere80ºC,70ºC,and60ºCfor zeaxanthin,β-cryptoxanthin, andβ-carotene, respectively. The effects of thevariousfactorswereanalyzedand,forallthecompounds,thetemperaturewasfound
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nottobesignificantasthemaineffect.Onthecontrary,thepressureofthesupercrit-icalfluidplaysanimportantrole,appearingtobesignificantforallthecarotenoids.
Atconstanttemperature,apressureincreasecausesanincreaseinfluiddensityandthuscouldhaveadoubleeffect: increasedsolventpowerofCO2andreducedinteraction between the fluid and the solid matrix, having as a consequence thedecreasingofthediffusioncoefficientathigherdensities.
Theamountofethanolwasalsosignificantforallthecarotenoids.Theincreaseinthepercentageoftheentrainerledtoanincreaseoftheextractionyields.Thiseffect is not only related to themodificationof thepolarityof the supercriticalfluidbutalso to the interactionof theethanolwith thesolidmatrixbecausethecarotenoidswithdifferentpolaritiesshowbetterrecoverieswhenethanolisaddedtothefluid.
Carerietal.[28]alsocomparedtheSFEobtainedatthebestconditionswiththatobtainedfromorganicsolventextraction.Intermsofyield,organicsolventextrac-tion showed a slight advantage, but because it is time-consuming (with multipleextractionandpurificationsteps)andveryexpensive,intheendSFE,provedtobeamoreeffectiveprocedure.
6.3 ConClusIon
The use of algae to obtain useful biochemicals for medical, pharmaceutical, anddietaryapplicationsshowsalargepotentialinthenearfuture.However,manyalgaealsoproduceharmfulcompounds.Althoughsomeofthesecanbedirectedtoobtainusefuldrugs,theymustbescreenedbeforehumanconsumptionoccurs.
Inmanycases,SFEshowsadvantagesovertheuseoforganicsolventstoobtaincompounds fromalgae,namelya shorter timeofextractionandbetteror similaryields,avoidingtheuseofexpensiveandpollutingsolvents.Moreover,SFEcanbeusedwithsupercriticalfluidfractionationandsupercriticalfluidchromatographytoseparateorpurifytheextractedcompounds.Becauseitisanexpensiveprocedure,SFEisonlyreservedforhigh-valuecompoundsincommercialapplications.
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80. Nakahara,T.etal.,Gamma-linolenicacidfromgenusMortierella,inIndustrial Appli-cations of Single Cell Oils,Kyle,D.J.andRatledge,C.,Eds.,AmericanOilChemists’SocietyPress,Champaign,IL,1992,61.
81. Kennedy, M.J., Reader, S.L. and Davies, R.J., Fatty acid production characteristicsof fungi with particular emphasis on gamma linolenic acid production, Biotechno. Bioengin.,42,625,1993.
82. Jaime, L. et al., Separation and characterization of antioxidants from Spirulina platensis microalgacombiningpressurizedliquidextraction,TLC,andHPLC-DAD,J. Sep. Sci.,28,2111,2005.
83. Certik,M.,Andrasi,P.andSajbidor,J.,Effectofextractionmethodsonlipidyieldandfattyacidcompositionoflipidclassescontainingg-linolenicacidextractedfromfungi,J. Am. Oil Chem. Soc.,73,357,1996.
84. Mendes,R.L.etal.,Solubilityofβ-caroteneinsupercriticalcarbondioxideandethane,J. Supercritical Fluids,16,99,1999.
85. Goto,M.,Sato,M.andHirose,T.,Extractionofpeppermintoilbysupercriticalcarbondioxide,J. Chem. Eng. Jpn.,26,401,1993.
86. Hart,D.J. andScott,K.J.,DevelopmentandevaluationofanHPLCmethod for theanalysis of carotenoids in foods, and the measurement of the carotenoid content ofvegetablesandfruitscommonlyconsumedintheU.K.,Food Chem.,54,101,1995.
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215
7 Application of Supercritical Fluids in Traditional Chinese Medicines and Natural Products
Shufen Li
Contents
7.1 Introduction................................................................................................. 2167.2 SpecialFeaturesofSFETechniqueinProcessingTCMand
NaturalProducts......................................................................................... 2177.3 StatusofSFEinProcessingTCMandNaturalProductsinChina............ 219
7.3.1 NationalSymposiumsonSCFTechnology..................................... 2197.3.2 SCFEquipmentMadeinChina....................................................... 2197.3.3 SummaryofApplyingSFEinProcessingTCMand
NaturalProducts..............................................................................2207.3.3.1 SFEwithPureSupercriticalCarbonDioxide....................2207.3.3.2 SFEwithCO2inPresenceofCosolvent............................2207.3.3.3 SFEwithCO2inPresenceofSurfactant............................ 2217.3.3.4 CombingSFEwithUltrasound-Enhanced
ExtractionMethod..............................................................2237.3.3.5 CombiningSFEwithEnhancedSeparationMethods.......2237.3.3.6 CombiningSFEwithOtherTechniquesto
MakeFullUseofHerbalMaterials....................................2247.4 SelectExamplesofSFEofTCMandNaturalProducts.............................225
7.4.1 ExtractionofEssentialOilfromCloveBudwithSC-CO2..............2257.4.2 ExtractionofMedicalIngredientsfromtheMixtureof
Angelica sinensisandLigusticum chuanxiongHortwithSC-CO2...2287.4.3 ExtractionofEdibleandMedicinalIngredientsfrom
GrapeSeedswithSC-CO2...............................................................2307.4.4 IsolationofOrganochlorinePesticidefromGinsengwithSC-CO2... 233
7.5 SummaryandProspect............................................................................... 236References.............................................................................................................. 237
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216 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
7.1 IntroduCtIon
TraditionalChinesemedicine(TCM)isascientificsummaryofrichexperiencesoftheChinesenation’sstruggleagainstdiseaseforthousandsofyears.Itisoneoftheoldestandstrongesttraditionalmedicalsystemsinthehistoryoftheworld.ClassicalChinese herbal medicines encompass a large number of herbal formulations withknownmildpharmaceuticaleffectsandminimumsideeffectsthatareusedforthetreatmentofawidevarietyofdifficult-to-treatdiseases.Overthecourseofmanycenturies,TCMhasgreatlyformedauniquetheoreticalsystemanddiagnosingandtreatingtechniquesthathavemadeanindelibleandsubstantialcontributiontoboththehealthandprosperityoftheChinesepeople[1,2].TCMhasnotonlyenjoyedanexcellentreputationinChinabutalsointherestoftheworld.Itwillplayamoreandmoreimportantroleincontributingtothehealthandlongevityofmankind.
InChina,morethan11,000plantsareconsideredtobemedicineherbs.Almost2,000ChinesetraditionalpatentmedicinesarelistedintheofficialChinesePharma-copoeia.ThesemedicinesarewidelyusedasTCMinChina,eveninSoutheastAsia[1–4].Mostofthemareprocessedwithmanykindsofmedicineplantsaccordingtothetheoryofprescriptioncomposition.However,somearecomposedofonlyasingleplant.TheefficacyofChineseherbalmedicines is considereda synergismofmanyeffectivecomponents, includingnotonly thesmallmoleculecompoundssuchasvolatileoils,alkaloids,flavonoids,andsaponinsbutalsobiologicalmacro-moleculessuchaspolysaccharides,proteins,andpeptides.
Themosttraditionalmethodforprocessingherbsinvolvesboilingtheminwaterforhourssothatmostoftheingredientsaredissolved.Anothermethodinvolvestheuseofconventionalorganicsolventsforextractioninsteadofboilingwater;themostcommonlyusedorganicsolventsareethanol,ether,chloroform,andmethanol.Whenthetraditionalextractionmethodsforprocessingherbsareused,theextractsconsistofvariouscompounds,includingsomeundesiredsubstancesthatdissolvewiththedesiredproducts.Therefore,furtherpurificationstepsarenecessarytoremovethecoextractedimpurities.Intheseprocesses,longprocessingtimesof2to7daysaregenerallyneeded.Highboilingorextractiontemperaturesoftenleadtodegradationofheat-sensitivecompounds.Moreover,tracesoftoxicsolventsarehardlyremovedfromtheextracts,whichdirectlyinfluencesthequalityoftheproducts.Hydrodistil-lation(steamdistillation) isgenerallyusedforobtainingvolatileoilsfromplants.Itshighprocessingtemperaturecanalsoleadtodegradationofheat-sensitivecom-pounds. Therefore, alternative extraction techniques with better selectivity andefficiencyarehighlydesirable.
In recent years, the catchphrase “modernization and internationalization ofTraditionalChinesemedicine”hasoftenbeenpresentedinChinesepapers,maga-zines, and symposiums, and this concepthasbecomeaveryhot topic [5,6].Forthis reason, the pharmaceutical study of natural products has become one of themost interesting and active research areas in China. Some new chemical separa-tiontechnologies,suchassupercriticalfluidextraction(SFE),membraneseparation,ultrasonic-assist extraction,molecular distillation (MD), andpolymeric adsorbenttechnology,havebeenconsideredtohelpimprovetheproductiveprocessofTCM
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Application of Supercritical Fluids in Traditional Chinese Medicines 217
[7,8].Theseeffortshavebeensuccessful.Amongthesenewtechnologies,SFEispresentlyconsideredasoneofthemostcleanandhighlyeffectivetechnologiesforprocessingTCM.
The high solvent power of supercritical fluid (SCF) was first reported over acentury ago. Demonstration of SFE technology for industrial applications wasreportedbyZoselin1970[9].Sincethen,thefundamentalandappliedaspectsofSCFandprocesseswithapplicationscoverawiderangeoftopicsinenergy,environment,medicine,chemicalindustries,andanalyticalfield.Ithasbeenalsorapidlyextendedtootherfields,suchaschemicalreaction,supercriticalfluidchromatography(SFC),andparticleformationinmaterialprocessing withSCF[10,11].
This chapter focuses on introducing the SFE techniques used in processingTCMandnaturalproducts.ThespecialfeaturesofSFEtechniquesandthestatusofSFEinprocessingTCMandnaturalproductsinChinaarebrieflyreviewed.FourtypicalexamplesofSCFapplicationinTCMandnaturalproductsfromourlabora-toryresearchhavebeenselectedtomakefurtherdescription.
7.2 speCIal Features oF sFe teChnIque In proCessIng tCM and natural produCts
Agas,whencompressedisothermallytopressuresgreaterthanitscriticalpressure,exhibits enhanced solvent power in the vicinity of its critical temperature. Suchfluid is called supercritical fluid. SCFs possess desirable specific characteristicsthatmakethemattractiveassolvents.Liquid-likedensitiesandgas-likeviscosities,coupledwithdiffusioncoefficientsthatareatleastanorderofmagnitudehigherthanthoseofliquids,contributetotheenhancementofmasstransfer.Inparticular,adjusting pressure and temperature can control the solvent density and hencesolventpower,becausethesolventpowerofaSCFrelatestothesolventdensityinthecriticalregion[12–14].
Among SCFs, supercritical CO2 (SC-CO2) remains the most commonly usedfluid for SFE application because of its mild critical properties (Tc = 31.1°C,Pc=7.38MPa),nontoxicity, chemical inertness, andavailability inhighpurity atlowcost.TheseexcellentpropertiesleadCO2tobeconsideredan“environmentallyfriendly”solvent forextractionofnaturalproducts,suchascoffee, tea,hops,andselectedspices[12–13].
Asisknown, thedipolemomentofCO2iszeroanditspolarizability isonly26.5× 10–25cm–3,which is less than thatofallofhydrocarbonsexceptmethane[14].Therefore, SC-CO2 is only a good solvent for extraction of nonpolar com-pounds,suchashydrocarbons,whileitslargequadripolemomentalsoenablesittodissolvesomemoderatelypolarcompounds,suchasalcohols,esters,aldehydes,andketones[9].WhenpureSC-CO2isemployedasasolventforprocessingnaturalproducts,mixturescontainingbothnonpolarandmoderatelypolarsubstancesaregenerallyextracted.
Both the properties of the solute and the solvent can affect the extractabilityof natural products. Vapor pressure, polarity, and molecular weight of solutesare themost important factors affecting the solubilityof solutes inSCF.Raising
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218 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
the temperature raises the vapor pressure or sublimation pressure of solutes andhence increases the solubility of the solute. However, increasing the temperaturealsocausesasimultaneousdecreaseinthedensityofCO2,whichtendstodecreasesolubility.Twocompetingfactorsneedtobeconsideredtofindthesuitabletempera-ture.RaisingthepressureincreasesthedensityofthesolventofSC-CO2andhencethesolventpowerofthesolutes.However,thebenefitofthisincreaseisoftenlimitedbymanufacturingabilityofhigh-pressureequipmentandcapitalcosts.Selectingasuitablecosolventorentrainerthatcanmaintainorimproveselectivityandincreasesolubilitymaybeoneof thekeystoexpandingtheapplicationofSFE.Theaddi-tionofacosolventcannotonlyshift thecriticalpropertiesfromthepuresolventcriticalpropertiesandhenceaffect thepropertiesof theSCFbutcanalso inducecosolvent-soluteinteractionsorassociations,suchasacid-baseinteractions,depend-ingonthepropertiesofthesolute,solvent,andcosolvent,anyofwhichmayenhancethesolubility[15–19].
Inmostcasesofsolventextractionfrombotanicalsubstances,fourstepsofmasstransportoccur:
1.Diffusionofsolventintothebotanicalsubstance 2.Solvationofsolute 3.Diffusionofsoluteintobulkfluidphase 4.Transportofsoluteandthebulkfluidphasefromtheextractionzone
Usually,thediffusionofthesolutesoutofthematrixisthelimitstep.Inordertoreducethediffusiondistanceofsolutesthroughthebotanicalsubstrateandfurtherrupturingofthecellwall,henceeliminatingsomediffusionbarriers,itisnecessarytogroundthenaturalrawmaterialintotheoptimumsizebecauseparticlesizehassomeeffectonyieldandrateofrecovery[20–25].
SFEprocessesneedtoconsiderbothextractionandseparation.Threebasicoper-ationmodelscanbeusedtoseparatesolutesfromSCFsolvents:pressurereduction,temperaturevariation,andadsorption.Eachoperationalmodelhas itsadvantagesandlimitations.SFEwithextractseparationbyvaryingthetemperatureisoperatedin isobaric state.Extract separationby adsorption allows theSFEprocess to runisobaricallyandisothermally,andsoSCFcanbecirculatedwithoutrecompression.Thesetwomodesrequirelessenergyconsumption.However,pressurereductionisthemostusedmodeinprocessingofTCMbecausetheoperationcanbemademorestablebyeffectivelycontrollingtheliquidCO2levelwhenreducingtheseparationpressureandtemperaturetobelowthecriticalvalues.
ForsuccessfulSFE,somefactorsmustbetakenintoconsiderationpriortotheexperiments.Thesefactorsincludethetypeofrawmaterials,methodoffeedprepara-tion,typeoffluid,choiceofcosolvents,methodoffeedingcosolvents,andextractionandseparationconditions,includingpressure,temperature,flowrate,andextractiontime.TooptimizeSFEconditions,astatisticalexperimentaldesignbasedonortho-gonalexperimentsiscommonlyusedandreported,wheretheyieldandthecontentoftheactivecompoundintheextractsareoftenconsideredasthetargetindex.
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Application of Supercritical Fluids in Traditional Chinese Medicines 219
7.3 status oF sFe In proCessIng tCM and natural produCts In ChIna
7.3.1 NatioNal SympoSiumS oN SCF teChNology
Asoneofthegreenchemistryandengineeringtechnologies,SCFscienceandtech-nologycallsgreatattentionfromthegovernment.Manynationalprojectsrelatingto SCF science and technology were supported by the government. Many enter-prisesalsocarryoutsomeresearchanddevelopmenttogetherwiththeresearchersofuniversitiesandinstitutes.Inaddition,nationalsymposiumsonSCFscienceandtechnologyhavebeenheldevery2yearssince1996.Uptofivesymposiumproceed-ingshavenowbeenpublishedinChina[26–30].
Table7.1summarizesthepresentationsfromthefiveChinesesymposiumsandarticlesappearingintheChinesecorejournalsinVIPChineseDatabank,whichwastheauthoritativeprofessionaldatabaseinChinathrough1998.ItcanbeseenfromTable7.1 that, although theapplicationofSCFhasalsobeen rapidlyextended tosupercriticalchemicalreaction,particleformation,SFC,andotherfields,theearliestandmostactiveresearchfieldcentersonSFE,especiallyitsapplicationsinextract-ingactivecomponentsfromChineseherbs.
7.3.2 SCF equipmeNt made iN ChiNa
Assupercritical statesoffluidsareathighpressures, the industrializationdesignandscale-upplantarethecoreissuesformostenterprisesandresearchinstitutions.Currently, at least seven setsof large-scale industrialSFEequipmentwere intro-duced fromEurope,ofwhich the largestonewasmade inGermany(UhdeHighPressure Technologies GmbH) and the extraction vessels’ configuration for eachof the threevesselsare3500L, respectively.Aside fromfor importing large-sizeinstruments, the domestic instruments for SCF technology especially in SFE arebeingdeveloped.Therearemorethan30setsofhomemadeSFEinstrumentswithextractorsizesover100L,and the largestsize is2000L.Additionally, thereare
table 7.1presentations in the Five Chinese symposiums and VIp on sCF technology
1st, 1996
2nd, 1998
3rd, 2000
4th, 2002
5th, 2004 VIp total
SFE 23 31(4) 42(1) 58 68 129 351
SCR 13(6) 16(4) 18(4) 17(6) 21 76 155
Particleformation 3 5 11 10 24 38 91
SFC 2 1 2 1 2 8 16
Others 1 2 7 8 13 21 52
Theoreticalstudy 12 13 19 18 16 37 125
Papersineachsymposium 48 68 99 112 144 309 790
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220 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
morethan150SFElaboratoryunitswithexactorvolumeslessthan25LlocatedinthemanyprovincesofChina[6].
There exist some obstacles to developing homemade SFE instruments. Forexample,theauto-controlsystemisstillrelativelybackwardandsomemanufacturelevelsforhigh-pressurepartscannotreachinternationalstandards.
7.3.3 Summary oF applyiNg SFe iN proCeSSiNg tCm aNd Natural produCtS
Atleast150kindsofChinesetraditionalherbalplantswereselectedasrawmaterialsto investigatewithSFE technology inChina. The research methods used canbeclassifiedintosixdifferentcases:
1.SFEwithpureSC-CO2
2.SFEwithCO2inpresenceofcosolvent 3.SFEwithCO2inpresenceofsurfactant 4.CombingSFEwithultrasound-enhancedextractionmethod 5.CombingSFEwithotherseparationmethods 6.CombiningSFEwithothertechniquestomakefulluseofherbalmaterials.
7.3.3.1 sFe with pure supercritical Carbon dioxide
Anoverviewof recentpublicationsonapplicationsofpureSC-CO2 inTCMandnaturalproductsisgiveninTable7.2.Thetargetextractsaremostcommonlyvola-tileessentialoils,whichareamixtureofnonpolarcomponentsandmoderatelypolarsubstances.Theextractiontemperaturesaregenerallyfrom30°Cto60°C,andtheinvestigatedpressureswerefrom8to40MPa,dependingonthepropertiesofboththerawmaterialusedandthedesiredextracts.Thesekindsofapplicationscanfullyshow the advantages of SFE over traditional solvent extraction, hydrodistillation,and steam distillation, such as higher yield with better quality, less hydrocarbonpollution,greatersafety,lowerproductioncost,andnodegradationofheat-sensitivecompounds.Asweknow,traditionalprocessingmethodsforthesekindsofherbalmaterialsaremostlyhydrodistillationandsteamdistillationorsolventextraction,inwhichhighboilingtemperaturesoftenleadtodegradationofheat-sensitivecom-pounds.Furthermore,tracesoftoxicsolventsarehardlyremovedfromtheextractswhensolventsareused,whichdirectlyinfluencesthequalityoftheproducts.
7.3.3.2 sFe with Co2 in presence of Cosolvent
SomeexamplesofusingSC-CO2inthepresenceofacosolventarelistedinTable7.3.Mostoftheextractsobtainedaremiddle-polarsubstances,suchasalkaloids,saponins,andflavonoids.Themostcommonlyusedcosolventsareethanolanddifferentcon-centrationsofaqueousethanolsolutions.Asisknown,comparedwithmethanolandchloroform, ethanol is less toxic andmoreacceptable forprocessingTCM.Wateris themost acceptableandcheapest solvent.By regulating the ratioofwater andethanol,onecanreadilymanipulatethepropertiesofthefluids.Usually,additionof
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Application of Supercritical Fluids in Traditional Chinese Medicines 221
asmallamountofaliquidcosolventcansignificantlyenhancetheextractioneffi-ciencyandconsequentlyreducetheextractiontimeorpressure.
7.3.3.3 sFe with Co2 in presence of surfactant
TheresearchandprogressofSC-CO2microemulsionverifythepossibilityofextract-ing polar compounds with SC-CO2. When an appropriate surfactant is added intotheSC-CO2,a reversemicroemulsioncanform,whichfacilitates thedissolutionofhydrophilic molecules in SC-CO2.The formation of SC-CO2 microemulsion needs
table 7.2overview on the extraction of active Compounds from Chinese herbals with sC-Co2
raw Materials Conditions extracts Yield (%) references
Arnebiaeuchroma(Royle)Johnst 35°C,27MPa Naphthaquinoniccompounds
4.1–4.6 [31]
AtractylodesmacrocephalaKoidz 50°C,28MPa Volatilecomponents 4.27 [32]
Beepollen 55°C,30MPa Lipophiliccomponents
5.0 [33]
Cortexalbiziae 35°C,30MPa Lipophiliccomponents
5.4 [34]
Curcumakwangsiensis 60°C,26MPa β-elemene 0.0271 [35]
EarofSchizonepetatenifoliaBriq. 50°C,20MPa Essentialoil 6.31 [36]
Figresidues 45°C,30MPa Anticancercomponents
2.53 [37]
GlycyrrhizauralensisFisch. 50°C,25MPa Essentialoil 1.69 [38]
LeavesofArtemisiaeargyi 32°C,15MPa Essentialoil 2.71 [39]
Liliumbrownii 50°C,18MPa Essentialoil 2.92 [40]
OcimumbasilicumL. 45°C,16MPa Essentialoil 4.96 [41]
Orris 55°C,26MPa Orrisoil 12.71 [42]
Perillafrutescens(L.)Britton 50°C,15MPa Essentialoil 2.5 [43]
RadixAngelicaedahuricae 35°C,25MPa Essentialoil 3.6 [44]
RadixLitseaeCubebae 55°C,30MPa Lipophiliccomponents
2.6 [45]
SalviacastaneaDielsf.tomentosaStib.
65°C,35MPa Tanshinones 2.9 [46]
Saposhnikoviadivaricata(Turcz)Schischk
35°C,22MPa Lipophiliccomponents
4–4.5 [47]
SchisandraChinensis(Turcz)Baill
50°C,25MPa Schizandrin Notavailable
[48]
StromaofCordycepskyushuensis 50°C,20MPa Essentialoil 9.72 [49]
Wheatplumule 35°C,20MPa Wheatplumuleoil Notavailable
[50]
Zanthoxylumseed 35°C,40MPa Zanthoxylumseedoil
10.32 [51]
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222 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
surfactantwithCO2-philicgroups,suchassiloxane,fluoroalkane,fluoroether,tertiaryamine,andalkyonol[69].
SC-CO2extractioninthepresenceofsurfactantandcosolventhasnotbeenwidelyusedyetinthefieldofTCM.However,someresearchersinChinahaveexploredthiskindofextraction.Forexamples,Chenetal.[70]observedtheeffectofsurfactantonenhancingtheefficiencyofSC-CO2extractionofephedrinefromephedra.Dioctylsodium sulfosuccinate (DSS), sodium dodecyl sulfate (SDS), 1-heptanesulfonate(SHS),andcarboxymethylcellulosesodium(CMC-Na)wereusedasthesurfactants,
table 7.3extraction of active Compounds from Chinese herbals by sC-Co2 in the presence of Cosolvent
raw Materials Condition Cosolvent extracts Yield (%) references
ApliniaOxyphyllaMiquelseeds
35°C,25MPa Ethanol Volatileoil 3.21 [52]
Astragalusroot 45°C,40MPa 95%ethanol AstragalosideIV 0.27 [53]
BranchesandneedlesofTaxusyunnanensis
40°C,34MPa Methanol Taxol 0.0057 [54]
CornusofficinalisSieb.etZucc.
45°C,35MPa Ethanol Urosolicacid 0.239 [55]
CorydalisyanhusuoW.T.Wang
40°C,15MPa 95%ethanol Tetrahydro-palmatine
0.039 [56]
Curcumalonga 55°C,25MPa Ethanol Curcumin 0.0024 [57]
Iristectorum 50°C,25MPa Chloroform Irone Notavailable
[58]
LigusticumchuanxiongHort.
45–65°C,30–50MPa
Ethanol Femlicacid 0.735 [59]
Polygonumcuspidatum
50°C,25MPa 95%ethanol Resveratrol Notavailable
[60]
PolygonummultiflorumThunb
50°C,30MPa Chloroform+methanol
Phospholip 3.11 [61]
PricklyashPeel 35°C,20MPa Ethanol Essentialoil 13 [62]
Propolis 40°C,35MPa 95%ethanol Flavonoids 34.9 [63]
PterissemipinnataL. 60°C,25MPa Ethanol Diterpenoids 0.117 [64]
RhizomeofCoptischinensisFranch
60°C,50MPa 1,2-propanediol Berberine 7.53 [65]
Salviamiltiorrhizabunge
60°C,25MPa Methanol TanshinoneIIA 0.038 [66]
Sinomeniumacutum(Thumb)RehdetWils
60°C,30MPa Methanol Sinomenine 0.747 [67]
Taxusmaireibark 45–50°C,30–35MPa
Ethanol Taxoids Notavailable
[68]
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Application of Supercritical Fluids in Traditional Chinese Medicines 223
andtheirinfluencesonextractionofephedrinefromephedrabySFEwerestudied.TheresultsindicatethatDSS,SDS,SHS,andCMS-NaenhancedtheefficiencyofSFEby246.8%,123.4%,83.0%,and53.2%, respectively,whichwasdue to theirmolecularconstitutions.Themoreliposolublepartsthesurfactantshave,thehighertheefficiency.TheapplicationofsurfactantsofferedavaluablewayforSFEofalka-loid.Geet al. [71] studied theuseofTween-80andSpan-80 in theextractionofmatrinesfromKuh-seng.Andtheyfoundthattheyieldis1.8–2.2timesmorethanthatof themethodwithout the surfactant.Satisfactory resultswerealsoachievedwhen Wang et al. [72] use nonionic surfactant, Span-80, and Tween-80, togetherwithwaterandethanol,inacertainproportionasmodifierstoextractlactonesfromatractylodesmacrocephalaKoidz.Thecontentofthelactonesintheextractivecanreachto87.78%atextractiontemperatureof15°Candpressureof30MPa.
7.3.3.4 Combing sFe with ultrasound-enhanced extraction Method
Most TCMs are solid materials and the mass transfer rate of the solid materialsis limitedby thediffusion inside theparticles.Thatmakes themass transfer rateslowintheSCFandleadstoalongerextractiontimeasaresult.ThisbottleneckofSFE,however,canbesolvedbyintroducingultrasoundintotheSCF.Anumberofphysicaleffects(turbulence,particleagglomeration,andbiologicalcellrupture)aswellaschemicaleffects(freeradicalformation)possessedbyultrasoundfacilitatethemasstransferinSCF.
Dingetal.[73]useddouble-frequencyultrasoundsalternatelytoenhanceSFEofflavonoidsfromToonasinensis.Ethanolwasalsousedasacosolvent.Dingetal.demonstrated in their research that thesuccessiveorderofdifferenteffect factorsontheyieldiscosolventamount>ultrasonicfrequency>extractiontemperature>extractionpressure>ultrasonicpower,andtheoptimumconditionsoftheextrac-tionincludetemperature50°C,pressure20MPa,cosolventamount2mL/g,ultra-sonicfrequency20kHz,andpower150W.Whenextractingoilandcoixenolidefromadlayseeds,Huetal.[74]foundenergysavingsaftertheintroductionofultrasoundbecausetheextractiontemperature,pressure,andrateofCO2couldbedecreasedandextractiontimecouldalsobeshortened.
7.3.3.5 Combining sFe with enhanced separation Methods
7.3.3.5.1 SFE Combined with Pressured Fractional DistillationSFEcoupledwithpressuredfractionaldistillationhasreportedlybeenusedtocon-centratew-3fattyacidsfromfishoils[75].Itisnowusedinseparationandpurifica-tionofTCMandnaturalproducts.Forexample,Liuetal.[76]usedSFEcoupledwithdistillationtoextractandconcentratevitaminEfromsoybeanoildeodorizer.Li[77]has successfully used this combined technology for extracting the lipid fractionsfromadlayseeds.Theindustrialscale-upofthisprocesshasledtoreplacementoftheoriginalsolventextractionmethodandhasobtainedapprovalfromChina’sFoodandDrugAdministrationforapplicationinthepharmaceuticalindustryformanu-facturingTCM.
The lipidfractionsofadlayseedsare theactivepharmaceutical ingredientsofKanglaiteInjection,aparenteraldrugapprovedinChinaandRussiafortreatmentof
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224 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
advancednon-small-celllungcancer.ExtractionofadlayseedoilintheextractiontankwasfirstlycarriedoutwithSC-CO2attemperaturesof30°Cto45°Candpressuresof22MPa.TheCO2withdissolvedcrudeadlayseedoiltheninturnenteredtothesepa-rationcolumnandtwoseparationtankstoremoveimpurities,includingfattyacids,moisture,andpigments.WhenSFEiscombinedwithpressuredfractionaldistilla-tion,thechangeoftemperatureandpressurecanresultinachangeofrelativesepara-tionfactor.Asaresult,thequalityofadlayseedoilextractedwithSC-CO2extractionhasattainedthestandardofrefinedoilintermsofitsqualityspecifications.
7.3.3.5.2 SFE Combined with MD or HSCCC TechniquesSFE extracts are generally not a single compound but rather a complex mixtureof effective components, including some impurities. Sometimes, in order to geta component of high purity, it is necessary to use other separation techniques tofurthertreatmentextractsofSFE.MD,high-speedcountercurrentchromatography(HSCCC),silicagelcolumnseparation,solidphaseextraction,andotherprocessesarepresentlybeinginvestigatedforthispurpose,withMDandHSCCCbeingthemostwidelyinvestigatedprocessesinChina[78–88].
MDisaliquid-liquidextractiontechniqueinahigh-vacuumcondition,whichhasthefeatureoflowdistillationtemperature,shortheatingtime,andhighselectivity.ThecombinedtechniqueofSFEandMDismostlyusedtoextracttheessentialcom-ponentsofTCM.AfterMDprocessing, thecomponentsof lowmolecularweightintheSFEextractscanbeconcentrated.Zhangetal.[78–83]usedthiscombinedtechniquetoextracttheeffectivecomponentsofrhizomaatractylodismacrocephala,garlic, forsythia suspense, radix angelicae pubescentis, Spirulina, and ligusticumwallichiiFranch,andalltheresultsweresatisfactory.Forexample,whengarlicwasextracted with SC-CO2, 16 compounds in the extractive were identified, whereasonly 4 active compounds (diallyl disulfide, 3-ethenyl-1,2-dithia-cyclohex-5-ene,2-ethenyl-1,3-dithia-cyloohex-5-ene,anddiallyltrisulfide)wereobtainedbymolecu-lardistillationoftheextractive[80].
HSCCCisauniqueliquid-liquidpartitionchromatographytechniquethatusesnosolidsupportmatrix.Iteliminatestheirreversibleadsorptivelossofsamplesontothesolidsupportmatrixthatoccurswithuseoftheconventionalchromatographiccolumn[84]. Cao et al. [85–86] used HSCCC to purify the catechins and free fatty acidsextractedbySFEfromcratoxylumprunifoliumDyerandgrapeseedsandobtainedpuritiesof98%and99%,respectively.Wangetal.[87]gotpsoralenandisopsoralenpurities of above 99% when they combined SFE with HSCCC. Similarly, Pengetal.[88]gotflavonoidsof97.6~99.2%purityfromPatriniavillosaJuss.Theliteraturementions[84]thatcountercurrentchromatographycouldbeusedinalittlelargerscale,whichsuggestsabrightfutureforcombinedSFEandHSCCCtechniques.
7.3.3.6 Combining sFe with other techniques to Make Full use of herbal Materials
Sometimes,medicalplantshavemorethanonekindofeffectivecomponent.Inordertomakefulluseoftheplant,researcherstrytoisolatedifferenteffectivecomponentswithdifferentmethods.SFEhastheadvantageofextractingnonpolarandmoder-atelypolarsubstances,soitisusuallyusedtoextractthelipophiliccompounds.
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Application of Supercritical Fluids in Traditional Chinese Medicines 225
Zhangetal.[89]obtainedlipophiliccomponentsoftanshinoneandhydrophiliccomponentsofdanshensuandprotocatechualdehydeatonetimebycombiningSFEwithwaterboiling.Ourlabalsodidsomeworktomakefulluseoftherawmateri-als.Yeetal.[90]usedSC-CO2toextractoilfromgrapeseeds,theresiduesofwhichwereextractedwithhotwater and thendepositedwithappropriatealcohol togetproanthocyanidin.Xiaoetal.[91]combinedSC-CO2extractionwithsolventextrac-tiontoobtainbothessentialoilandalkaloidsfromNelumboNuciferaGaertn.
Another case is that the active compounds with stronger polar in herbs aredesired,buttheherbsalsocontainacertainamountoflipophilicsubstances,whichwereonceremovedasimpuritieswithtraditionalsolventextractionmethods.Withthefeatureofconvenientoperation,highsafety,highremovalratio,andeasyiso-lationofsolvent,SFEisnowadopted toremovethe lipophiliccompoundsbeforeextractionofeffectivecomponentswithothertechniques.
Inorder toextractpolysaccharidefrommongoliamushroom,Wangetal.[92]firstinvestigatedtheeffectofpretreatmenttodegreaseanddecolarmongoliamush-roombySC-CO2orbysolventextraction.Theexperimentalresultsindicatedthat,whensuitableextractionconditionswereusedwithCO2,theeffectofdegreasinganddecolarwasexcellent.Moreover,thepretreatmentprocessfavoredtheextractionofpolysaccharide.TheextractionyieldofthepolysaccharidewithpretreatmentbySFEis1.8foldthatwithsolventpretreatmentand4.2timesthatwithoutpretreatment.
7.4 seleCt exaMples oF sFe oF tCM and natural produCts
Inourlaboratory,morethan20kindsofherbalplantswereselectedasrawmaterialstoinvestigateSFEprocessing.Fourtypicalexampleswerebrieflyreported,whichindicatethatdifferentSFEprocessesandparameterscanbedevelopeddependingonprocessingpurposeandthepropertiesoftherawmaterials.
7.4.1 extraCtioN oF eSSeNtial oil From Clove Bud with SC-Co2
Clove(Eugenia caryophyllataThunb.) iswidelycultivated in thesouthofChina.Clove bud oils contain high contents of eugenol, which give it strong biologicalactivity and antimicrobial activity. Beside eugenol, clove bud oils also containsomeamountofotheractivecompoundsofeugenolacetateandβ-caryophyllene.Clovebudoilhasseveraltherapeuticeffects,includingantiphlogistic,antivomiting,analgesic,antispasmodic,carminative,kidneyreinforcement,andantisepticeffects.Italsoisusedasaflavoringagentandantimicrobialmaterialinfood[93–95].
Extraction of clove oils from clove bud with SC-CO2 was investigated [96].TheherbalmaterialsofclovebudweregroundbyaFW80SampleMillmachinein different periods to get different particle distribution, which was measured bymechanical sieving after extraction and calculated by weight of different size ofclovebud particle.Gradesof particle size were classifiedon the following scale:1=<10mesh;2=10~20mesh;3=20~40mesh;4=40~60mesh;5=60~80mesh;6=80~100mesh;7=100~120mesh;8=>120mesh.Particlesizeindexwascalcu-latedbythefollowingformula:
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226 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
Particle size indexweight of each grade= × gradetotal weight highest grade×∑ (7.1)
Theparticlesize indexesofmaterial in thisexperimentwere0.7944,0.6430,and0.5223,namedas1#,2#,and3#respectively.
Thefollowingparameterswereused:temperature,30°C,40°C,and50°C;pres-sure,10MPa,20MPa,and30MPa;andparticlesize,1#,2#,and3#.Alltheselectedfactorswereexaminedusingathree-levelorthogonalarraydesignwithanOA9(33)matrix,asshowninTable7.4.Itcanbeseenfromtheorderofthemaximumdiffer-encesthatparticlesizehadthemostinfluenceontheoilyield,thentemperatureand
table 7.4three-level orthogonal design and experimental results for extraction of Clove oil with sC-Co2 [96]
run no.Factor a (t/°C)
Factor b (p/Mpa)
Factor C (particle size/#)
Yield (kg extract/
kg feed)
eugenol Content
(%)
1 1(30) 1(10) 1(1#) 0.2056 53.69
2 1(30) 2(20) 2(2#) 0.1943 54.22
3 1(30) 3(30) 3(3#) 0.1830 55.64
4 2(40) 1(10) 3(3#) 0.1910 56.20
5 2(40) 2(20) 1(1#) 0.2224 54.52
6 2(40) 3(30) 2(2#) 0.2043 55.30
7 3(50) 1(10) 2(2#) 0.1956 58.77
8 3(50) 2(20) 3(3#) 0.1827 57.83
9 3(50) 3(30) 1(1#) 0.2395 56.97
Yield(%)
K1 0.5830 0.5923 0.6676
∑=1.8186
K2 0.6178 0.5995 0.5942
K3 0.6179 0.6269 0.5568
K1/3 0.1943 0.1974 0.2225
K2/3 0.2059 0.1998 0.1981
K3/3 0.2060 0.2090 0.1856
R 0.0117 0.0116 0.0369
Eugenolcontent(%)
K1 163.55 168.66 165.18
∑=503.14
K2 166.02 166.57 168.29
K3 173.57 167.91 169.67
K1/3 54.52 56.22 55.06
K2/3 55.34 55.52 56.10
K3/3 57.86 55.97 56.56
R 3.34 0.25 1.04
ReprintedfromFood Chemistry,101,1558–1564,©2007.WithpermissionfromElsevier.
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Application of Supercritical Fluids in Traditional Chinese Medicines 227
pressure.However,thesequenceoftheinfluencesoftheparametersontheeugenolcontentintheoilswastemperature,particlesize,andthenpressure.Thefactoroftemperatureshowsthemaximuminfluenceontheeugenolcontentintheoils.
Basedontheabovedatafromthethree-levelorthogonalarraydesign,Figure7.1furthershowstheeffectoftemperature,pressure,andparticlesizeontheyieldandeugenolcontentofcloveoilextractedbySC-CO2.Itcanbeobservedthatincreaseoftemperaturefrom30°Cto40°Cresultsinanincreaseoftheextractionyieldandhigheugenolcontentintheoils,whiletheincreaseoftemperaturefrom40°Cto50°Cdoesnotresultinanincreaseoftheoilyield,andthereisanincreaseofeugenolcontentincloveoil.Theextractionyieldenhancedsignificantlywith increaseofpressureduetotheincreaseofthesolubilityoftheoilcomponents.Thisincreaseisattrib-utedtotheincreaseoftheCO2density,whichresultsinanincreaseofitsdissolvingability.However,becausethehigh-molecular-weightcompoundsinclovebuds(fattyacids,fattyacidsmethylesters,sterols,etc.)werealsocoextractedwithincreaseofpressure,theeugenolcontentofthecloveoildoesnotobviouslychange.Theextrac-tionyieldincreasesbydecreasingtheparticlesizeofthecomminutedclovebudsduetothehigheramountofoilreleasedwhenthebudcellsaredestroyedbymilling,andthisamountofoiliseasilyextractedfordirectexposuretotheSC-CO2.However,theeugenolcontentinthecloveoilincreasesasparticlesizeincreases.Therefore,theparticlesizeshouldnotbetoosmallinordertoavoidcoextractionofmorecom-poundswithhigh-weightmolecules.
Gas chromatography with Mass spectrometry (GC/MS) analysis was used toidentifythecompoundsinthecloveoilsextractedwithSC-CO2.Twenty-threecom-poundswereidentified.Comprehensivecomparisonofthecloveoilsobtainedbydif-ferentmethodsislistedinTable7.5.ThecontentofthemainbiologicalingredientsofeugenolpluseugenolacetateinthecloveoilbySoxhletextractionislowest,althoughitsyieldofcloveoilishighestamongthefourextractionmethods.Furthermore,the
Pressure (MPa) Particle Size (index)Temperature (°C)50
60
58
50
52
54
56
2# 3#1#4030
0.23
0.18
0.19
0.20
0.21
0.22
Euge
nol i
n O
il (%
)
Yield
(kg
extra
ct/k
g fe
ed)
10 20 30
Eugenol Content Yield
FIgure 7.1 Effectoftemperature,pressure,andparticlesizeonyieldandeugenolcontentofcloveoilextractedbySC-CO2.(ReprintedfromFood Chemistry, 101,1558–1564,©2007.WithpermissionfromElsevier.)
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228 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
extractbySoxhletmethodisbrownointment,whichmeansmoreundesiredimpuri-tiesandorganicsolvent residuemayhaveexisted.SFEoffers themost importantadvantagesoverothermethods.ExtractionyieldofSFEwasabouttwotimesashighasthatobtainedbysteamandhydrodistillation.Thehighestcontentofeugenolpluseugenolacetateintheextractedoilwasalsoobtained.PaleyellowoilisdesiredandshortestextractiontimeisneededforSFEcomparedwiththeotherthreeextractionmethods.
7.4.2 extraCtioN oF mediCal iNgredieNtS From the mixture oF AngelicA sinensis aNd ligusticum chuAnxiong hort with SC-Co2
Angelica sinensis (Oliv.)DielsandLigusticum chuanxiong horthavebeenwidelyusedasTCMtotreatpathologicalconditionssuchasatherosclerosisandhypertension.TheirphytochemicalprofilesanalysessuggestthatAngelica sinensisandLigusticum chuanxionghortcontainsimilarsubstances,suchasferulicacidandessentialoil.FerulicacidisoneofthemostimportantmedicalcomponentsinAngelica sinensisandLigusticumchuanxiongbecauseitpossessesantioxidativepropertiesbyvirtueofthephenolichydroxylgroupinitsstructure.Studieshaveshownthatferulicacidcouldinhibitmalondialdehyde(MDA)productionfromplatelets,inhibiterythrocytelysesinducedbyMDAandhydroxylradical,andinhibitlipidperoxidationinducedbyH2O2andO2[97–99].
ExtractionofferulicacidfromamixtureofsimilarportionsofAngelica sinensisand Ligusticum chuanxiong hort was firstly carried out with SC-CO2. As ferulicacid was considered to be the active component for preventing heart disease, itscontentinextractswasanalyzedbyhigh-performanceliquidchromatographyandtheanalyzingresultswereusedasthequalitycontrolindexformedicalefficiency.
Theeffectsofextractiontemperature,extractionpressure,particlesize,andmate-rialsourcesontheextractyield(E)andthecontentofferulicacidinextracts(C)usingpureCO2wasfirstexperimentallyinvestigated,aslistedinTable7.6.AsshowninTable7.6,theextractyieldsincreasedfrom2.95%to3.95%andthecontentofferulicacidinextractsincreasedfrom0.25%to0.28%whenpressureincreasedfrom20to50MPaatconstanttemperatureof65°C.Thesechangesoccurredbecauseincreas-ing the pressure at constant temperature increases the density of SC-CO2, whichfurtherincreasesthesolvationpoweroftheSCF.Whentemperaturesincreasedfrom
table 7.5Characteristic of the Clove oils obtained by different Methods [96]
Clove oil Yield (%)
eugenol plus eugenol acetate
(%)extraction period (h)
Color and texture
organic solvent used
SFE(50°C,10MPa) 19.6 58.8+19.6 2 Paleyellowoil No
Steamdistillation 10.1 61.2+10.2 8–10 Paleyellowoil Yes
Hydrodistillation 11.5 50.3+3.2 4–6 Brownyellowoil Yes
Soxhletextraction 41.8 30.8+9.3 6 Brownointment Yes
ReprintedfromFood Chemistry,101,1558–1564,©2007.WithpermissionfromElsevier.
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Application of Supercritical Fluids in Traditional Chinese Medicines 229
35°Cto65°Catapressureof30MPa,theextractyieldsincreasedfrom3.18%to3.63%andthecontentofferulicacidinextractsincreasedfrom0.18%to0.26%.
TheeffectofparticlesizeontheextractyieldisalsoshowninTable7.6.Extractyields increased from 3.63% to 5.52% with particle size decreasing from 20–40meshesto60–80meshes.Therefore,itisnecessarytogroundthenaturalrawmate-rialsintotheoptimumsizeinordertoreducethediffusiondistanceandtoimproveextractionefficiency.However,thecontentofferulicacidintheextractsdecreasedfrom0.26%to0.21%,indicatingothercomponentsmayalsobeextracted.
AlthoughSC-CO2hasbeenwidelyinvestigated,itisapoorextractantforpolarsubstances.Inordertoincreasethepowerofsolventforextractingpolarferulicacid,threecosolventswereemployed:ethanol,ethylacetate,andn-butylalcohol.Differ-ent ratiosofcosolvents to rawmaterial (w/w)werealsostudied.Cosolventsweredirectlyadded into the rawmaterialsandsoaked for4hoursbeforecarryingoutSC-CO2extractionundertheconditionsof65°Cand30MPa.AftertheSFEprocess,theextractswerevaporizedwithanevaporatorinavacuumtoremovethesolvent.TheexperimentalresultsarelistedinTable7.7.
Table7.7 shows that all of three cosolvents not only greatly enhanced thecontentsofferulicacid in theextractsbutalso increased theextractyieldgreatly
table 7.6experimental data of extraction of Ferulic acid from the Mixture with pure Co2
temperature (°C)
pressure (Mpa)
particle size (mesh) e(%) C(%)
65 20 20–40 2.95 0.25
65 30 20–40 3.63 0.26
65 40 20–40 3.79 0.28
65 50 20–40 3.95 0.28
35 30 20–40 3.18 0.18
45 30 20–40 3.32 0.23
55 30 20–40 3.50 0.24
65 30 20–40 3.63 0.26
65 30 40–60 4.40 0.22
65 30 60–80 5.52 0.21
table 7.7experimental results of different Cosolvents
Cosolvent t (°C) p(Mpa) e (%) C (%)
PureCO2 65 30 3.63 0.26
Ethanol 65 30 5.16 0.58
Andn-butylalcohol 65 30 5.13 0.52
Ethylacetate 65 30 4.69 0.31
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230 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
compared with pure CO2extraction. Because ethanol is one of the few acceptedorganicsolventsinthefoodandmedicineindustries,itwasemployedtostudytheratioofcosolvent.Theexperimentalresultsshowthattheextractyieldof7.12%andthecontentofferulicacidof0.83%inextractivewereobtainedwhentheratiooftheethanoltorawmaterialswas1.6.
ThepercolationmethoddescribedinthePharmacopoeiaofPeople’sRepublicofChina(2000year)wasemployed.Ethanolwithconcentrationof95%wasusedasasolvent.Beforepercolation,thepoweredmixturewassoakedwithsolventfor24hours.Thepercolationflowratewas15drops/minandthepercolationtimewasabout8hours.Afterpercolation,theextractswerevaporizedwithavacuumrotatoryevaporatortoremovethesolvent.Thematerialwasgroundusingamixer-grinder.ComparisonsofSFEwithpercolationmethodarelistedinTable7.8.
It can be seen that both the extract yields and the content of ferulic acid inextractsbypureCO2arethelowestamongthethreeprocessingmethods.Addingasuitablecosolvent,suchasethanolinthisstudy,couldgreatlyincreasethecontentofferulicacidinextracts,whichissuperiortothetraditionalpercolationmethodofextractingpolarferulicacidfromthemixtureofAngelica sinensisandLigusticum chuanxiong hort. This method may be one key way to make use of a suitablecosolventforincreasingthesolventpowerofCO2andtoexpandtheapplicationofSFE.However,SFEextractsfromtheherbsofthemixtureofAngelica sinensisand Ligusticum chuanxiong hortaregenerallyacomplexmixtureof thecomponents,whichmayhavesomedifferencesinbothcompositionandcontentscomparedwiththeextractiveobtainedbytheoriginalpatentedtraditionalmethods.Therefore,fur-therresearchonpharmacologyandmedicineefficiencyisneededforthesafeandeffectiveuseofthismethodforTCM.
7.4.3 extraCtioN oF ediBle aNd mediCiNal iNgredieNtS From grape SeedS with SC-Co2
Inourlab,wealsoinvestigatedtheuseofSC-CO2intheextractionofactivecom-poundsfromgrapeseeds.Grapeseedscontainseedoilandprocyanidins,whicharegenerallynamedplantpolyphenol.Theweightproportionofgrapeoilinthetotalgrapeseedisabout10–15%andthatoil is rich in linoleicacid,whichbelongs tounsaturatedfattyacid.Inthefieldsoffood,cosmetics,andmedicine,itisconsid-eredtobebeneficialtouseoilshighinlinoleicacid.Grapeprocyanidinshavebeenincreasinglypaidmuchattentionasoneofthetenmostpopularherbalmedicinesintheworldandcanbeusedformedicine,hygienicfood,andcosmeticsduetotheir
table 7.8Comparison of sFe with percolation Method
extraction Methodtemperature
(°C)pressure (Mpa) e (%) C (%)
SFEwithpureCO2 65 30 3.63 0.26
Percolation 4.54 0.61
SFEwithethanol 65 30 7.12 0.83
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Application of Supercritical Fluids in Traditional Chinese Medicines 231
biological and pharmacological actives, such as anti-oxidation and anti-mutation[101–103].
The influencesof extraction temperatureandpressureon theextractionyieldoftheseedoilwereinvestigated[104].Figure7.2illustratestheeffectofextractionpressureontheextractionyieldofseedoilat45°C.WithpureCO2,weobtainedthequalifiedgrapeoils.Theresultsshowthattheyieldofoilseedwasupto9.5%at45°Cand30MPa.However,whenusing seeds supplied fromanother source area, theresultshowsthattheyieldofoilseedwasupto13.51%at55°Cand30MPa.GC-MSanalysisshowsthattheunsaturatedfattyacidintheextractedoilconstituentwasupto90.1%.Therefore,itisimportanttoinvestigatesourceareaoftherawmaterialsandmakesomenecessaryanalysisfortheactivecomponents.
Afterextractingoil from thegrape seedswithSC-CO2, theextractionofpro-cyanidinswasfurtherstudiedwithSC-CO2inthepresenceofthecosolventofethanol.ThreekindsofmethodsforaddingcosolventwereinvestigatedinordertoenhancethesolventpowerofCO2forincreasedyieldandpurityofprocyanidins.Thethreemethodsincludedaddingcosolventtorawmaterialinstaticmode,addingcosolventtoSC-CO2inflowingmode,andacombinationofthetwomodes.Theeffectsofextrac-tiontemperature,pressure,theconcentrationanddosageofcosolvents,andsoakingtimeontheextractionyieldandpurityofprocyanidinswerestudied.Theexperimen-talresultsshowthat,whenthemassratioofcosolventaddedtotherawmaterialwas1.2:1(w:w)andsoakedfor60minutesbeforetheSC-CO2extractionwascarriedout.Attemperatureof55°Cand30MPaandwhenaconcentrationof60%cosolventinCO2wasappliedinflowingmode,ayieldof10.9%withapurityof95.9%ofpro-cyanidinscouldbeobtained (Figure7.3).Whenaconcentrationof60%cosolventinCO2wasusedasextractionsolventsinflowingmodetomakeSFEatextraction
0
2
4
6
8
10
0Amount of CO2 Used (L)
Yiel
d of
Gra
pe S
eed
Oil
(%)
20 MPa30 MPa40 MPa
200 400 600 800
FIgure 7.2 Plotofextractionyieldofgrapeseedoilvs.SC-CO2amount(45°C).
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232 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
temperatureof55°Candextractionpressureof35MPa,ayieldof10.9%withapurityof95.9%ofprocyanidinscouldbeobtained.Whenthemassratioof1.2:1(w:w)forcosolventaddedtotherawmaterialwasusedandsoakedfor60minutes,andthen25%concentrationofcosolventinSC-CO2inflowingmodewasusedasextractionsolvents for SFE at the extraction temperature of 55°C and extraction pressure of35MPa,thehighestyieldof11.73%,withapurityof96.6%ofprocyanidins,couldbeobtained(Figure7.4).Therefore,addingcosolventtorawmaterialinstaticmodecombinedwithaddinga suitableconcentrationof cosolvent toSC-CO2 inflowingmodemaybethebestmethodamongthethreeforaddingcosolvents.
0
1
2
3
0.5 1.0Mass Ratio of Entrainer to Material
Yiel
d, %
0
20
40
60
80
100
Purit
y, %
YieldPurity
1.5
FIgure 7.3 Influenceofmass ratioofcosolvent tomaterialonyieldandpurityofpro-cyanidins(T=55°C,P=30MPa,soakingtime=60min).
8
10
12
10Concentration of Cosolvent in Solvent
Mixture (wt%)
Yiel
d (%
)
70
80
90
100
Purit
y (%)
YieldPurity
20 30 40 50
FIgure 7.4 Effectofdosageofcosolventflowingonyieldandpurityofprocyanidins(T=55°C,P=35MPa,massratioofcosolventrestingtomaterial=1.2:1,soakingtime=90min).
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Application of Supercritical Fluids in Traditional Chinese Medicines 233
Forcomparison,anotherintegratedtechnologywasalsoinvestigatedforobtain-ing procyanidins from grape seeds by using the combined method of SFE andmacroporous resinadsorption technology [105].SFEwithSC-CO2wasfirstusedtoremovethegrapeseedoil.Thenmacroporousresinadsorptiontechnologywasusedtopurifythecrudeprocyanidinsextractedbyhotwaterextractionwithalcoholdeposition.Theexperimentalresultsshowthatayieldof4.88%havingapurityof95%ofprocyanidinscouldbeobtained,whichisfarlessthantheyieldobtainedbyaddingcosolvent to rawmaterial in staticmodecombinedwithaddinga suitableconcentration of cosolvent to SC-CO2 in flowing mode. It reveals that extractingnaturalproductswithSFEhasobviouspredominance.
7.4.4 iSolatioN oF orgaNoChloriNe peStiCide From giNSeNg with SC-Co2
ManyChinesetraditionalandherbaldrugsarebeingexportedabroadasfoodaddi-tivesorplantdrugs.RadixginsengisarareChinesetraditionalmedicinematerialwhichhastherapeuticeffectsthatcanbeusedtotreatmanydiseasesandcanalsobeusedasahealthfood.However,ahighcontentofresiduesofprohibitoryorgano-chlorinepesticides,suchashexachlorocyclohexanes(BHC),existsinradixginseng,whichexceedsthelimitedlevelgreatlyaccordingtotheinternationalstandardregu-lation[106–108].
ThesafetyissueofChineseherbalmedicinesisasubjectofscientificinterest.SCFs as “environmentally friendly” alternatives to liquid solvents for samplepreparation inanalyticalchemistryhave receivedmuchattention in thepast fewyears.SFEhasbeenshown tobeanefficientand rapidmethod for the isolationoforganochlorinepesticidesfromvegetables[109].However,nostudiesreportontheprocessof removalofBHCfrom radixginsengwithSFE.The feasibilityofremovingBHCpesticideresiduesfromradixginsengwithSC-CO2wasexploredinourlab[110–113].
The rootswithhairsof radixginsengwerepowderedandsiftedoutprior toextraction, in which radix ginseng with sizes of 550 to 1120 µm was selected.ExtractionswereperformedwithaSpe-edSFE instrument (AppliedSeparationsInc.,Allentown,PA).
ForthedeterminationofBHC,GasChromatographwithElectricalConductivityDetector(GC-ECD)analysiswascarriedoutusinganAgilent6890plus(U.S.)gaschromatographequippedwith63Nielectron-capturedetector,usingaBPX608cap-illarycolumn(25m×0.32mm).Thechromatographicconditionswereasfollows:injectortemperature,280°C;detectortemperature,320°C;andnitrogenflow-rates,10.0mL/min(carriergas).Thecolumntemperaturewasprogrammedasfollows:increasedatarateof25°C/minfrominitialtemperatureof50°Cto150°C,retainedfor1min,andthenincreasedatarate6°C·min–1to240°C.
DeterminationofBHCinradixginsengsampleswascarriedout.TheBHCcon-tentwas2.380mg·kg–1feeds.Accordingtotheinternationalstandardregulationforfoodanddrugs,thecontentofthepesticideresiduesofBHCinradixginsengshouldbelowerthan0.1mg·kg–1.Therefore,atleast95.2%ofBHCshouldberemovedfromtheradixginsengsamplesinthiswork.
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234 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
ExtractionofBHCfromradixginsengwasfirstinvestigatedwithpureSC-CO2intemperaturesrangingfrom60°Cto80°Candpressurerangingfrom25MPato50MPa.However,theresultsofGC-ECDanalysesindicatedthatpureCO2couldnotreduceBHC contenttothelevelof0.1mg·kg–1tomeettheBHCpermissionlimit,sothatacertainmodifierwasnecessary.
Removal of BHC residues from radix ginseng with CO2 in the presence ofcosolventwasinvestigatedusingthreekindsofsolvents:water,ethanol,andhexane.Whenasuitableamountofwaterisaddedintothefeedstockbeforeextraction,BHCcontentinradixginsengcouldbereducedto0.0394mg·kg–1at60°Cand30MPa,whileadditionofthesameamountofethanolorhexaneascosolventsdidnotservesuchapurpose.
The effect of extraction pressure on removal of BHC from radix ginseng bySC-CO2inthepresenceofcosolventwaterat60°CisshowninFigure7.5.TheBHCcontentinradixginsengwasreducedtolevelslowerthan0.1mg·kg–1,whichwere0.08mg·kg–1and0.04mg·kg–1separatelyinthepressuresof20MPaand30MPa.
00.020.040.060.08
0.10.120.140.160.18
20Extraction Pressure (MPa)
BHC
Isom
ers C
onte
nt(m
g/kg
)
α-BHC
β-BHC
γ-BHC
δ-BHC
BHC(∑)
30 40 50
FIgure 7.5 EffectofpressureonremovalofBHCwithSC-CO2inthepresenceofwaterat60°C.(FromLi,S.F.andQuan,C.,Chinese Journal of Chemical Engineering, 13,433,2005.Withpermission.)
00.020.040.060.08
0.10.120.140.16
40Extraction Temperature (°C)
BHC
Isom
ers C
onte
nt (m
g/kg
)
β-BHC
γ-BHC
δ-BHC
BHC(∑)
α-BHC
60 80
FIgure 7.6 EffectoftemperatureonremovalofBHCwithSC-CO2inpresenceofwaterat30MPa.(FromLi,S.F.andQuan,C.,Chinese Journal of Chemical Engineering, 13,433,2005.Withpermission.)
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Application of Supercritical Fluids in Traditional Chinese Medicines 235
However,furtherincreaseinpressureincreasedtheBHCresidues,sotheoptimalpressureobtainedinthisstudywas30MPa.
Theeffectofextractiontemperaturewasstudiedataconstantpressureof30MPawithwater as cosolvent at temperatures ranging from40°C to80°C (Figure7.6).IncreasingtemperatureresultedindecreasingcontentofBHCinradixginseng.Atatemperatureof40°C,BHCresiduesinradixginsengwerehigherthan0.1mg·kg–1.However,theBHCcontentwasreducedto0.04mg·kg–1whentemperatureincreasedto60°C.However,inlightofthethermo-sensitivepropertiesofthenaturalplant,theoptimaltemperatureis60°C.
Toseektheminimumcosolventdosage,influencesofdosageofwateronSFEofBHCinradixginsengwerestudiedatatemperatureof60°Candpressureof30MPa.ThecontentofBHCinextractedradixginsengdecreasedrapidlyfrom1.43mg·kg–1to0.04mg·kg–1(Figure7.7)whenthedosageofwaterincreasedfrom0to0.5[water(g)/ginseng(g)],sodosageofcosolventhadsignificanteffectonremovalofBHCwithSFE.Whenthedosageofwaterwas0.4,contentofBHCwas0.11mg·kg–1,andwhenitwas0.5,contentofBHCwas0.04mg·kg–1.Therefore,thedosageofwatermustbemorethan0.4.However,anexcessiveamountofwaterdoesnothaveaposi-tivefunctionbecauseitcannotbeabsorbedbythepowderedginseng,sothesuitabledosageofwaterwasabout0.5gpergramofginseng.
At60°C,30MPa,andadosageofwaterof0.5g,influencesoftheamountofCO2onremovalofBHCfromradixginsengwereinvestigated.AsshowninFigure7.8,α-BHC, β-BHC, and γ-BHC change less while δ-BHC decreased obviously withincreasingamountofCO2forcertainginsengmaterial.Asthetotalresults,about30 to50standard literCO2pergramginsengcouldmatch the totalBHCresiduepermissionlimit—thatis,150LormoreofCO2couldreducethecontentofBHCtolessthan0.1mg·kg–1for5gginseng.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
0Modifier Dosage (water(g)/ginseng(g))
BHC
Isom
ers C
onte
nt (m
g/kg
)α-BHC
β-BHC
γ-BHC
δ-BHC
BHC(∑)
0.1 0.2 0.3 0.4 0.5
FIgure 7.7 Influences of dosage of water on removal of BHC from radix ginseng at atemperatureof60°Candpressureof30MPa.(FromLi,S.F.andQuan,C.,Chinese Journal of Chemical Engineering, 13,433,2005.Withpermission.)
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236 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
7.5 suMMarY and prospeCt
Fromtheabove-statedexamples,wefindthatSFEhassufficientlyadvancedtech-nologyandsuperiorefficiencywhenextractingnonpolarorlow-polarcompounds.Withthemodificationofcosolventandsurfactant,thelimitationofSFEinextract-ingmoderatelypolar and intensivelypolar compounds is somewhat improved. Inaddition, thecombinationofSFEwithother techniqueswidenstheapplicationofSFE in thefieldsofTCMandnaturalproducts.Asa cleanandgreen separationtechnique,SFEhasapromisingfutureinitsapplicationinthefieldsofTCMandnaturalproducts.However,wealsolearnedthatSFEextractsfromherbsaregener-allynotasinglecompoundbutacomplexmixtureofcomponents,whichcommonlyhavesomedifferencesincompositionorincontentscomparedwiththeextractiveobtainedbyothertraditionalmethods.Therefore,mostoftheresearchresultsstatedin this chapter aregenerally in the laboratory level, fewof themhave scaledup.FurtherresearchonpharmacologyandmedicineefficiencyisnecessarytoensurethesafetyandefficacyofcombinedextractionprocessesforTCM.
Additionally,somefactorsthatlimitedtherapiddevelopmentofSFEshouldnotbeoverlooked.AslackofadeeperunderstandingtoSCFstateitself,muchexperi-mentalworkisneededtodeterminetheprocessconditionsthatcannotbepredicted.TheoreticalresearchofthethermodynamicanddynamiccontrolmechanismsoftheSFEprocessalsoneedtobeinvestigatedbecausethisresearchcanprovideimpor-tant guidance for optimizing the industrial production of SC-CO2 extraction. AsoperatingSFEprocessisathighpressurewhichdemandsequipmenttoguaranteesafety,howto increase thequalityof theequipmentsand toreduce theoperatingcostsalsoneedthemanufacturerstotaketheirefforts.
SCFtechnologyisapromisingtechnologywithexcitingcommercialpotential.It is replacing older solvent technologies and creating new technologies for pro-cessingTCMandnaturalproductsbecauseitissafe,environmentallybenign,and
0
0.02
0.04
0.06
0.08
0.1
0.12
20Amount of CO2(L/g)
BHC
Isom
ers C
onte
nt (m
g/kg
)α-BHC
β-BHC
γ-BHC
δ-BHC
BHC(∑)
30 40 50
FIgure 7.8 ImpactontheamountofCO2onBHCcontent.(FromLi,S.F.andQuan,C.,Chinese Journal of Chemical Engineering,13,433,2005.Withpermission.)
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Application of Supercritical Fluids in Traditional Chinese Medicines 237
cost-effective.Itfascinatesmanyresearchersanddeservesfurtherinvestigationtoexpanditsapplication.
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243
8 Extraction of Bioactive Compounds from Latin American Plants
M. Angela A. Meireles
Contents
8.1 Introduction:ExamplesofLatinAmericanBioactiveCompounds............ 2438.1.1 ExamplesofSFEfromNativeLatinAmericanPlants....................244
8.2 ExtractingBioactiveCompoundsbySFE................................................... 2528.2.1 RelevantProcessInformationforCOMEstimation........................2548.2.2 SelectingParametersIntendedforCOMEstimation......................254
8.2.2.1 PressureandTemperatureProcesses................................. 2558.2.2.2 TheKineticParameters...................................................... 257
8.2.3 TheCOMforLatinAmericanPlants..............................................2608.3 Conclusions................................................................................................. 262References.............................................................................................................. 262
8.1 IntroduCtIon: examples of latIn amerICan BIoaCtIve Compounds
In this chapter, a brief review of supercritical fluid extraction (SFE) of bioactivecompoundsfromsolidsubstratumispresented.ThestateoftheartofSFEinLatinAmerica is described. Examples of research in development, embodying experi-mentalandmodelingofmasstransferandthermodynamics,forseveralsystemsarediscussed.Forpreliminarystudiesof technicalandeconomical feasibility,averysimpleempiricalmodelcanbeusedtodescribethemasstransferintheextractorcell.Tocalculatethecostofmanufacturing(COM),nosolubilitydataarerequired,andconsideringtheflashseparatorideal,therequiredinformationistheglobalyieldinextractatagivenconditionoftemperatureandpressurealongwithanestimatetimeintervalforanextractioncycle.COMestimatedthiswayisprovidedforsomeLatinAmericanplants.
LatinAmerica(LA)isformedby33countries:AntiguaandBarbuda,Argentina(AR),Bahamas,Barbados,Belize,Bolivia(BO),Brazil(BR),Chile(CH),Colombia(CO), Commonwealth of Dominica, Costa Rica, Cuba, Dominican Republic,Ecuador, El Salvador, Granada, Guatemala, Guyana, Haiti, Honduras, Jamaica,Mexico,Nicaragua,Panama,Paraguay(PA),Peru(PE),SaintKitts,SaintVincentand the Grenadines, Santa Lucia, Suriname, Trinidad and Tobago, Uruguay, and
7089_C008.indd 243 10/15/07 5:45:46 PM
244 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
Venezuela.FewofthesecountriesareintheAmazonianregion;thosethatareincludeBrazil,Bolivia,Colombia,Ecuador,Guyana,Peru,Suriname,andVenezuela.Therichnessof theAmazonianbiodiversitymayhelp the regiondevelopmentaswellas its devastation. Presently, the governments of the Amazonian countries havedemonstrated their concernwith thedevastationof theirnatural resources.Manyinitiatives of sustained development are available from governmental agencies,nongovernmental organizations, and private companies. These initiatives includesustainedharvestingofnativeplantsbylocalcommunities.Addingvaluetotherawmaterialbyprocessingithasalsobeenstimulated.
Besidesthat,LAcountriesareproducersofcondiments,aromaticherbs,roots,andtropicalfruitsusedbythefood,pharmaceutical,andcosmeticindustries.Someof theseproducts areused locally, andothers are exported.Among the exportedproductsareblackpepper,clovebuds,andginger.Essentialoilsandoleoresinsofvetivergrass,eucalyptus,cinnamon,mint,andotherplantsarealsoexported.BrazilandParaguayarelargeproducersofstevia,aplantwhoseaqueousextracthasbeenusedforyearsasasucrosesubstituteinspecialdiets[1].Inaddition,severalotherplantspossess lipids, starches,andcellulose thatcanpotentiallybeeconomicallyexplored.Examplesof these are turmeric, saffron, andbacuri. In addition,Chilecultivates certain microalgae, such as Spirulina maxima, which maybe used assourceoffattyacids[2–3].
Questionsrelatedtotheuseoftechniquesthatavoidorminimizedamagestotheenvironmentarecurrentlybeingdebated.Consumers’demandsindicatethat, inthenearfuture,productsofbetterqualitywillberequestedmoreandmore.ThistendencycanbeexploredthoroughlybytheLAcountries.Totakeadvantageoftheirpotential,thesecountriesneedtodeveloporadapttechnologiesthatareeconomicallyviableandecologicallyresponsible.ProductsobtainedbySFEarefreefromtoxicresiduesandgenerallypossesshigherqualitythanproductsobtainedbyconventionaltechniques.
Therefore, rawmaterials fromLAcountries represent abusinessopportunityforproducersofvegetableextracts,moreoveriftheseextractsarepreparedbySFE.Combining this rich biodiversity with an ecologically correct technology wouldrepresenttheidealmarriage!
8.1.1 ExamplEs of sfE from NativE latiN amEricaN plaNts
Compilations of literature on SFE were done recently by Meireles [5], Rosa andMeireles[6],Diáz-Reinosoetal.[7],anddelValleetal.[8].Therefore,theinforma-tionpresentedinthischapterrepresentsanupdateofthepreviousworks.Inspiteof that, thecompilationof literaturedatawasnotmeant tobeexhaustive; insteadit focused on including information that was not easily accessible. Table8.1 toTable8.3 listLatin-Americanplants (spontaneousor cultivated) studied; theSFEstudiesonthemicroalgaeSpirulina maximawerealsoincluded[2,3].Thecommonnameswereconfirmed in theU.S.DepartmentofAgriculturePlantDatabase[9],theRainforestDatabase[10],w3TROPICOSofTheMissouriBotanicalGarden[11],SearchableWorldWideWebMultilingualMultiscriptPlantNameDatabase [12],andCropINDEX[13].ThescientificnamespellingswereconfirmedusingthesamedatabasesandtheFlorabrasiliensis[14].Theregionsofoccurrence(spontaneousor
7089_C008.indd 244 10/15/07 5:45:47 PM
Extraction of Bioactive Compounds from Latin American Plants 245
taB
le 8
.1B
ioac
tive
Com
poun
ds fr
om l
atin
am
eric
a pl
ants
(sp
onta
neou
s an
d In
trod
uced
): v
olat
ile o
il, o
leor
esin
, and
o
ther
aro
ma
Com
poun
ds
Com
mon
nam
esc
ient
ific
nam
epa
rt u
sed
Bio
acti
ve C
ompo
und(
s)
reg
ion
of
occ
urre
nce
(spo
ntan
eous
or
Cul
tiva
tion
)sf
e C
ondi
tion
s/
mpa
/K/C
osol
vent
Yiel
d (%
)r
efer
ence
Agu
arib
aySc
hinu
s m
olli
sFr
actio
natio
nof
ste
am
dist
illat
ion
vola
tile
oil
β-Pi
nene
,α-p
inen
e,a
ndli
mon
ene
LA
9/32
3—
[15]
Ann
atto
Bix
a or
ella
naSe
eds
Vol
atile
oil
and
oleo
resi
n:b
ixin
and
no
rbi
xin
BR
-N20
–30/
313–
333/
EtO
H1–
45[1
6,1
7]
Bam
boo
pipe
ror
pi
men
ta-l
onga
Pip
er a
dunc
umL
eave
sα-
hum
ulen
e,a
sari
cin,
β-
cary
ophy
llene
SA10
–30/
303–
313
1.4–
1.8
[18]
Bas
il(s
wee
t)O
cim
um g
rati
ssim
umL
eave
sE
ugen
olB
R-S
E10
–30/
313
1–1.
8[1
9]
Bla
ckp
eppe
rP
iper
nig
rum
L.
Seed
sβ-
cary
ophy
llene
,lim
onen
e,
3-δ-
care
ne,s
abin
ene
BR
,PA
15–3
0/30
3–32
30.
5–2.
1[2
0–22
]
Bus
hyli
ppia
Lip
pia
alba
Lea
ves
Car
vone
and
lim
onen
eB
R-S
E/N
E8–
12/3
13–3
231.
5–5
[23]
Cha
mom
ileC
ham
omil
la r
ecut
ita
[L]
R.
Flow
ers
Azu
lene
and
cha
maz
ulen
eB
R-S
10–2
0/30
3–31
30.
82–4
.3[2
4]
Citr
onel
laC
ymbo
pogo
n w
inte
rian
us,J
owitt
Lea
ves
Citr
onel
lal,
citr
onel
lolg
eran
iol
BR
-SE
7–16
/289
–298
0.45
–1[2
5]
Clo
veb
uds
Eug
enia
car
yoph
yllu
sFr
uit
Eug
enol
,β-c
aryo
phyl
lene
,and
α-
hum
ulen
e
BR
-NE
6.7–
10/2
83–3
08~
14[2
6,2
7]
cont
inue
d
7089_C008.indd 245 10/15/07 5:45:48 PM
246 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
taB
le 8
.1 (c
onti
nued
)B
ioac
tive
Com
poun
ds fr
om l
atin
am
eric
a pl
ants
(sp
onta
neou
s an
d In
trod
uced
): v
olat
ile o
il, o
leor
esin
, and
o
ther
aro
ma
Com
poun
ds
Com
mon
nam
esc
ient
ific
nam
epa
rt u
sed
Bio
acti
ve C
ompo
und(
s)
reg
ion
of
occ
urre
nce
(spo
ntan
eous
or
Cul
tiva
tion
)sf
e C
ondi
tion
s/
mpa
/K/C
osol
vent
Yiel
d (%
)r
efer
ence
Cof
fee
Cof
fea
arab
ica
Frui
tA
rom
aco
mpo
unds
(py
razi
nes,
py
ridi
nes,
and
fur
and
eriv
ativ
es)
LA
24–3
1/31
5–37
1—
[28]
Cor
iand
erC
oria
ndru
m s
ativ
umL
.Se
eds
Vol
atile
oil,
phe
nolic
com
poun
dsL
A20
–30/
298–
331
0.8–
2.3
[29,
30]
Cro
ton
Cro
ton
zehn
tner
iPa
xet
Hof
fL
eave
s(E
)-A
neth
ole
BR
-NE
/S6.
7–7.
9/28
3–30
12.
1–3.
8[3
1]
Erv
aba
leei
rao
rw
ilds
age
Cor
dia
verb
enac
eaL
eave
sβ-
cary
ophy
llene
and
α-h
umul
ene
BR
-SE
7.8–
30/2
99–3
230.
11–5
.5[3
2]
Euc
alyp
tus
Euc
alyp
tus
citr
iodo
ra,
Hoo
kL
eave
sC
itron
ella
land
citr
onel
lol
BR
-SE
7–16
/289
–298
0.31
–0.6
8[2
5]
Euc
alyp
tus
Euc
alyp
tus
tere
tico
rnis
Lea
ves
Aro
mad
endr
ene,
1,8
-cin
eola
nd
glob
ulol
BR
-NE
6.7–
7.9/
283–
298
0.45
–1.1
3[3
3]
Fenn
elFo
enic
ulum
vul
gare
Seed
sA
neth
ole,
fen
chon
e,a
ndf
atty
aci
dsB
R-S
E10
–30/
313
3–12
.5[3
4]
Gin
ger
Zin
gibe
r of
ficin
alis
R.
Rhy
zom
eβ-
pine
ne,m
-die
thyl
-ben
zene
,o-
diet
hyl-
benz
ene,
ar-
curc
umen
e,
α-zi
ngib
eren
e,β
-ses
quip
hella
ndre
ne
BR
-SE
15–3
0/29
3–31
32–
3[3
5–37
]
Gre
enp
eppe
rba
sil
Oci
mum
sel
loi
Lea
ves
Vol
atile
oil
BR
-SE
10–3
0/30
3–32
30.
71–2
.2[3
8]
Hor
seta
il(g
iant
)E
quis
etum
gig
ante
umL
.A
eria
lpar
tsO
leor
esin
BR
-S12
–30/
303–
313
1.44
[39]
Kho
aSa
ture
ja b
oliv
iana
Lea
ves
Pule
gone
,iso
men
thon
e,ty
mol
BO
6.5–
7/28
9–29
4/E
tOH
2–
4.6
[40]
7089_C008.indd 246 10/15/07 5:45:49 PM
Extraction of Bioactive Compounds from Latin American Plants 247
Lem
onv
erbe
naA
loys
ia tr
iphy
lla
Lea
ves
Ner
al(
orZ
-citr
al)
and
gera
nial
(o
rE
-citr
al),
and
spa
thul
enol
BR
-SE
10–3
5/30
8–31
80.
6–1.
5[
41]
Lem
ongr
ass
Cym
bopo
gon
citr
atus
Aer
ialp
arts
Ner
ala
ndg
eran
ial
BR
(N
E,S
E,
and
S)6.
9–7.
4/28
8–29
70.
21–4
2[4
2]
Lip
pia
sido
ides
Lip
pia
sido
ides
C.
Lea
ves
Tim
olB
R-N
E6.
7–7.
9/28
3–29
82.
2–3.
3[4
3]
Mac
ela
Ach
yroc
line
sa
ture
ioid
esa
nd
A. a
lata
Lea
ves
α-hu
mul
ene,
β-c
aryo
phyl
lene
,qu
erce
tin
BR
-SE
10–3
0/30
3–31
31.
2–4.
2[4
4]
Mar
igol
dC
alen
dula
offi
cina
lis
Flow
ers
Ole
ores
inB
R-S
12–2
0/29
3–31
3K
2–2.
8[4
5]
Mas
tran
toH
ypti
s su
aveo
lens
Lea
ves
Spat
ulen
e,G
erm
acre
neB
,C
aryo
phyl
lene
V
E8–
9/30
8–31
80.
1–0.
3[4
6]
Ora
nge
(sw
eet)
Cit
rus
sine
nsis
(L
.)Sh
ells
Vol
atile
oil
SA20
/313
0.6–
0.15
[47,
48]
Ore
gano
Ori
ganu
m v
ulga
reL
.L
eave
sC
is-s
abin
ene
hydr
ate,
thym
ol,
carv
acro
lL
A10
–20/
293–
313
0.4–
1.3
[49]
Palm
aros
aC
ymbo
pogo
n m
arti
ni
Rox
b.L
eave
sG
eran
iol,
linal
ool
BR
-AM
7–16
/289
–298
0.07
–0.2
[25]
Pipr
ioca
Cyp
erus
ses
quifl
orus
Rhy
zom
esSp
athu
leno
l,tr
ans-β-
guai
ene,
ge
rmac
rene
D
BR
-AM
10–1
2/33
3–35
30.
3–0.
4[5
0]
Ros
emar
yR
osm
arin
us o
ffici
nali
sL
eave
sC
amph
or,c
arno
sic
and
rosm
arin
ic
acid
s,p
heno
licd
iterp
enes
BR
-SE
10–3
0/30
3–31
31–
5[5
1,5
2]
Stev
iaSt
evia
reb
audi
ana
B.
Lea
ves
Aus
troi
nulin
,n-t
etra
cosa
ne,
n-pe
ntac
osan
eB
R/P
A25
/303
1.4–
1.6
[53,
54]
Vet
iver
gras
sVe
tive
ria
ziza
nioi
des
(L.)
Nas
hR
oots
Khu
zym
olB
R-N
E/S
E20
/313
3.2
[50,
55,
56]
Xyl
opia
ar
omat
ica
Xyl
opia
aro
mat
ica
Frui
tβ-
Phel
land
rene
,β-m
yrce
ne,α
-pin
ene
LA
7.5/
318
1.5
[57]
LA
:L
atin
Am
eric
a;S
A:
Sout
hA
mer
ica;
BR
-AM
:A
maz
onia
nre
gion
of
Bra
zil;
BR
-NE
:N
orth
east
of
Bra
zil;
BR
-S:
Sout
hof
Bra
zil;
BR
-SE
:So
uthe
ast
ofB
razi
l;A
R:A
rgen
tina;
BO
:Bol
ivia
;PA
:Par
agua
y;V
E:V
enez
uela
7089_C008.indd 247 10/15/07 5:45:49 PM
248 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
taB
le 8
.2B
ioac
tive
Com
poun
ds fr
om l
atin
am
eric
a pl
ants
(sp
onta
neou
s an
d In
trod
uced
): l
ipid
s an
d li
pid-
solu
ble
Com
poun
ds
Com
mon
nam
esc
ient
ific
nam
epa
rt u
sed
Bio
acti
ve C
ompo
unds
reg
ion
sfe
Con
diti
ons/
m
pa/K
/Cos
olve
ntYi
eld
(%)
ref
eren
ce
Bac
uri
Pla
toni
a in
sign
isSh
ell
Free
fat
tya
cids
BR
-AM
6.3–
7.0/
289–
294
0.15
–0.4
7[5
8]
Bur
itiM
auri
tia
flexu
osa
L.
Frui
tC
arot
enoi
ds,t
ocop
hero
ls,a
nd
lipid
s:f
atty
aci
ds,e
tc.
BR
-AM
20–3
0/3
13–3
28
4.7–
7.8
[59]
Cup
uass
uT
heob
rom
a gr
andi
floru
mSe
eds
Lip
ids:
fat
tya
cids
,etc
.B
R-A
M24
.8–3
5.2/
323–
353
Solv
ents
:CO
2/E
than
e2–
6/5
–6[6
0]
Jojo
baSi
mm
onds
ia c
hine
nsis
Seed
sFa
ttya
cids
AR
40/3
13–3
530.
34–0
.4[6
1]
Oliv
ehu
sk—
Hus
kV
eget
able
oil
CH
30/3
13
7.5–
12.5
[62]
Palm
E
laei
s gu
inee
nsis
L.
Pres
sed
palm
fib
ers
ork
erne
lC
arot
enoi
ds,t
ocop
hero
ls,
fatty
aci
ds,e
tc.
BR
-AM
15–3
0/31
8–32
81.
8–4.
9[6
3–66
]
Papr
ika
pow
der
Cap
sicu
m a
nnuu
m—
Car
oten
oids
AR
30/3
330.
9[6
7]
Pass
ion
frui
tPa
ssifl
ora
edul
isSe
eds
Lip
ids,
fat
tya
cids
,etc
.B
R-A
M/S
E20
–30/
317–
343
13.7
–27.
7[6
8]
Pejib
aye
orp
upun
haG
uili
elm
a sp
ecio
sa o
r B
actr
is g
asip
aes
Frui
tFa
ttya
cids
SA-A
M8–
30/2
93–3
23
9–13
[69,
70]
Rap
esee
dB
rass
ica
napu
sSe
eds
Veg
etab
leo
ilC
H30
/313
6–
12[6
2]
Ric
ebr
anO
ryza
sat
iva
Parb
oile
dri
ceb
ran
Toco
trie
nola
ndto
coph
erol
sB
R-S
/SE
15–3
0/29
8–33
38
[71,
72]
Ros
ehip
Ros
a ca
nina
L.
Seed
sC
arot
enoi
dsa
ndf
atty
aci
dsC
H10
/301
4–6.
5[6
2]
Tuc
uman
Ast
roca
ryum
vul
gare
Seed
sFa
ttya
cids
BR
-AM
20–3
0/31
3–34
331
[73]
Ucu
uba
Viro
la s
urin
amen
sis
Seed
sT
rim
irys
tinSA
20–2
5/32
344
[74]
SA:S
outh
Am
eric
a;S
A-A
M:A
maz
onia
nre
gion
of
Sout
hA
mer
ica;
BR
-AM
:Am
azon
ian
regi
ono
fB
razi
l;B
R-S
E:S
outh
east
of
Bra
zil;
AR
:Arg
entin
a;C
H:C
hile
7089_C008.indd 248 10/15/07 5:45:50 PM
Extraction of Bioactive Compounds from Latin American Plants 249
taB
le 8
.3B
ioac
tive
Com
poun
ds fr
om l
atin
am
eric
a pl
ants
(sp
onta
neou
s an
d In
trod
uced
): m
isce
llane
ous
Com
mon
nam
esc
ient
ific
nam
epa
rt u
sed
Bio
acti
ve c
ompo
und(
s)r
egio
nsf
e co
ndit
ions
/ m
pa/K
/Cos
olve
ntYi
elds
/%r
efer
ence
And
read
oxa
And
read
oxa
flava
Lea
ves
8-m
etho
xy-N
-met
hyl-
flind
ersi
neB
R-N
E20
.4–3
0.6/
313
—[7
5]
Arn
ica
Soli
dago
chi
lens
isL
eave
sIs
oque
rcet
in,r
utin
,vite
xin
BR
10/3
33/E
tOH
12.5
[76]
Aro
eira
Schi
nus
ther
ebin
thif
oliu
sL
eave
s,
flow
ers
and
stem
s
Ana
card
ica
cids
BR
-NE
13.6
–27.
2/31
3–32
3—
[77]
Arr
uda
das
erra
or
arru
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etia
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enon
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peno
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—[7
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ican
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7089_C008.indd 249 10/15/07 5:45:51 PM
250 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
taB
le 8
.3 (c
onti
nued
)B
ioac
tive
Com
poun
ds fr
om l
atin
am
eric
a pl
ants
(sp
onta
neou
s an
d In
trod
uced
): m
isce
llane
ous
Com
mon
nam
esc
ient
ific
nam
epa
rt u
sed
Bio
acti
ve c
ompo
und(
s)r
egio
nsf
e co
ndit
ions
/ m
pa/K
/Cos
olve
ntYi
elds
/%r
efer
ence
Cas
hew
Ana
card
ium
occ
iden
tale
L.
Nut
sC
arda
nol,
anac
ardi
cac
id,
cate
quin
BR
-NE
9.8–
30/3
13–3
33
2–22
[8
6,8
7]
Chi
lean
hop
H
umul
us lu
pulu
sL
eave
sA
lpha
-aci
dsC
H20
/313
6–
14[8
8]
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aE
ryth
roxy
lum
coc
a L
am.
Lea
ves
Coc
aine
CO
17–2
2/31
3/M
eOH
+
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0.17
–0.6
0[8
9]
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aiba
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aife
ra s
p.L
eave
sPh
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ompo
unds
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-AM
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30.
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[90]
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peVi
tis
vini
fera
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tski
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esve
ratr
olA
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ecov
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00%
of
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pefr
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rus
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%o
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in
[92]
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coM
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Rec
over
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fein
e[9
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frui
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arpu
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tero
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ves
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5,1
01]
7089_C008.indd 250 10/15/07 5:45:52 PM
Extraction of Bioactive Compounds from Latin American Plants 251
Mat
eIl
ex p
arag
uari
ensi
s A
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Hil.
Lea
ves
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rm
aypo
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ssifl
ora
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buia
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,BR
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ecov
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[103
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AR
:Arg
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H:C
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;CO
:Col
ombi
a
7089_C008.indd 251 10/15/07 5:45:52 PM
252 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
cultivationplaces)wereindicated;thisinformationwasgatheredfromthepublica-tionsorthedatabasespreviouslymentioned.Plantswereclassifiedasproducersofvolatileoilsand/oroleoresins(Table8.1)[15–57],lipids,andlipid-solublesubstances(carotenoids, tocopherols, etc.) (Table8.2) [58–74]. Plants that produce miscella-neouscompoundssuchasphenoliccompounds,isoflavones,andsoonaregroupedinTable8.3[75–111].TheexperimentaldatainTable8.1toTable8.3wereobtainedfocusedintheSFEprocess,thus,yields,kineticbehavior,andchemicalcompositionoftheextracts(orthecontentofthetargetcomponent)and,infewcases,thebio-logicalactivities(antioxidant,anticancer,antimycobacterium)ofthevarioussystemsweredetermined.Someplants,inspiteoftheireconomicalimportancefortheLAcountries(suchastheorangeforBrazil)havereceivedlittleattention;nonetheless,aninterestingstudyonthefractionationoftheoily-fractionofconcentrated-frozenorangejuiceoilwasdonebyMarques[112].
Table8.4summarizesthephaseequilibriumdata[113–133]measuredforsomeofthesystemsinTable8.1toTable8.3;someentirelypredictivestudieshavealsobeenincluded[122,125–127].Phaseequilibriummeasurementandmodelingweremostlydoneforlipidsystems[5,118,132].Alkaloids+CO2phaseequilibriumdatawere measured for caffeine in CO2 and CO2 + cosolvent [115] and predicted forpurinealkaloids[130].PhaseequilibriumdataforartemisinininCO2weremeasuredandfittedtodensitybasedmodelandcubicequationofstate[113].Thephaseequi-libriumofquercetin+CO2+ethanolwasmeasuredandthedatawerefittedtogroupcontributionandequationofstate(EOS)models[131].ThesolubilitiesofoleoresincompoundssuchasboldineandcapsaicininCO2weremeasuredandthedatawerefitted todensity-basedmodels [114,117].ThephaseequilibriaofSFEextractsofclove and fennel in CO2 [119, 120, 133] showed liquid-vapor and liquid-liquid-vaporphasesplit;thephaseequilibriumofvetivergrassSFEextract+CO2showedliquid-vaporsplit.Thephaseequilibriaofcamphor+CO2,camphor+propane,andcamphor + CO2 + propane were measured and fitted to the Peng-Robinson EOS(PR-EOS)[116].Thesehighlyasymmetricalsystemsshowliquid-vaporphasesplitaswellasliquid-liquid-vaporthatwerequantitativelydescribedbythePR-EOS.Theexperimentalphaseequilibriumoflimoneneoxidationproducts+CO2wasalsowelldescribedbythePR-EOS[123,124].
BecauseoftheimportanceofSFEasananalyticaltool,Table8.5isgivenheretopresentsomeanalyticalapplicationsindevelopmentinLA[134–146];thesestudiesareconcentratedinBrazilandChile.
Otherapplicationsofsupercriticalfluids(SCFs)arerelatedtotheuseofcurcumi-noidstoimpregnatePolyethyleneTerephthlate(PET)films[156],tohydrolyzestarchymatrices such as ginger and turmeric [111, 157], to hydrolyze cellulosic matrices[99,104],andtofractionvolatileoilsthatcoupleSFEandmembranes[158].
8.2 extraCtIng BIoaCtIve Compounds BY sfe
SFEfromvegetablematricesiscomplexbynature.Therefore,hardlyanycharacter-isticofthesystemcanbedescribedbyasimplemodel.Nonetheless,averysimpli-fiedmodelcanbeextremelyusefulforCOMestimation.Inthissimplifiedmodel,thevegetablematerialdescribedhasbeenformedbyacellulosicstructure(CS)and
7089_C008.indd 252 10/15/07 5:45:53 PM
Extraction of Bioactive Compounds from Latin American Plants 253
asolutemixture.TheCScontainsallinsolublematerials,includingproteins,carbo-hydrates, and salts, and is insoluble in the solvent but strongly interactswith thesolutemixture.Thesoluteisformedbyamulticomponentmixturecontainingcom-poundsfromavarietyofchemicalfunctionsfromlow-molecular-masssubstancessuch as, for instance, ethanol (this substance occurs naturally in orange oil fromcertain varieties cultivated in Brazil [112]), terpenoids, and high-molecular-masssubstances such as stevia glycosides [53, 54] and curcuminoids [51, 107–109]. Inthis definition, the presence of cosolvent modifies the composition of the solutemixtureaswellasthatoftheCS.Yet,thesolidmatrixcanbeviewedaspreviouslydescribedbyincorporatingintothesolutethesubstancesthatarenowsolubleduetothepresenceofthecosolvent.Analogously,intheCSphaseremainsonlythesolvent(SCF+cosolvent)insolublematerial.Therefore,intheextractorvessel,themixtureCS + solute + solvent can be treated as a pseudoternary system. The solute is a
taBle 8.4phase equilibrium (or solubility) for Bioactive Compounds at High pressures
pure Component or mixture solvent type of equilibrium/mpa/K reference
Artemisinin CO2 Solubility/10–25/308.2–328.2 [113]
Boldine(Hydro-alcoholicboldleafextracts)
CO2 Solubility/8–40/298–333 [114]
Caffeine CO2+EtOHandIsoC3 Solubility/15–30/323–343 [115]
Camphor CO2/propane LV/3.2–13.6/304–354 [116]
Capsaicin CO2 Solubility/6–40/298–318 [117]
Castoroilandtheirfattyacidethylesters
CO2 LV/1.7–25.4/313–343 [118]
CloveSFEextract CO2 LV,L1L2V/303–328/5.8–13.2 [119]
FennelSFEextract CO2 LV,L1L2V/4.7–22/303–333 [120]
Fishoil CO2 Solubility/14.7–29.4/301–323 [121]
L-dopa CO2 * [122]
Limoneoxidation CO2 LV/4.9–14/313–343 [123,124]
Orangepeeloil CO2 LV/4–11/313–333 [125–127]
Palmfattyaciddistillate CO2 Solubility/20–35/313–363 [128,129]
Purinealkaloids CO2 * [130]
Quercetin CO2+EtOH Solubility/8–12/313 [131]
Soybeanoilanditsfattyacidethylesters
CO2 LV/1.3–26.4/313–343 [118]
Triglycerides CO2 LV/3.3–14/278.3–368.5 [132]
VetivergrassSFEextract CO2 LV/7.7–30.8/303–333 [133]
EtOH:ethanol;IsoC3:isopropylalcohol;LV:Liquid-vaporequilibrium;L1L2V:Liquid-Liquid-Vaporequilibrium;Sol:Solubilitydata*Noexperimentaldataareavailable.
7089_C008.indd 253 10/15/07 5:45:54 PM
254 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
multicomponentmixturewhosenaturedependsonthevegetablematerialused.TheCSisalsoamulticomponentmixtureand,thus,apseudocomponentthatisentirelyinert to theactionof thesolvent(orsolventmixture);nonetheless, itdoesinteractwiththesolutemixture.Thesysteminaverysimplifiedconceptioncanbeconsid-eredasatwo-phasesystem.Thelight,orsolvent,phaseconstitutesofsolutemixture+solvent,andtheheavyphasecontainsthecellulosicstructure+solutemixture.
8.2.1 rElEvaNt procEss iNformatioN for com EstimatioN
ThehugeamountofdataonSFE fromseveraldifferent solidmatricespublished[5–8]indicatesthatSFEhasbeenproventobetechnicallyfeasibleforvirtuallyanysolidsubstratum.Nonetheless,inspiteoftherecentdevelopmentofnewindustrialplantsallovertheworld,LAhasnone.OneofthereasonsistherestraintimposedbythefixedcostofinvestmentofanSFEunit.Therefore,tofulfillthecurrentpursuitofcleantechnology,onemustshowinvestorsthatinadditiontobeingtechnicallyviable,SFEisindeedanattractivechoiceforanextractionprocess.Inordertodoso,wemustbuildabenchmarkfortheCOMofSFEtobecomparedwiththeCOMofconventionalprocesses.So,asimpleandyetreliablemethodtoestimateCOMforpreliminaryanalysisorbusinessplananalysisisneeded.Forthis,asimpleproce-durethatemploysminimumexperimentalinformationwouldbeadequate.RosaandMeireles[147]havedemonstratedsuchmethodologybasedonthemethoddescribedbyTurtonetal. [148]; theseauthorsestimatedCOMforclovebudoilandgingeroleoresin. In the next section, we describe in detail the required information toemploytheprocedureadoptedbyRosaandMeireles[147].
8.2.2 sElEctiNg paramEtErs iNtENdEd for com EstimatioN
For a business plan, that is, at the very early stages of process development, thefollowingquestionsmustbeansweredforeachvegetablematrix:
taBle 8.5analytical applications
substratum Bioactive Compound reference
Honey Pesticides [134]
Fish Mercury [135]
Lactealmatrices Fatsolublevitamins [136]
Organotin Organotin [137]
Sausages N-nitrosamines [138]
Soilsamples Butyltin [139]
Soilsamples Pesticides [140–142]
Vegetableoils Polycyclicaromatichydrocarbons [143]
Humanhair Cadmium [144]
Urine Chromium [145]
Urine Nitrofurantoin [145]
7089_C008.indd 254 10/15/07 5:45:55 PM
Extraction of Bioactive Compounds from Latin American Plants 255
1.Whatisthebestprocessforobtainingthedesiredextract?(Atthispoint,thealternativeextractionprocessesshouldbeconsidered,includingSFE,low-pressure solvent extraction [LPSE]withavarietyof solvents, and steamdistillation[forvolatileoilonly].)
2.Foragivenextractwithspecifiedfunctionalproperties,wouldSFEbeagoodchoice?
3. IfSFEisanalternative,whatarethepressureandtemperatureofextraction? 4.Atthispressureandtemperature,whatistheprocessyield? 5.Howlongdoesittaketoobtainsuchyield?
Inordertoanswerthesequestions,twotypesofexperimentaldatamustbeavailable:
1.Globalyield,ortotalamountofsolublesubstancespresentinthevegetablematrixforagivenconditionoftemperatureandpressure.
2.SFEkineticsforthesystemunderconsideration.
To develop mass transfer and phase equilibrium models, several types ofinformationarerequired[36,61,97,149–151].Forinstance,thecharacteristicsofthe solidmatrix, suchashumidity,contentof solublematerial, structure,particlesize,anddistribution,are required forevaluationof themass transferparametersfromdifferentmodels.Tochoosea thermodynamicmodelsuitable fordescribingphaseequilibrium,experimentaldatamustbeavailable.Theseparametersmustbemeasured,estimated,orboth,preferablyusingstandardprocedures.Nonetheless,forthebusinessplan,theneededinformationisless.Forinstance,withtheknowledgeofthebedapparentdensity,therequiredbedvolumeforagivenproductioncanbeestimated.Or,ifextractorsofagivenvolumeareavailable,itispossibleusingthebedapparentdensitytocalculatetherawmaterialdemandtobeprocessed.Addingtothissimpleinformation,theglobalyieldandthetimeforanextractioncycleisenoughforCOMestimation[147].
Additionally, the compositionof the extract in termsof itsmajor compoundsandonefunctionalpropertywouldhelpthedecisionmakers.Toobtaintherequiredinformation for process design, identification of the solute mixture is necessary;therefore, the chemical composition of SFE extracts must be determined byappropriate methods, such as gas chromatography with flame ionization detector(GC-FID), Gas Chromatography-Mass Spectrometry (GC-MS), high-performanceliquidchromatography,orultraviolet spectrophotometry.Forextracts thatwillbeusedasnutraceuticals,biologicalactivitymustalsobemonitored.ThiscanbedoneusingasimpletechniquetoaccesstheantioxidantactivityoftheSFEextract.OnesuchmethodisthatofHammerschmidtandPrat[152],whichhasbeenadaptedtobeusedforSFEextracts[51].AnotherkeyissueistheoptimizationoftheseparationstepthatcanbesimulatedusingasimplecubicEOS,suchasthePR-EOS.
8.2.2.1 pressure and temperature processes
Theselectionoftheprocessconditions,suchastemperature,pressure,solventflowrate, cosolvent (if required), solid matrix preparation, and so on, is required forprocessoptimization.Nonetheless,beforeselectingtheparametersrelatedtomass
7089_C008.indd 255 10/15/07 5:45:56 PM
256 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
transfer(i.e.,tothekineticsthatwillultimatelybeusedforprocessoptimization),itisinterestingtochoosethepressureandtemperatureofextraction.Thiscanbedoneconsidering the thermodynamicsof the systemaswell as the composition of theextractatagivenconditionoftemperatureandpressure.Inordertoselectthepres-sureandtemperatureofprocess,thephaseequilibriumorsolubilityofthesystemsolute+SCF,thesolubilityofthepseudoternarysystemCS+solute+SF,ortheglobalyieldcanbeused.Atthispoint,itisimportanttorememberthatinspiteoftheCSbeinginerttothesolvent,itstronglyinteractswiththesolute;therefore,theinteractionofthesolutewiththesolvent,aswellaswiththeCS,mustbeconsidered.Thus, thesolubilityof thesolute insolventmeasured in thepseudobinarysystemformedbysoluteandsolvent,andthesolubilityofthesolutemeasuredforthesolidmatrix-solvent system will be quite different. In the second case, as reported byBrunner[4],thesolubilitycanbeanorderofmagnitudesmallerthanthesolubilitymeasured for thefirst case.As theCSstrongly interactswith the solutemixture,itcan be expected that the various compounds that form the solute mixture willhavedifferentaffinitiesfortheCS;therefore,thechoiceofprocesstemperatureandpressurewillbebetterdoneconsideringparametersmeasuredforthepseudoternarysystem.Thephaseequilibriumofseveralpseudobinarysystems(solute/solvent)hasbeensystematicallyreportedinliterature[5];thesedataareveryhelpfulinoptimiz-ingtheSFEseparationstep.Otherauthorshavemeasuredthesolubility(Y*)ofthepseudoternarysystemCS+solute+SFusing thedynamicmethod.AsdiscussedbyRodriguesetal.[27],toobtaincorrectvaluesofY*wouldrequireatediousandcostlyworktodeterminethesolventflowrate thatcanbesafelyusedtomeasurethisparameter.Thesolubilityhasalsobeenreportedbyseveralauthorsastheinitialslopeofanoverallextractioncurve(OEC)intermsoftotalyieldasafunctionoftheratioofsolventmass(S)tothefeedmass(F);thisparameterisreferredasY*S/F.ThedifferencebetweenY*andY*S/Fcanbeunderstoodbyrecallingthatinordertomea-sureY*trueequilibriumforthepseudoternarysystemisexpectedtobeobtainedwhileY*S/Fismeasuredatagivenratioofsolventmasstofeedmass.TheresultsofMouraetal.[34]forfennel+CO2haveshownthatY*S/Fcanbeafunctionofthefixedbedgeometry,i.e.,theratioofthebedheight(HB)tothebeddiameter(DB).TheseauthorsobtainedincreasingvaluesofY*S/FastheratioHB/DBincreasedfrom2.21to8.84.Ontheotherhand,similarexperimentsdonebyCarvalhoetal.[52]forrosemary+CO2showedthatY*S/FvariedinanarrowrangeasHB/DBincreasedfrom0.67to8.4.Evenso,asreportedbyMouraetal.[34]andCarvalho[52],theseresultsweredependentonthesolventflowrate.Moreprecisely,theinterstitialvelocityinthefixedbedplaysanimportantroleintheprocess,thusaffectingthemeasurementofY*S/F.Additionally,toobtainreliableresultsofeitherY*orY*S/F,theexperimentalassaysmustbeperformedinSFEunitscontainingextractorvesselswithvolumesofatleast50mL,sinceanOECmustbebuilt.Alternatively,thechoiceoftheoperatingtemperatureandpressurecanbedoneconsideringtheglobalyieldsisotherms(GYI)asreportedbyRodriguesetal.[154]andMouraetal.[34],amongothers.Thetotalorglobalyield(Xo)canbeobtainedthroughoutexhaustiveextractioninaSFEunit.ThereisnoneedtobuildanOEC;therefore,extractorvesselsofsmallvolumes(VE<50mL)andsmallamountsoffeedarerequired.Thisisaveryconvenientchoicewhenonlysmallamountsofthevegetablematerialareavailable,whichisoftenthe
7089_C008.indd 256 10/15/07 5:45:57 PM
Extraction of Bioactive Compounds from Latin American Plants 257
caseforspontaneouscrops.Atrickquestioniswhatshouldbetheratioofsolventmass tofeedmass?Excesssolventmustbeusedinorder toobtainthetruevalueofXo.Consideringthatthetotalyieldshouldbeanintensivepropertyasdiscussedelsewhere[43,154],itshoulddependonlyontemperatureandpressure,therefore,itisenoughtoestablishasuitablevaluefortheratioS/Ftomeasuretheyieldinsuchawayastoguarantyitsusabilityforselectingtheoperatingpressureandtempera-ture.SincetheglobalyieldwasdeterminedataselectedratioofS/FitshouldbedenotedasXo,S/F.Basedonourresults[100],valuesofS/Fgreaterthan15aregoodenoughfortheselectionoftheoperatingtemperatureandpressure.Consideringthatan extraction experiment to build an OEC for some vegetable matrices can takeseveralhourswhileglobalyieldsassaysareshorter,theusageoftheGYItoselecttheoperatingtemperatureandpressureinsteadcansavehoursofexperimentalwork.IfOECsareavailableandglobalyieldinformationismissing,thentheglobalyieldcanbeestimatedusingthesplinefittingoftheOEC,aswillbediscussednext.
8.2.2.2 the Kinetic parameters
Fortheproductionofessentialoils,oleoresins,vegetableoilsfromexoticvegetableseeds,sweeteners,andsoon,ingeneral,amultipurposeplantcontainingatleasttwofixed-bedextractorswillbeemployed.Atthelaboratorylevel,theanalysisofsuchaprocesscanbedoneconsidering theOEC.Theeffectsof theprocessvariablespressure, temperature, and solvent flow rate on the total yield as well as on thechemicalprofileoftheextractarenoteasilyseenfromtheOEC.Therefore,forfirstapproximations,itwouldbeinterestingtoestablishasimpleproceduretoanalyzetheeffectsoftheprocessvariables.Afterward,theconditionscanbeoptimizedcon-sideringtheglobalprocess.
AnOECisobtainedconsideringtheamountextracted(massofextractoryield)asafunctionoftime.TheinformationprovidedbyanOECisthetimerequiredforanextractionbatch.AtypicalOECcanbedescribedbythreesteps:
1.Aconstantextractionrateperiod(CER) 2.Afallingextractionrateperiod(FER),whichrepresentsthestepforwhich
bothconvectionanddiffusioninthesolidsubstratumcontrolstheprocess 3.Adiffusion-controlledrateperiod(DC)
PriortoSFE,thesolidsubstratumrequirespreprocessingthatatleastincludescomminution.Inordertoavoidchanneling,theparticlesizeusedinSFEwilldependon the ratio of bed diameter to particle diameter, which has been reported to bebetween50and250[5].BecauseofthestronginteractionbetweenwaterandCS,solute dehydration is required if the water content is more than 20%, wet basis.Therefore, thepreprocessingof the solid substratumpromotes the ruptureofcellwalls;thus,thesolidmatrixsubjectedtoSFEwillcontainrupturedaswellasunrup-turedcells.Evenso,forcertainsolidmatrices,theseveretreatmentsufferedduringthe pretreatment results in about 100% of ruptured cells, as for instance, for therecoveryofcarotenoidsfrompressedpalmoilfibers[63,64].However,forspecialsolidmatricesforwhichthesoluteislocatedverysuperficially,nocomminutionis
7089_C008.indd 257 10/15/07 5:45:58 PM
258 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
needed.Indeed,Silva[17]hasshownthatextractionofbixinandnorbixinfromuru-cum(Bixa orellana)seedsismoreeffectiveusingwholeseedsthanmilledseeds.
TheCERperiod is characterizedby theextractionof the solutecontained inthesurfaceofthesolidsubstratumparticlesorincellsthatwerebrokenduringpre-processing.Sovová [149]called thesolute removedduring theCERperiodeasily accessible solute. Themasstransferintheexternalfilmneartheparticle’ssurfaceiscontrolledbyconvection.TheCERperiodischaracterizedbythefollowingkineticparameters: (1) themass transfer rate (MCER), (2) thedurationof theCERperiod(tCER),(3)theyieldduringtheCERperiod(RCER),and(4)massratioofsoluteinthefluidphaseattheextractorvesseloutlet(YCER).About70%toasmuchas90%ofthesolublematerialcanbeextractedfromthesubstratumduringtheCERperiodifcarefulpretreatmentisused[20,26].IntheFERperiod,aconsiderableportionofthesolidparticlesisnolongercoatedwithsoluteorthenumberofbrokencellsisnolongeruniform.Thus,themasstransferratediminishesasaresultofthedecreaseintheeffectivemasstransferareaaswellastheincreaseinimportanceofthedif-fusionalmechanism.IntheDCperiod,thesolutecoatingofthesolidparticleshasbeencompletelyremovedand,thus,theextractionprocessiscontrolledbythediffu-sionofthesolventtotheinnerpartsoftheparticlesfollowedbythediffusionofthesolute-solventmixturetothesurfaceoftheparticles.
8.2.2.2.1 Describing the OEC by a SplineAnOECcanbedescribedbyafamilyofstraightlines.Themassofextract(ortheyield)canbeobtainedfromthefollowingequations.
ForNlines:
m b C b b tExt i i
i
i N
i
i
i N
= −
++=
=
=
=
∑ ∑0 1
1 1
(8.1)
Fortwostraightlines:
m b C b b b tExt = −( ) + +( )0 1 2 1 2 (8.2)
Forthreestraightlines:
m b C b c b b b b tExt = − −( ) + + +( )0 1 2 3 3 1 2 3 (8.3)
wherebifori=0,1,2arethelinearcoefficientsoflines1,2…andCifori=1,2aretheinterceptsoftheselines(forinstance,C1istheinterceptofthefirstandsecondlines,andC2istheinterceptofthesecondandthirdlines),mExtisthemassofextract(ortheyield),andtistime.
Figure8.1 shows that two straight lines can quantitatively describe the OECforginger+CO2 [36],whereasFigure8.2shows that three linesare required forchamomile+CO2[24].ThefirstlinerepresentstheentireCERplusthebeginningoftheFERperiod.Theslopeofthelinerepresentsthemass-transferrateoftheCERperiod,MCER.Thetimecorrespondingtotheinterceptionofthetwolinesisdenoted
7089_C008.indd 258 10/15/07 5:46:01 PM
Extraction of Bioactive Compounds from Latin American Plants 259
0.00
0.50
1.00
1.50
2.00
2.50
0 60 120 180 240
tCER
300 360 420
Extraction Time/min.
Yiel
d/%
fIgure 8.1 OECforginger+CO2:20MPa,308K,5.91×10–5kg/s[36].
0.00
0.50
1.00
1.50
2.00
2.50
3.00
3.50
4.00
0 100 200 300 400
tFERtCER
500 600Extraction Time/min.
Yiel
d/%
fIgure 8.2 OECforchamomile+CO2:20MPa,313K,6.67×10–5kg/s[24].(FromPovh,N.P.,Marques,M.O.M.andMeireles,M.A.A.,J. Supercrit. Fluids, 21,245,2001.Withpermission.)
7089_C008.indd 259 10/15/07 5:46:04 PM
260 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
bytCERandroughlyrepresentstheminimumtimeaSFEcycleshouldlast.Themassratioofsoluteinthesupercriticalphaseatthebedoutlet(YCER)isobtainedbydivid-ingMCERby themeansolventflowrate for theCERperiod.Theyieldrelative totheCERperiodisRCER,orminimumyieldexpectedfromSFEprocessatagiventemperature,pressure,solventflowrate,andsolidsubstratumpreprocessing.IftheGYIismissingbutanOECisavailable,thentheglobalyieldcanbeestimatedusingEquation8.1orEquation8.2bycalculatingthemExtatt=3tCER;thisapproximationwasusedbyPovhetal.[24]forchamomile+CO2.
ThedatafittingcanbeperformedusingthesplinemethodofFreudandLittle[153]andSAS6.12software.Oncethisisestablished,thekineticparametersshouldbeassociatedwithaphenomenologicalmodeltodescribetheOEC.
Forcertainsolidsubstrataassociatedwithunusuallylowsolventflowrates,theOECwillshowalag-phasebeforethesystemreachesthepseudosteadystate;thistimeintervalwillbeidentifiedbytLAGandiscalculatedfromthesplinemodelbysettingthemassofextractequaltozero(mExt=0)(Figure8.3).
8.2.3 thE com for latiN amEricaN plaNts
Table8.6 shows the COM for selectedLA plants. COM was estimated using theprocedureofRosaandMeireles[147]thatappliesthemodelofTurtonetal.[148],inwhichCOMiscalculatedasasumofdirectcosts(DMC),fixedcosts(FMC),andgeneralexpenses(GE):
COM DMC FMC GE= + + (8.4)
DMC C C C C FCI COMRW WT UT OL= + + + + +1 33 0 069 0 03. . . (8.5)
0.00
0.20
0.40
0.60
0.80
1.00
1.20
1.40
1.60
1.80
0 60 120 180 240 300Extraction Time/min
Yiel
d/%
tLAG tCER
fIgure 8.3 OECforginger+CO2:20MPa,313K,1.60×10–5kg/s[36].
7089_C008.indd 260 10/15/07 5:46:08 PM
Extraction of Bioactive Compounds from Latin American Plants 261
taB
le 8
.6C
om
for
sfe
extr
acts
from
sel
ecte
d pl
ants
raw
mat
eria
lB
otan
ic n
ame
targ
et C
ompo
nent
sfe
Con
diti
ons
mpa
/K/C
osol
vent
Yiel
d(%
)*t
ext(m
in.)
Co
m
(us
$/kg
)r
efer
ence
Ani
seP
impi
nell
a an
isum
Ane
thol
e10
/303
7.9
100
21.2
1[1
60]
Bra
zilia
ngi
nsen
gP
faffi
a gl
omer
ata
Ecd
yste
rone
20/3
030.
614
01,
648.
00[8
3]
Clo
veE
ugen
ia c
aryo
phyl
lus
Vol
atile
oil
10/2
8812
.970
9.18
[147
]
13.5
909.
88
14.1
120
10.9
7
Fenn
elFo
enic
ulum
vul
gare
Ane
thol
e25
/303
12.5
808.
81[1
60]
Gin
ger
Zin
gibe
r of
ficin
alis
Ole
ores
in20
/313
2.7
150
99.8
0[1
47]
Ros
emar
yR
osm
arin
us o
ffici
nali
sV
olat
ileo
il30
/313
510
042
.69
[160
]
Tabe
rnae
mon
tana
Tabe
rnae
mon
tana
cat
hari
nens
isC
oron
arid
ine
and
voac
angi
ne35
/308
–318
/EtO
H1.
490
440.
31[1
61]
*tE
xt:e
xtra
ctio
ntim
e
7089_C008.indd 261 10/15/07 5:46:08 PM
262 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
FMC C FCIOL= +0 708 0 168. . (8.6)
GE C FCI COMOL= + +0 177 0 009 0 16. . . (8.7)
whereCRWisthecostrawmaterial,CWTisthecostofwastetreatment,COListhecostofoperationallabor,andFCIisafractionoftheinvestment.
COMestimationwasdoneusingthesoftwareTecanalysisv.2.0developedinLASEFI–DEA/FEA-UNICAMP.Incalculatingthecost,theyieldsandthemini-mumtimeoftheSFEcycleswereestimatedasdiscussedinSection8.2.2.2.1.TheSFEunitchosenasthebenchmarkcontainstwoextractorvesselsof400L(approx-imate cost U.S. $2 million). The total annual operating time was assumed to be7920h,whichcorrespondsto330daysperyearbasedona24-hshift.ThecostofoperationallaborwasestimatedtobeU.S.$3.00/h.TheSFEunitismultipurpose;therefore,itshouldbeoperatingtheassumed7920hperyearregardlessoftherawmaterialused.Thecostofwastetreatmentwasassumedtoequalzero.Table8.7andTable8.8showtheTecanalysisreportfortheinputdataandresults,respectively,forCOMestimationofclovevolatileoil.Theextractiontimes(tExt)wereassumedequalor1.10to1.90tCER.
8.3 ConClusIons
SFEcanbeatruealternativeforobtaininghighqualityextractsfromseveralLAplants.ComparingtheCOMestimatedwiththesellingprices,itisclearthatthereisabusinessopportunity.
referenCes
1. Meireles,M.A.A.,Wang,G.-M.,Hao,Z.-B.,Shima,K.andTeixeiradaSilva,J.,Stevia(SteviarebaudianaBertoni):Futuristicviewofthesweetersideoflife,inFloriculture, Ornamental and Plant Biotechnology: Advances and Topical Issues, TeixeiradaSilva,J.,Ed.,GSB,2006,chap.46,pp.415–425.
2. Canela,A.P.R.F.,Rosa,P.T.V.,Marques,M.O.M.andMeireles,M.A.A.,SupercriticalfluidextractionoffattyacidsandcarotenoidsfromthemicroalgaeSpirulina maxima, Ind. Eng. Chem. Res., 41,3012–3018,2002.
3. Valderrama,J.O.,Perrut,M.andMajewski,W.,Extractionofastaxantineandphyco-cyaninefrommicroalgaewithsupercriticalcarbondioxide,J. Chem. Eng. Data, 48,827–830,2003.
4. Brunner,G.,Gas Extraction: An Introduction to Fundamentals of Supercritical Fluids and the Application to Separation Processes, Springer,NewYork,1994.
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6. Rosa,P.T.V.andMeireles,M.A.A.,SupercriticaltechnologyinBrazil:Systeminvesti-gated(1994–2003),J. Supercrit. Fluids, 34,109–117,2005.
7. Diáz-Reinoso, B., Moure, A., Dominguéz, H. and Parajoá, J.C., Supercritical CO2extraction and purification of compounds with antioxidant activity, J. Agric. Food Chem.,54,2441–2469,2006.
7089_C008.indd 262 10/15/07 5:46:11 PM
Extraction of Bioactive Compounds from Latin American Plants 263
taBle 8.7technical-economical analysis report: Input data
raw material: Clove Buds
Initial Investment
Price(US$):SFEunitwithtwoextractors 2,000,000.00
Extractorvolume(m³) 0.40
Totalannualoperationtime(h) 7920
Operationlaborcost(US$/h) 3
Rawmaterialcost(US$/MT) 505
Initialhumidity(%) 10
Finalhumidity(%) 10
Grindinganddryingcost(US$/MT) 30
CO2cost(US$/kg) 0.1
LossofCO2(%oftotalusedinacycle) 2
Electricalpowercost(US$/Mcal) 0.0703
Coolingwatercost(US$/Mcal): 0.0837
Saturatedsteam(5barg)Cost(US$/Mcal) 0.0133
Depreciation(%/Year) 10
Seafreightcost(US$/MT⋅km) 0.01
Seafreightdistance(km) 0
Totalroadfreightcost(US$/MT⋅km) 0
operational data
Extractiontime(min) 120
Extractiontemperature(°C) 15
Extractionpressure(MPa) 10
Flashtankpressure(MPa) 4
CO2flowrate(kg/h) 90
Beddensity(kg/m³) 520
scale-up model
Yield(kgextract/kgfeed) 0.1414
Waste treatment Cost
Solidwaste(US$) 0
Liquidwaste(US$) 0
Gaswaste(US$) 0
7089_C008.indd 263 10/15/07 5:46:12 PM
264 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
taBle 8.8technical-economical analysis report: results
raw material: Clove Buds
fraction of Investment
Totalinvestment(US$)-IT 2,000,000.00
Columnvolume(m³)-Cv 0.40
operational labor Cost
equipment Hmo/Hop total Hmo (h) Cost (us$)
Extractor 1 7920 23,760.00
Flashdistillation 0.1 792 2,376.00
Condenser 0.1 792 2,376.00
CO2tank 0.5 3960 11,880.00
Pump 0.05 396 1,188.00
Heatexchanger 0.1 792 2,376.00
Total 43,956.00
Hmo/Hop:Man-laborhoursperequipmentperhourofoperationofthesystem;Hop:Annualoperatinghoursoftheequipment
raw material Cost
Solidmattercost(US$) 415,958.40
CO2usedinprocess(kg) 712,800.00
LossofCO2(%) 2.00
CO2-specificcost(US$/kg) 0.10
CO2cost(US$) 1,425.60
Preprocessingcost(US$) 24,710.40
Seacargocost(US$) 0.00
Roadcargocost(US$) 0.00
Rawmaterialcost(US$) 442,094.40
utility Cost
equipment energy (mcal) specific
Cost (us$/mCal) Cost (us$)
Flashdistillation 33,854.79 0.0133 450.27
Condenser –36,385.97 0.0837 3,045.51
Pump 1,013.96 0.0703 71.28
Heatexchanger 1,922.80 0.0133 25.57
Total 3,592.63
Waste treatment Cost
Solidwaste(US$) 0.00
Liquidwaste(US$) 0.00
7089_C008.indd 264 10/15/07 5:46:12 PM
Extraction of Bioactive Compounds from Latin American Plants 265
8. delValle,J.M.,delaFuente,J.C.andCardarelli,D.A.,ContributionstosupercriticalextractionofvegetablesubstratesinLatinAmerica,J. Food Eng., 67,35–57,2005.
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taBle 8.8 (continued)technical-economical analysis report: results
Waste treatment Cost (continued)
Gaswaste(US$) 0.00
Total(US$) 0.00
Cost of manufacturing
variable value (us$) value in Com (us$) % of Com
Investment(US$)-IT 2,000,000.00 607,407.40 47.53
Rawmaterial(US$)-CRM
442,094.40 545,795.55 42.71
Operationlabor(US$)-COL
43,956.00 120,200.67 9.41
Utilities(US$)-CUT 3,592.63 4,435.34 0.35
Wastetreatment(US$)-CWT
0.00 0.00 0.00
Costofmanufacturing(US$)-COM
1,277,838.95
Massofextract(kg) 116,468.30
Specificcost(US$/kg) 10.97
7089_C008.indd 265 10/15/07 5:46:13 PM
266 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
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151. Ruetsch, L., Daghero, J. and Mattea, M., Supercritical extraction of solid matrices.Modelformulationandexperiments,Latin Ame. Appl. Res., 33,103–107,2003.
152. Hammerschmidt,P.A.andPratt,D.E.,Phenolicantioxidantsofdriedsoybeans,J. Food Sci., 43,556–559,1978.
153. Freud,R.J.andLittle,R.C.,SAS System for Regression, SAS Series in Statistical Appli-cations, 2nd Ed.,SASInstitute,Cary,NC,1995,211.
154. Rodrigues,V.M.etal.,Supercriticalextractionofessentialoilfromaniseed(Pimpinella anisum L)usingCO2:Solubility,kinetics,andcompositiondata,J. Agric. Food Chem., 51,1518–1523,2003.
155. Germain,J.C.,delValle,J.M.anddelaFuente,J.C.,Naturalconvectionretardssuper-criticalCO2extractionofessentialoilsandlipidsfromvegetablesubstrates,Ind. Eng. Chem. Res., 44,2879–2886,2005.
156. Herek, L.C.S., Oliveira, R.C., Rubira, A.F. and Pinheiro, N., Impregnation of PETfilmsandPHBgranuleswithcurcumininsupercriticalCO2,Braz. J. Chem. Eng., 23,227–234,2006.
157. Moreschi,S.R.M.,Petenate,A.J.andMeireles,M.A.A.,Hydrolysisofgingerbagassestarch in subcritical water and carbon dioxide, J. Agr. Food Chem., 52, 1753–1758,2004.
158. Castelan,L.H.etal.,Extractionoflemongrassessentialoilwithdensecarbondioxide,J. Supercrit. Fluids, 21,33–39,2001.
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159. Pokrywiecki, J.C. et al., Separation of active principles from the essential oil ofmedicinalplantswithsupercriticalcarbondioxideandreverseosmosismembrane,inV Braz. Meet. Supercritical Fluids, Ferreira,S.R.S.,Ed.,UFSC,Florianópolis,Brazil,2004,CD-ROM.
160. Pereira,C.G.andMeireles,M.A.A.,Manufacturingcostofessentialoilsobtainedbysupercriticalfluidextraction,inProceedings of the Eighth Conference on Supercritical Fluids Application, Reverchon,E.,Ed.,ISASF,Ischia,Italy,2006,77–82.
161. Pereira,C.G.,Rosa,P.T.V. andMeireles,M.A.A.,Extraction and isolationof indolealkaloids from Tabernaemontana catharinensis A.DC: Technical and economicalanalysis,J. Supercrit. Fluids, 40,232–238,2006.
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275
9 Antioxidant Extraction by Supercritical Fluids
Beatriz Díaz-Reinoso, Andrés Moure, Herminia Domínguez, and Juan Carlos Parajó
Contents
9.1 Introduction................................................................................................. 2759.2 TypesofAntioxidantsandRegulationAspects.......................................... 2769.3 NaturalAntioxidantsandSources.............................................................. 277
9.3.1 PhenolicCompounds.......................................................................2809.3.2 Terpenoids........................................................................................2809.3.3 Carotenoids...................................................................................... 2819.3.4 VitaminE......................................................................................... 2819.3.5 OtherNaturalAntioxidants.............................................................282
9.4 BiologicalPropertiesofAntioxidantCompounds......................................2829.4.1 PhenolicCompounds.......................................................................2829.4.2 Terpenoids........................................................................................ 2839.4.3 Carotenoids......................................................................................2849.4.4 VitaminE.........................................................................................2849.4.5 AntioxidantPropertiesofSC-CO2Extracts....................................284
9.5 DeterminationofAntioxidantActivity.......................................................2859.6 Supercritical-CO2ExtractionofAntioxidants............................................286
9.6.1 ProcessingSchemes.........................................................................2879.6.2 EffectsoftheMostInfluentialOperationalVariables.....................288
9.6.2.1 PressureandTemperature..................................................2899.6.2.2 Modifier..............................................................................290
9.6.3 SC-CO2ExtractsversusConventionalSolventExtracts.................292References.............................................................................................................. 293
9.1 IntroduCtIon
According toawidelyuseddefinition,anantioxidant isanysubstance that,whenpresentatlowerconcentrationsthanthoseofanoxidizablesubstrate(suchaslipids,proteins,deoxyribonucleicacid[DNA]orcarbohydrates),significantlydelaysorpre-ventsoxidationof thatsubstrate [1,2].Neither thisdefinitionnorotherdefinitions[3]restrictantioxidantactivitytoaspecificgroupofcompoundsortoanyparticularmechanismofaction.Naturalantioxidantsplayadecisiveroleindifferentsystems:i)inplants,theyactasprotectingagentsagainstradiationormicrobialinfections,ii)in
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276 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
foods,theydelayorinhibittheformationoftoxiclipidoxidationproducts,maintain-ingnutritionalqualityandincreasingshelflife,andiii)inbiologicalsystems,alongwithendogenousdefenses(enzymes,vitamins,proteins,andothers),dietaryantioxi-dantsmayhelppreventorslowtheoxidativestressinducedbyfreeradicals[4].Sinceconsiderableevidenceindicatesthatoxidativedamagemaycontributetothedevel-opmentofage-relatedanddegenerativediseases,theprotectiveeffectsofbeneficialcompoundshavebeenascribedtotheirantioxidantactivity,althoughmanyantioxi-dantsin vivoprobablyactbyothermechanismsthanin vitro assaysorareunlikelytohavesucheffectsattheconcentrationsavailableinplasma[5,6].
Duetoanincreasingconsumerdemandtoreplacecontroversialsyntheticanti-oxidants,suchasbutylatedhydroxytoluene(BHT),butylatedhydroxyanisole(BHA),tertiary butyl hydroquinone (TBHQ), and gallates, the preservation of foods is apromisingapplicationofnaturalantioxidants,whichcouldconferadditionalbiologi-calactivitiestotheproducts.Althoughnaturalantioxidantsareassumedtobesafeandinnocuous,theirlackoftoxicityshouldbeconfirmed.
Greateffortisbeingdevotedtothesearchforalternativeandcheapsourcesofnaturalantioxidants,aswellastothedevelopmentofefficientandselectiveextrac-tiontechniques.Extractionwithconventionalsolvents issometimescharacterizedbypoorselectivityandrequireshightemperatures,whichcouldresultindegradationofthedesiredcompounds.Supercriticalfluidextraction(SFE)ismoreselectivethanconventionalextractionandisoptimalwhenproductsfreefromresidualsolventsarerequired (for example, for food, cosmetic, and pharmaceutical purposes). Carbondioxide(CO2)isthemostsuitedsolventforSFEofthermolabilecompounds,owingto its nontoxic andnonflammable character andhigh availability at lowcost andhighpurity, allowinganoptimal reproductionof thephysicochemical,biological,and therapeutic properties of the target compounds. Supercritical CO2 (SC-CO2)extracts are regarded as “natural”; are free from pathogenic and spoilage micro-organisms,spores,andenzymes;theabsenceoflightandoxygenpreventsoxidationreactions.FuturedevelopmentsinextractionofantioxidantswillprobablyberelatedtoSFE[7],whichiswellpositionedwithrespecttoincreasinglyrestrictiveenviron-mental,toxicological,andhealthregulations.
Theoretical and practical aspects of the SFE of compounds with recognizedantioxidantactivityhavebeenrevised[8–11]andparticularizedfortheextractionofantioxidants[12–14].
9.2 types of AntIoxIdAnts And regulAtIon AspeCts
Lipid oxidation is important in food deterioration—for example when oxygenreactswith lipids ina seriesof free radicalchain reactions [15]or in theoxida-tivemodificationoflow-densitylipoproteins(LDLs).Accordingtothefreeradicaltheoryofaging,variousoxidativereactionsoccurringintheorganism(mainlyinmitochondria)generatefreeradicalsasby-products,whichdamagenucleicacids,proteins,andlipidsandresultinagingandage-associatedpathologies.Thestagesoftheclassicalnonenzymaticfreeradical–mediatedchainreactionsare:1)initiation(byheat,light,ionizingradiation,metalions,ormetalloproteins),2)propagation,
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Antioxidant Extraction by Supercritical Fluids 277
3)branching, and 4)termination. The main features of the mechanisms of lipidoxidationandantioxidantactionhavebeendetailedintheliterature[3,4,15–19].
Antioxidants have traditionally been divided into two groups: primary andsecondary. Primary antioxidants (such as phenolic compounds or vitamin E) aredestroyedduringtheinductionperiod,whentheydelayorinhibittheinitiationstepbyreactingwithradicals.Secondary,orpreventative,antioxidantsslowtheoxida-tionrate,removingsubstratebybindingoxygenfromair,complexingwithtransitionmetalions(acetates,citrates,tartrates,andphosphates),quenchingsingletoxygen,bindingcertainproteinswithprooxidanteffects,absorbingultraviolet(UV)radia-tion or (in the case of phospholipids) creating a protective layer between oil andairsurface.Antioxidantscanactaccordingtoseveralmechanisms,andsynergismamongdifferentoxidationinhibitorscanoccur[15,17].
Othernonmechanisticclassificationshavebeenestablishedforantioxidants.Forexample,accordingtotheirorigin,theycanbeclassifiedasnaturalproducts,naturalidentical(α-tocopherol),orartificial.Dependingontheirchemicalstructure,anti-oxidantshavebeengroupedintophenolics(BHA,BHT,TBHQ,gallates),quinones(hydroquinone,tocopherols,hydroxychromanes,hydroxycoumarins),organicacids(ascorbic, citric, tartaric, and lactic acids and their salts and ethylenediaminetetraacetic acid and its salts), sulfur compounds (inorganic: sulfites, bisulfites, andmetasulfites;organic:methionine,cisteine),andenzymes(catalases,peroxidases,superoxidedismutase).Thenatural antioxidants foundwithinbiological systemsinclude four general groups: enzymes, large molecules (albumin, ceruloplasmin,ferritin, other proteins), small molecules (ascorbic acid, glutathione, uric acid,tocopherol,carotenoids,polyphenols),andsomehormones(estrogen,angiotensin,melatonin)[20].
Technological requirements for food antioxidants include low volatility andstability (to avoid losses during processing and storage), ability to protect fromoxidation at low concentrations, solubility and compatibility with other compo-nentsoftheoxidizablesubstrate,nontoxicandnonirritantcharacterattheeffectiveconcentration, and ability to not confer color, odor, or taste to the final product.Foodutilizationof syntheticantioxidants suchasBHT,BHA,andgallates isnotpermitted in theEuropeanUnion (EU) for some special foods, suchas those forinfants and young children [21, 22], and it is generally restricted to levels thatdependon theconsideredapplication.However,antioxidants fromnaturalorigins(suchasspices)donotneedtobedeclaredandareallowedathigherdoses[12,17].Table9.1summarizesdataonthemajorfoodantioxidantsaccordingtotheEUandU.S.regulations,aswellasmaximumlevelsofacceptabledailyintake(ADI)estab-lishedbytheCodex Alimentarius.
9.3 nAturAl AntIoxIdAnts And sourCes
ThemoststudiedantioxidantsextractablefromvegetalbiomassbySFEwithCO2arephenolics,terpenoids,carotenoids,andtocopherols.Astheavailabledatashowacomparativelyhigherantioxidantactivityforphenolics,amoredetaileddiscussionisprovidedforthesecompounds.
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278 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
tAb
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7089_C009.indd 278 10/8/07 12:13:04 PM
Antioxidant Extraction by Supercritical Fluids 279
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280 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
9.3.1 Phenolic comPounds
Phenoliccompoundsareoneofthemainclassesofsecondarymetabolitesinplants,responsibleforcolordevelopment,pollination,andprotectionagainstUVradiationandpathogens.Infoods,thesecompoundscontributetosensoryproperties(color,astringency). Phenolics refer to monomeric, oligomeric, or polymeric compoundswith an aromatic ring bearing one or more hydroxyl substituents and functionalderivatives(esters,methylethers,glycosides,etc.).
Phenolicsincludesimplephenols,coumarins,flavonoids,stilbenes,lignans,andhydrolyzableandcondensedtannins.Flavonoids(alargeandcomplexgroupofcom-poundscontaininga three-ring structurewith twoaromaticcenters andacentraloxygenated heterocycle) are common antioxidants. The six major subclasses offlavonoidsareflavones,flavonols,flavanones,catechinsorflavanols,anthocyanidins,and isoflavones. Most flavonoids present in plants are conjugated with sugars,althoughoccasionally theyare foundasaglycons [23].More than4,000differentnaturallyoccurringflavonoidshavebeendiscovered,andmorethan36,000differentflavonestructuresarepossible.
Phenoliccompoundshavepowerfulantioxidantactivitiesin vitro[24],basedontheirstructure,hydrogen-donatingpotential,andabilitytochelatemetalions.Theymay show higher efficacy than endogenous or synthetic antioxidants [25]. Theirantioxidant activity [26–28] and their structure-activity relationships have beenexamined[17,29–32].
The most-studied sources of phenolic antioxidants are fruits and vegetables[33–36],grainsandcereals[37],andteas[38,39].Agriculturalandindustrialwastesarerenewable,cheap,andhighlyavailablesourcesofphenolicantioxidants.
9.3.2 TerPenoids
Terpenoids,alsoknownasisoprenoids, aresecondaryplantmetabolitesaccountingforthelargestfamilyofnaturalcompounds,widespreadinplantsandlowerinverte-brates. The isoprenoid biosynthetic pathway generates primary and secondarymetabolitesofecologicalrelevancetoplantgrowthandsurvival.Thesecompoundsare involved in interactions between plants, between plants and microorganisms,and between plants and insects, acting as allelopathic agents and attractants orrepellants in plants [40]. They are involved in the defense, wound sealing, andthermotoleranceoftheplantsaswellasinthepollinationofseedcrops,theflavoroffruits,andthefragranceofflowers,determiningthequalityofagriculturalproducts.Some terpenoids or their precursors act as scavengers for external aggressivemoleculesinthegaseousphase(i.e.,ozone).Thetermterpenesisusedforagroupofcompoundswith thebasicC5 isopreneunit.According to thenumberof theseunits (1 to6), terpenoidsareclassified intohemiterpenoids,monoterpenoids (C10)(limonene,carvone,carveol);sesquiterpenoids(C15);diterpenoids(C20)(retinoids);sesterterpenoids(C25);tri-(C30);andtetraterpenoids(carotenoids),havingeightiso-prenoidC5residues.
Terpenoid compounds (monoterpenes, sesquiterpenes, andditerpenes) are themaincomponentsofessentialoils,whichalsocontainoxygenatedderivativesandother compounds (including aldehydes, ketones, phenolic, acetates, and oxides).
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Antioxidant Extraction by Supercritical Fluids 281
Theantioxidantactivityofdifferentessentialoilsindifferentmodelsystemsiswellknown[41,42],andsynergisticeffectswithphenolicshavebeenreported[40,43].Essential oils are the commercial sources of terpenoids, whereas enzymes andextractsfrombacteria,cyanobacteria,yeasts,microalgae,fungi,plants,andanimalcellshavealsobeenusedfor theproductionandbioconversionof terpenes.Theirbiotechnologicaltransformationsappearespeciallypromisingbecauseapplicationssuchasfragrancesandflavorsincosmeticsandfoodsdependontheabsolutecon-figuration(differentenantiomerspresentdifferentproperties).
9.3.3 caroTenoids
Carotenoids are a group of more than 600 different compounds, with isoprenoid(tetraterpenoid) structure, synthesized by plants, photosynthetic organisms, andsomenonphotosyntheticbacteria,yeasts,andmolds.Theycanbefoundaspigmentsinfruits,flowers,andanimalspecies(birds,insects,fish,andcrustaceans)andplayanimportantroleintheprotectionagainstphotooxidativedamage.Mostcarotenoidsarecomposedofacentralcarbonchainofalternatingsingleanddoublebonds(3to15conjugateddoublebonds)withdifferentcyclicoracyclicendgroups.Theyareclassified as carotenes (α- and β-carotene, lycopene), composed only of carbonandhydrogenatoms,orxanthophylls(zeaxanthin,lutein,α-andβ-cryptoxanthin,canthaxanthin,astaxanthin),withatleastoneoxygenatom.Carotenoidspredomi-nantly occur in their all-trans configuration, although cis-isomers can be formedduringfoodprocessing[44].Lycopeneexhibitsthehighestantioxidantactivity,anditsplasmalevelisslightlyhigherthanthatofβ-carotene[45].Theresultsreportedfortheantioxidantactivityofβ-carotenedifferwidelyduetothevarioustestsystemsandtheexperimentalconditionsused[46].Theconjugateddouble-bondsystemisresponsiblefortheantioxidantpropertiesofcarotenoids,whichcanactbyquenchingsingletoxygenformedduetotheeffectsofUVlight,scavengingperoxylradicals,hydrogentransfer,orelectrontransfer[47–49].
Major sourcesof lycopene include tomatoes, rosehip,apricots,guavas,water-melons,papayas,andpinkgrapefruits;α-caroteneisfoundincarrots,tomatoes,andgreenvegetables;β-caroteneispresentinthesamematerialsasα-caroteneaswellas in paprika and sweet potatoes;β-cryptoxanthin is present in mangos, papaya,peaches, paprika, oranges, lutein in bananas, egg yolks, spinach, parsley, andmarigoldflowers;zeaxanthininpaprika;astaxanthininsalmon, theyeastPhaffia rhodozyma,andthealgaeHaematococcus pluvialis;andcanthaxanthinincarrots.
9.3.4 ViTamin e
VitaminEincludesafamilyoftocopherols(havingaphytyltailattachedtotheirchromanolnuclei), tocotrienols (withanunsaturated tail),andsomeof theiresterderivatives(suchassuccinateandacetate).VitaminEeffectivelyinhibitstheperoxida-tionoflipidsbecauseitcanscavengetheperoxylradicals.Theradical-scavengingcapacityofα-tocopherolandα-tocotrienol issimilar inhexane,butα-tocotrienolis more active in membrane systems andα-tocopherol shows higher bioactivity.Themajor sourcesofvitaminEareplant species,and itscontentvariesbetweentissues,withpreferentialaccumulation inseeds.Due to theiramphipathicnature,
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282 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
tocopherolsareassociatedwithmembranelipidsorlipidstoragestructures.VitaminEisthemostimportantnaturalantioxidantinvegetableoil–derivedfoods,foundinricebran,palmoil,andwheatgerm[50].Therichestsourceisaby-productofsoy-beanprocessing(theoildeodorizerdistillate).
9.3.5 oTher naTural anTioxidanTs
Othercompoundswithantioxidantactivitysuitableasfoodadditivesarepeptidesandproteins[51,52],Maillardproducts[53,54],oligosaccharides,sugarsandpolyols[55],andmicrobialmetabolites[56].
9.4 bIologICAl propertIes of AntIoxIdAnt CoMpounds
9.4.1 Phenolic comPounds
Avarietyofbiologicaleffectshavebeenreportedforphenolicacids,includingalleviationofhyperuricemiaandprotectionagainstLDLoxidation,anti-inflammatory,antitumor,andautoimmune-relatedeffects[57–61].Caffeicandferulicacidsprovideprotectionagainstcarcinomas[62],ferulicacidestersprotectagainstUVradiation[63],andtrans-cinnamicacidcanbeusedinthepreventionortreatmentofdiabetes[64].
Research in flavonoids has increased since the discovery of the low cardio-vascularmortalityrateinMediterraneanpopulationsthatisassociatedwithredwineconsumptionandhighdietarysaturatedfatintake(“Frenchparadox”).Theirstrongantioxidantpowermakesflavonoidsabletoquenchfreeradicalsandtoactagainstthe oxidation of LDLs, attenuating the development of atherosclerosis, reducingthrombosis, and promoting normal endothelial function [65–68]. Flavonoids areexcellentcandidatesashealth-promoting,disease-preventing,andchemopreventiveagentsbecause theyare extremely safe andassociatedwith low toxicity [69,70].Protectiveactionhasbeenpostulatedforchronicdiseases[71,72],cardiovasculardiseases[71,73–77],stroke[77],hyperlipidemia[71],diabetes[74], inflammation[74,78–81],allergies[74,78,79],immunesystemdisorders[72,82],mutagenesis[74,83],andcataracts[72]aswellasforneurologicaldisorders[72,73,84],particularlythoserelatedtoaging,suchascognitive,motoric,andmooddecline[85].Flavonoidshavebeenrecognizedtoexertavarietyofbiologicalactivities(includingestrogenic,antimicrobial,antiviral,andanalgesic)[78,79]andtohavehepatoprotective,cyto-static, and apoptotic properties [79]. Some of these protective effects have beenconfirmedbyepidemiologicalstudies[75,76,83,86].Allinflammatoryprocessesincludeoxygen-activatingprocessesthatproducereactiveoxygenspecies,andfreeradicalscavengersorquenchersofactivatedstateswarrantmetaboliccontrolwithincertainlimits.Cardiovasculardiseaseisrelatedtoinflammationand,consequently,isamenabletointerventionviamoleculeswithanti-inflammatoryeffects[67].Withregardtotheimmunesystem,flavonoidsmaypreserveTcell–mediatedimmunity[82].Flavonoidsinthehumandietmayreducetheriskofvariouscancers,includinghormone-dependentbreastandprostatecancers[79],intestinalneoplasia[83],andskincancer[87].
In vitro flavonoids can bind electrophils, inactivate oxygen radicals, preventlipidperoxidation,andinhibitDNAoxidation.Incellcultures,theyincreasetherate
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Antioxidant Extraction by Supercritical Fluids 283
ofapoptosis,and inhibitbotcellproliferation,andangiogenesis [83],butadirectextrapolationtohumanscannotbemadeonthebasisofthesedata.Flavonoidsarepresentinthedietasglycosylated,esterified,orpolymerizedderivatives,andhumaninterventionstudieshaveprovidedevidencethatflavonoidsarepartlyabsorbed.Duetothelowassimilationrateandthehighconcentrationspresent,significantflavonoidintakemightresultindirecteffectswithinthegastrointestinaltract,suchasbindingofprooxidant iron;scavengingof reactivenitrogen,chlorine,andoxygenspecies;andperhapsinhibitionofcyclooxygenasesandlipoxygenases[6].
A growing body of in vivo studies is beginning to provide insight into thebiologicalmechanismsofflavonoidaction[77].Thenatureofpolyphenolconjugatesin vivohasbeenidentified,showingthatthebiologicalfateofflavonoids,includingtheirdietaryforms,ishighlycomplexanddependentonalargenumberofprocesses[88]. The forms reaching the blood and tissues are, in general, neither aglycons(exceptforgreenteacatechins)northesameasthedietarysource.Asaconsequence,thepolyphenolconjugatesarelikelytopossessdifferentbiologicalpropertiesanddistributionpatternswithintissuesandcellsthanpolyphenolaglycons.Ontheotherhand,polyphenolconcentrationstestedshouldbeofthesameorderasthemaximumplasma concentrations achieved after a polyphenol-rich meal [89]. The biologicaleffectsofthesepolyphenolsdependontheextentandwayinwhichthecirculatingmetabolitesinteractwithandassociatewithcells[73].
Antioxidantpropertiesalonearenotsufficienttoexplainthebiologicalpropertiesofflavonoids.Withinthelastdecade,reportsonflavonoidactivitieshavebeenlargelyassociatedwithenzymeinhibitionandantiproliferativeactivity,whicharedependentonparticularstructures[75].Althoughtheactionmechanismsarenotfullyunder-stood,recentstudieshaveclearlyshownthattheroleofflavonoidsasmodulatorsofcellsignallingmaybeattributedtotheireffectsasanticanceragents,cardioprotec-tants,andinhibitorsofneurodegeneration[90].Certainflavonoids,especiallyflavonederivatives,expresstheiranti-inflammatoryactivityatleastinpartbymodulationofproinflammatorygeneexpression[80,81].Thepotentialneuroprotectiveeffectsofdietaryflavonoidsandtheirroleinmodulatingoxidativestressmayberelatedtocellsignallingcascades,geneexpression,anddown-regulationofpathwaysleadingtocelldeathandneuronalapoptosis[85,91].
9.4.2 TerPenoids
Someterpenoidsarethebioactivecompoundsoftraditionalherbalremediesusedinthetreatmentofpain,colds,bronchitis,andgastrointestinaldiseases.Terpenoidsarepresentinalmosteverynaturalfoodandhavebeenassociatedwithprotectionfromoxidative stress and chronic diseases [92]. Some exhibit cardioprotective action,such as ginkgolides A and B and bilobalide from G. biloba [93]. Other relevantpropertieshavebeenreported,includingantibacterial[94],anti-inflammatory[95],anticarcinogenic[40],antimalarial,antiulcer,antimicrobial,anddiureticactivities.Protectionagainstavarietyofinfectiousdiseases(viralandbacterial)andacaricidalactivityhavebeenreportedformonoterpenes[96].Thepresentcommercialimpor-tanceofterpene-basedpharmaceuticalsisexpectedtoplayamoresignificantroleinhumandiseasetreatmentinthefuture[97].
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9.4.3 caroTenoids
Themajorbiologicalfunctionsofcarotenoidsarerelatedtointercellulargapjunctioncommunication,celldifferentiation,immunoenhancement,andinhibitionofmuta-genesis.Somecarotenoids(α-andβ-carotene,β-cryptoxanthin)areprecursorsofvitaminAandprotectagainstchemicaloxidativedamage,severalkindsofcancer,andage-relatedmaculardegeneration.Noconvincingevidenceexistsoftheirprotec-tiveactionagainstcardiovasculardisease[47,48,98–100].In vitrostudiesevidencedthatcarotenoidscaninteractwithseveralreactivespeciesandcanactasprooxidants,althoughnodocumentedevidencetodateindicatestrueprooxidantactivityin vivo[101]. The maximum antioxidant effectiveness of carotenoids in human cells isrelatedtoanoptimaldose,becausehigherdosescanbelesseffectiveorresult incelldamage.Therelationshipbetweencarotenoidintakeandcancerhasbeenevalu-ated, showing an inverse association for lung, colon, breast, and prostate cancer,althoughnegativeeffectsofsupplementationshavebeenfound[49]anditisnotcleariftheassociationbetweendietanddiseaseisduetothespecificcarotenoid,othermicronutrientspresentinthespecificdiet,orthecombinedeffectofseveraloftheseactive ingredients.Studieson themechanismof cancer cell growth inhibitionbycarotenoidsattheproteinexpressionlevelmayinvolvechangesinpathwaysleadingtocellgrowthorcelldeath,includinghormoneandgrowthfactorsignaling,regula-torymechanismsofcellcycleprogression,celldifferentiation,andapoptosis[102].
9.4.4 ViTamin e
Theactionsoftocopherolsandtocotrienolshavebeenextensivelystudied.VitaminEprotectsvitaminA,sparesseleniumandvitaminC,andisthemosteffectivelipid-solubleantioxidant,whichprotectsunsaturatedfattyacidsinmembranes.Othernon-antioxidantfunctionsincludeenhancedimmuneresponseandregulationofplateletaggregation [50, 103]. The effects of Vitamin E have been observed at the levelofmessenger ribonucleicacid (mRNA)orproteinandcouldbe related to regula-tionofgenetranscription,mRNAstability,proteintranslation,andproteinstability.Landviketal.[103]publishedacompilationofhumanepidemiologicalstudiesonvitaminE,carotenoids,andcancerrisk.Thisvitaminalsoprotectsagainstcoronaryheartdisease[104],aging,cataracts,UVradiation,airpollution,andlipidperoxida-tionassociatedwithstrenuousexercise.VitaminEbioavailabilityandmetabolismis influenced by intestinal absorption, plasma lipoprotein transport, and hepaticmetabolism[105].DifferentdistributionofvitaminEisoformsintissueshasbeenreported, being an essential part of the antioxidant defense systems, particularlyintheskin,wheretocotrienolsarepreferentiallydistributed.Tocotrienolsaremoreeffective than tocopherolsat inhibitingneuronalcelldeath. Ithasbeensuggestedthatneithertheanticarcinogeniceffectsoftocotrienolsnortheneuroprotectionarerelatedtotheantioxidantpropertiesoftocopherolsandtocotrienols[50].
9.4.5 anTioxidanT ProPerTies of sc-co2 exTracTs
SinceSFEisarelativelynovelapplication,studiesonthebiologicalpropertiesoftheseextractswillprobablyincreaseinthefuture.Antimicrobialactivityofseveral
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extractshasbeenobservedforwhitegrapeseedfractions[106],spices[107],andmarjoram [108]. Protection from ischemic damage was reported for cocoa hullextracts [109]. Antimutagenic and antineoplastic properties have been claimedforSC-CO2extractsofplantandspices[107,110]aswellasantimutagenicityforTerminalia catappaleaveextracts[111].
9.5 deterMInAtIon of AntIoxIdAnt ACtIvIty
Theactivityofnaturalantioxidants,whicharemixturesormultifunctionalsystemsacting in complex media, cannot be evaluated satisfactorily by a simple test, andcontradictoryresultsusingdifferentassayshavebeenreported.Acomparativeevalu-ationofantioxidants isdifficultbecause, in foodstuffsandbiological systems, theactivitydependsonthesubstrate,themedium,theoxidationconditions,interfacialphenomena,andthepartitioningpropertiesoftheantioxidantbetweenphases.Theaffinity of antioxidants toward air, oil, water, and interfaces explains why polarantioxidantsaremoreactiveinbulkoilsandnonpolarantioxidantsaremoreactivein emulsions, a behavior known as the “polar paradox.” The need for approved,standardizedmethodsisespeciallyimportantforcomparingfoodornutraceuticalsinordertoprovidequalitycriteriaforregulatoryissuesandhealthclaims.Evalua-tionoftheantioxidantactivityatdifferentlevelshasbeensuggested[112],including:i)quantificationandidentificationoftheactivecompounds,ii)evaluationoftheradicalscavengingactivitywithmorethanonemethodindifferentsolvents,iii)evaluationofprotectionagainstlipidoxidationinmodelsystems,andiv)studiesofrelevanceforfoodapplicationsandhumanstudieswithmarkersforoxidativestress.
Many in vitro methods are performed in the absence of lipids and the parti-tioningofantioxidantsisnotevaluated,orthesemethodsdonotpredicttheabilityto inhibit oxidation of foods or in biological systems. More realistic informationcanbeachievedbyperformingseveraltestsandfollowingsomegeneralrecommen-dations:i)substratesandoxidationconditionsshouldsimulatechemical,physical,andenvironmentalconditionsinfoodorbiologicalsystems;ii)lowlevelsofoxida-tionshouldalsobeconsidered; iii)both initialandsecondaryproductsshouldbemeasured;andiv)theconcentrationsofcatalyst,antioxidants,andsubstratesshouldbecarefullyestablishedand thecompositionaldata shouldbeknown tocomparesamples[3,16,18,20,113,114].Theresultsarealsoinfluencedbythespecificityandmethodsusedtoanalyzetheprogressofoxidationandbythedegreeofoxida-tionchosenasend-pointfortesting[16,17,113,114].Acceleratedoxidationofoils,fats,oil-wateremulsions,andmusclefoodsarerelevantduringfoodprocessingordomestic use [114]. However, under some testing conditions (temperature, partialpressureofoxygen,metalcatalystsandotherinitiators,lightorUVradiation),theoxidationmechanismsmaychange[18].Methodsofexpressingantioxidantactivity,summarized by Antolovich et al. [18], include the induction period, percentageinhibitionofrates,IC50(concentrationtoachieve50%inhibition),andscalereadings(absorbance,conductivity).
Asfreeradicalgenerationisdirectlyrelatedtooxidation,variousmethodshavebeendevelopedbasedontheabilitytoscavengefreeradicals[5,19,115,116].Huangetal.[3]andPrioretal.[20]comparedtheperformanceandbiologicalrelevanceof
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286 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
differentmethodsandestablishedaclassificationbasedonthemechanisms:hydrogenatom transfer (HAT, measuring the ability to quench free radicals by hydrogendonation),singleelectrontransfer(SET, measuringtheabilitytotransferoneelectrontoreduceanycompound),oracombinationofHATandSET.AmongthemethodsbasedonHAT,themostfrequentlyemployedaretheOxygenRadicalAbsorbanceCapacity (ORAC)and theTotalPeroxylRadical-TrappingAntioxidantParameter(TRAP).Bychangingtheoxidantsourcesofperoxylradicals,thereactioncandif-ferentiate quenching of specific oxidants (O2
·-, HO·, HOCl, LO(O)·, ·OONO, and·O2).OtherHATtestsandtheirtargetapplicationsaretheTotalOxidantScavengingCapacitytest(TOSC,towardhydroxylradicals,peroxylradicals,andperoxynitrite);carotenoidsbleachingviaautoxidation (towardoxidation inducedby lightorheatoroxidationinducedbyperoxylradicals);andLDLOxidationinitiatedbyCu(II)orAAPH(withrelevancetooxidativereactionsthatmightoccurin vivo).Methodsbased on SET reactions are the Ferric Reducing Antioxidant Power (FRAP) andCopperReductionAssay(CUPRAC).Themostfrequentlyemployedmethodsbasedon both HAT and SET mechanisms are Trolox Equivalent Antioxidant Capacity(TEAC)andDPPH(2,2-diphenyl-1-picrylhydrazylradical).Bothofthemareoper-ationally simple andwidelyused, although the radical anionABTS·+ used in thefirstisnotfoundinhumanbiology,andthesecondhasseveraldrawbacks[20].TheFolin-Ciocalteutest,usedtoquantifyphenoliccontent,alsomeasurestheeffectiveoxidation/reductionefficiencyofalltheantioxidantspresentinthemedium.
Extrapolationofantioxidantmechanismsestablishedinfoodormodelsystemsto in vivo situations isnotdirect.Bioavailabilityandmetabolismofantioxidantsmustbeaddressedtoknowifthesecompoundsreachtargettissuesbecausetheirbiologicaleffectsmaybeaffectedbyavarietyoffactors,includingdigestion,absorp-tion,metabolism,andthepresenceofcompetitiveenzymesandotherantioxidantsor prooxidants. Although in vitro assays do not reflect the cellular physiology,metabolismandin vivoassays(withanimalsorhumans)arelesssuitedforinitialscreeningofantioxidantsthancellculturemodelsbecausetheyareexpensiveandtime-consuming [117]. However, cells in culture behave differently from thosein vivoduetothe“cultureshock”andtotheoxidativestresscausedbytheprocess[118].Apartfromthecriticalgeneralworksontheanalyticalmethodstodetermineantioxidant activity [3, 16, 17, 18, 20, 112, 113, 116], specific revisions concern-ingfoodapplications[19,114,115]havebeenpublished.Theavailablemethodstomeasurefreeradicalsandotherreactive(oxygen[ROS]/nitrogen/chlorine)speciescontributingtothedevelopmentofseveraldiseasesbyoxidativedamagehavebeenrevised[4,119].
9.6 superCrItICAl-Co2 extrACtIon of AntIoxIdAnts
Dependingonthephysicalstate(solidorliquid)ofthephasecontainingthetargetcompounds,SFEcaninvolvesolid-liquidorliquid-liquidmasstransfer.Solid-liquidextraction is a heterogenous operation involving the transfer of solutes from thevegetalmatrixtoafluid.Theextractionratedependsontheexternalmasstransfer,effectivesolutediffusivity in thesolid, solute solubility in thesolvent,andsolutebindingtothesolidmatrix.Batchextractionandsemicontinuousextractionarethe
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Antioxidant Extraction by Supercritical Fluids 287
most commonly used experimental methods. Extraction by solvent flow throughafixedbedof solid particles allows the recoveryof fractionsobtained along theextractionperiod.WhenaliquidstreamhastobeprocessedbySFE,bothsolubilityand interphasemass transfer are relevant.Operation is similar to extractionwithconventionalsolvents,andcontinuousoperationcanbecarriedoutinsingle-stageormultistagecontact(cross-floworcountercurrent).
9.6.1 Processing schemes
Different processing schemes have been proposed for SFE of compounds fromnatural sources.Figures9.1a to9.1dpresent simplifiedflowdiagramsof themostusualalternatives,including:
1.Singleextractionstageandfractionalseparationinseveralseparators.Theextractobtainedinasingleextractionstepcanbefractionatedbyreleasingpressure in theseparators.Thisdispositioniswidelyusedforprocessingsolidsandforanalyticalpurposes[120–122].
2.Stagewiseextractionatprogressivelyincreasedseverity.Afterafirststageat low severity (< 15 MPa, no modifier) to extract nonpolar compounds(essentialoilandwaxes),furtherSFEofthesolidresidueisperformedatincreasedseverity(upto50MPa,40%modifier)toextractmorepolaranti-oxidants [123, 124]. Stepwise extraction needs more solvent than simpleextractionwithstagewisefractionationofextracts[12,125],althoughtheextractionyieldscanbesimilar.
3.CombinationofconventionalsolventandSFEofsolidsamples.AfirstSFEstageunder lowseverityconditionscanbeperformed to removevolatilecompoundsandwaxesfromthesolidsubstrate[126,127]beforeextractionwithconventional solvents.Ahydrothermal treatment,withenvironmen-talandoperationaladvantagesderivedfromthenontoxiccharacterofthesolvent,hasbeenusedforextractingbiologicallyactivecompoundsfromSFE-extractedbamboo[128].
4.SFEofdryextractsorsolidresidues.Solid-liquidSC-CO2extractioncanbeemployedtopurifycommercialextracts,driedextractsfromconventionalsolvent extraction (CSE), or compounds remaining in the solid residuefromCSE.The twofirst schemeshavebeenproposed forenhancing theantioxidantactivityand improving theorganolepticproperties (dearoma-tization)ofextracts [129,130]. Improvedbenefitshavebeenreportedforhigh-molecular-weightcompounds,probablyduetotheirlowerconcentra-tionandinteractionswiththematrix[131].
AntioxidantshavebeenalsoobtainedbySFEofliquidfeedstreams,includingoilsanddistillates[132]andjuices[133].
Usually,naturalrawmaterialsforSFEshowbothlimitedcontentsofthetargetcompoundsandlowbulkdensity,makingtheutilizationoflargevolumeextractorsnecessary[134].Becauseofthis,processes involvingCSEandfurtherpurificationof
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288 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
thecrudebySFEarecomparativelyadvantageous,astheyprovidehigheryieldsand/orlowerspecificCO2consumptionthandirectextractionofthevegetablefeedstock.
9.6.2 effecTs of The mosT influenTial oPeraTional Variables
Previous conditioning of the starting material and the experimental conditionsemployedinextractionandseparationinfluenceSFEperformance.Thenatureandpropertiesofvegetablefeedstocksor theirprocessingstreams(includingmaturitystage,cultivar,variety,edaphoclimaticconditions)stronglyinfluencetheextractionofterpenoidsandphenolics[135,136],carotenoids[137–139],andtocopherolsfrom
RawMaterial
SCFE10–45 MPa
Antioxidant Compounds
R2R1
(a) E1
Crustacean, Micro-algae, TomatoRaw Material Flowers, Fruits, Leaves, Spices, Medicinal
Plants, Seeds, Hulls, Roots
Phenolics and Terpenoids
Olive Leaves, MedicinalPlants, Wheat Germ, Seeds
FRACTIONA-TION (1–3 separators)
RawMaterial
RawMaterial
RawMaterial
SCFE9–15 MPa
SCFE9–15 MPa
SCFE20–50 MPa
Antioxidant Compounds
R1
(b)
PaprikaRaw materialCarotenoids
FRACTIONATION(1–3 separators)
E1Oil
E2
R2
R1
(c)
E1
HydrothermalTreatment
Conventional SolventExtraction
ConventionalSolvent Extraction
R2
R3
(d)
DryingSCFE
10–35 MPa35–80°C
R1
Antioxid. CompoundsR2
Raw Material
E1Aroma
AntioxidantCompounds
Antioxidant Compounds
Raw MaterialAntioxidant Compounds
Antioxidant Compounds
Antioxidant Compounds
Antioxidant Compounds
Carotenoids
Vitamin E
Leaves, Medicinal Plants, SeedsPhenolics and Terpenoids
Medicinal Plants, StalksPhenolics and Terpenoids
Grape Seeds, Grape PomacePhenolics and Terpenoids
fIgure 9.1 ProcessingschemesforextractionofantioxidantcompoundsinvolvingSFEstages.Nomenclature:E1,E2:extracts;R1,R2,R3:solidresidues.
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Antioxidant Extraction by Supercritical Fluids 289
solidsamples.Whenprocessingsolids,mechanical-thermalconditioningisdecisivetofacilitatetheextractionofintracellularsolutes.Reducedparticlesizefavorsmasstransfer,buttoo-smallparticlescouldlimittheperformanceoffixedbedsandgrind-ingmayresultinlossesbyvolatilizationanddegradationofactivecompounds.ThemajorvariablesinfluencingSFEofantioxidants(pressure,temperature,solventflowrate, solvent-to-feed ratio, modifier type, and concentration) should be optimizedbeforeoperation.ThosemostinfluentialandspecificforSFEincomparisontoCSEarefurthercommented.
9.6.2.1 pressure and temperature
ThesolvatingpowerofSFEwithCO2dependsonpressureandtemperature.Densitygives an estimate of the joint effects of both variables on the solvating power.Besidestheoperationalconditions,theequilibriumsolubilityofpurecompoundsisinfluencedbymolecularweight,polarity,andpresenceoffunctionalgroups.Whenconsideringextractionfromasolidsubstrate,kineticsandyieldsalsodependontheinteractionwiththesolidmatrix.
Effect on the solubility of antioxidant compounds. Equilibriumsolubilitiesarebasicinformationforaddressingthedesignofextractionandseparationprocesses.Solubilitydataforsyntheticantioxidants[140],fat-solublevitamins[141],andmanyphenolic compounds have been obtained by different groups and were recentlycompiled[14,142].Solubilitydatahavebeenreportedforpurecompoundsandtheirmixtures[143,144]andforterpenoidsfromcitrusoils[145],aswellasforessentialoils[9]andtheircomponents[8].Mostsolubilitydatarefertoauniquesolute,andscarceinformationexistsfornaturalextracts,whicharemulticomponentmixtures.Since pioneer data on the solubility of tocopherols were published by Chrastil[146], severalother studies [147–149]havebeen reported.Data are alsoavailableformixtureswithmethyloleatetosimulatetheesterifiedby-productfromsoybeanoildeodorizerdistillate [150,151], formixturesof thisby-product [152,153]andforcrudepalmoil[132].Additionalliteratureconcerningpurecarotenoids,suchascapsaicin[148],β-carotene[148,154–156],andtheirmixtures[157],andfornaturalβ-carotene from carrots [156] has been published. Solubility data forβ-caroteneand tocopherols were compiled by Guglü-Üstündag and Temelli [137]. Table9.2summarizesdatafromreviewpapersonthesolubilityofdifferentcompoundshavingantioxidantactivity.
Yield and selectivity of antioxidant extraction. Increased pressure results inincreased solvent density, allowing higher extraction yields. Increasing pressurebeyond a threshold point results in higher fluid viscosity and reduced diffusioncoefficients.Pressuresover50MPa[160]havebeenreportedfortheextractionofantioxidants.Operatingathighpressure,increasedtemperaturesmaydecreasetheextractionyieldduetothereductionindensityandthesolventpowerofthefluid.Operatingatpressuresclose to thecriticalpoint,where thedensity showshigherinfluenceonthesolventpowerthanthevaporpressure,increasedtemperaturesmaydecreasetheextractionyieldduetothereductionindensityandthesolventpowerofthefluid.Athigherpressures,theincreasedinfluenceofthesolutevaporpressuregenerallyleadstoincreasedsolubility.Temperatureandpressureshowacrossover
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290 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
effectwherebyhighertemperaturesimproveextractionathighpressuresandlowertemperaturesfavorextractionatlowpressures.Thecrossoverregionsinsupercriticalfluids,orthepointwheretheslopeofsolubilityvs. temperaturechanges,arealsofavorabletodesignseparationprocesses.
Theeffectsofpressureandtemperatureontheextractionyieldofsomeanti-oxidantsareshowninFigure9.2toFigure9.4.Effectsoftemperatureontextureandcoloroftheextractshavebeenreportedformoso-bambooextracts[128].Inothercases,slightchangesinappearance[176]andsignificantonesincompositionhavebeenreported.Thislattereffectisduethesolventpower(whichcontrolstheabilitytodissolvedifferentmolecules)andtothethermalstabilityofthesolutes.Highlythermal-sensitive compounds require mild extraction conditions (temperaturesbelow50ºC)toavoidalteration.Undertheseconditions,SFEoffershigheryieldsofactivecompounds—forexample,carnosicacidfromrosemary[122],anacardicacid from cashew nut shell [177], hyperforin from Hypericum perforatum [178],carnosol frommarjoram[179],antioxidants fromaloe [165],andmatricine fromchamomile[180].
9.6.2.2 Modifier
PureCO2undersupercriticalconditionsisagoodsolventforlipophiliccompoundsbutispoorforphenolics.Extractioncanbeenhancedusingamodifierabletointeractwith
tAble 9.2solubility of selected Antioxidant Compounds in supercritical Co2
Compound p (Mpa) t (K) solubility ref.
tocopherols(alpha,delta) 8–35
8–35.219.5–351–2.52
292–353298–353303–353298–313
(y2·104)———
2.59–7.31
[141][137][143][12]
Carotenoids(astaxanthin,canthaxanthin,capsanthin,β-carotene,lycopene,lutein,zeaxanthin)
0.15–502–3.55–1805–80
288–343313–353288–353288–353
(y2·106)—
0.09–3.24—
0.019–0.989
[141][12][137][58]
terpenoids(monoterpenehydrocarbons,
sesquiterpenehydrocarbons,oxygenatedderivatives,aldehydes,ketones)
3–118–10
0.8–13
295–335313–333310–333
(mg/g)—
1.6–CMa
—
[59][8]
[143]
phenolic Compounds(benzoicacid,cinnamicacids,flavonoids) 2–50
0.91–2.532–40.479–5000.26–50
308–473308–318308–473308–373308–373
(y2·104)—
0.0788–5.61——
0.08·10–4–1730
[59][12][143][142][14]
a CM=Completemiscibility
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Antioxidant Extraction by Supercritical Fluids 291
thetargetcompounds,possiblyimprovingyieldandselectivity.However,highmodi-fierconcentrationsmaydecreaseselectivitydependingonthesizeofphenolics[181].Alcoholsarewidelyusedasmodifiers,ethanolbeingthemostrecommendedoneonthebasisoftoxicologicalandenvironmentalconsiderations.Ethanolhasbeenemployedtoincreasethesolubilityofginsenoids[182],phenols[183],flavonoids[163],terpenoids[184],andcarotenoids[166,170,185–187].Methanolhasbeenusedforextractingphe-nolics[188],flavonoids[106,131,135,180,187],and isoflavones[189](Figure9.2).Othermodifiersandtheirtargetcompoundsareisopropanolforterpenoids,phenolicketones,andcurcuminoids[190,191];propyleneglycolforpolyphenols[180];waterforphenolicditerpenesandphenolicacids[129];andacetone;2,2-dimethoxypropane,chloroformandn-hexane[138,168,192]forcarotenoids.Vegetableoilshavealsobeenproposedforextractinglycopene[139,193]andcaprylicacid[194].Mixturesoftwomodifiershavebeensuccessfullyassayed[107,190].Oppositely,ethylacetate,chloro-form[187],andaceticacid[129]werenotsuitableasmodifiers.
Phenolics and Terpenoids
0 10 15 20 Ethanol (%)
Yiel
d (m
g/g)
0
2
4
6
8
10
12
14
16
P (MPa)
0
1
2
3
4
5
6
7
5 10 15 20 25 30 35
Yiel
d (%
)
0
10
20
30
40
50
60
70
80
90
Yiel
d (%
)
Rosemary ( 30°C), ( 40°C) [122]
[161]
From:
Coriander ( 38°C), ( 58°C) [162]
Black Pepper ( 45°C), ( 50°C),
G. biloba ( ) [163]
Savory ( ) [126] Sweet Tamarind ( ) [164]
Naringin from citrus peel [165] ( Fresh citrus peel), ( Dry citrus peel)
Products from grape seed Conc.[131] Syringic acid ( ) Protocatechualdehyde ( )
Epicatechin ( )
5
( 55°C), ( 60°C), ( 65°C)
Catechin ( )
fIgure 9.2 Effectsofpressure,temperature,andethanolconcentrationontheextractionyieldofphenolicsandterpenoidsfromvarioussources.
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292 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
9.6.3 sc-co2 exTracTs Versus conVenTional solVenT exTracTs
Dataconcerning theantioxidantactivityofSC-CO2extracts fromsolidbotanicalsamples,commercialandcrudeextractsproducedwithconventionalsolvents,andliquidstreamsaresummarizedinTable9.3.Optimizationoftheoperationalvariablesisrequiredforeachprocess,owingtothewidevarietyofstartingmaterials,targetcompounds,andconditionsemployedforextractionandseparation.Tocomparetheextracts,boththeproductionconditionsandtheassayusedtoquantifytheantioxidantactivitymustbeconsidered.AgeneralcomparisonbetweenSFEandCSEcannotbe established beforehand. Even though conventional, less-selective solvents mayallowhigherextractionyields[121,188],theisolatedfractionscouldhaveunpleasantaromas.FurtherfractionationbySFEcanbeusedtopurifytheextract,preservingtheantioxidantactivity[195].Similarcompositionoftheextractsobtainedusingthese
Carotenoids A
bsor
banc
e
0 5 10 15 20 Ethanol (%)
0
0.1
0.2
0.3
20
40
60
80
Yiel
d(%)
P (MPa)
Abs
orba
nce
Yiel
d (%
)
20
40
60
80
10 20 30 40 50 60 0
0.2
0.4
0.6
Astaxanthin ( ) [166]; ( ) [167]
β-Carotene ( 35°C), ( 45°C), ( 55°C), ( 65°C) [170]
Lycopene ( 35°C), ( 45°C), ( 55°C), ( 65°C) [170]
Astaxanthin ( ) [167]
Astaxanthin (+ 40ºC), ( 50°C), ( 60°C) [166]
β-Carotene ( ) [168]
Carotenoids ( 40°C), ( 50°C),
Lycopene ( 45°C), ( 60°C) [139] Lycopene ( ), β-Carotene ( ) [170]
( 60°C) [169]
fIgure 9.3 Effects of pressure, temperature, and ethanol concentration on absorbanceandextractionyieldofselectedcarotenoids.
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Antioxidant Extraction by Supercritical Fluids 293
technologieshasbeen reported forchamomile [180]andmarigold [160],whereasdifferent compositionwas found inextracts fromoregano [125] and fennel seeds[176].Reports indicate thatSFE results in higher extraction yields and enhancedselectivityofactivecompoundsthanCSE[124,183,196].SuperiorityofSFEwithrespecttoconventionalmethodshasbeenreportedforeucalyptusleaves[196],blackpepperoleoresin[161]andLippia albastemsandleaves[197],owingtothehigherconcentrationsofactivecompounds.SFEmayresultinextractswithhigheractivitywhen processing substrates with high contents of thermally unstable active com-pounds and in better odor and color of the isolates [122]. Short processing timeand lowsolventconsumptionareadditionaladvantagesofSFE [182].Oppositely,thelowerselectivityofconventionalsolventscouldfavortheantioxidantactivityofextractedfractionsshowingsynergismamongcomponents,asobservedforturmeric[191],tamarindseedcoat[164],marjoram[108],andblacksesame[198].
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Tocopherols
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Tomato Seeds and Skins ( 32ºC), ( 50°C), ( 68°C), ( 86°C) [173]
Silybum Marianum Seeds ( 25°C),( 40°C), ( 60°C), ( 80°C) [171]
From:
Soy Deodorizer Distillate ( 40°C),( 50°C), ( 60°C) [172]
Wheat Germ ( 40°C), ( 45°C)( 50°C) [174]
fIgure 9.4 Effectsofpressure,temperature,andethanolconcentrationontheextractionyieldsoftocopherolsfromvarioussources.
7089_C009.indd 293 10/8/07 12:13:17 PM
294 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
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305
10 Essential Oils Extraction and Fractionation Using Supercritical Fluids
Ernesto Reverchon and Iolanda De Marco
Contents
10.1 Introduction.................................................................................................30510.2 SolidsProcessing........................................................................................307
10.2.1 SelectionoftheOperatingParameters..........................................30810.2.2 Examples........................................................................................ 312
10.2.2.1 Leaves............................................................................ 31210.2.2.2 Flowers.......................................................................... 31410.2.2.3 Seeds.............................................................................. 31410.2.2.4 OtherMatrices............................................................... 31410.2.2.5 FlowerConcretesFractionation..................................... 316
10.3 LiquidFeedProcessing............................................................................... 31810.3.1 SelectionoftheOperatingParameters.......................................... 31910.3.2 Examples........................................................................................ 320
10.4 AntisolventExtraction................................................................................ 32210.4.1 SelectionoftheOperatingParameters.......................................... 32210.4.2 Examples........................................................................................ 323
10.4.2.1 ProteinsandAromaExtractionfromTobacco.............. 32310.5 MathematicalModelling.............................................................................324References.............................................................................................................. 328
10.1 IntroduCtIon
The extraction from natural sources is the most widely studied application ofsupercriticalfluids(SCFs),andseveralhundredscientificpapersonthetopichavebeenpublishedandreviewed[1–9].Indeed,supercriticalfluidextraction(SFE)hasimmediateadvantagesovertraditionalextractiontechniques;itisaflexibleprocessduetothepossibilityofcontinuousmodulationofthesolventpower/selectivityoftheSCF,anditallowstheeliminationofpollutingorganicsolventsandoftheexpensivepostprocessingoftheextractsforsolventelimination.
Several compounds have been examined as SFE solvents, including hydrocarbonssuchashexane,pentaneandbutane,nitrousoxide,sulfurhexafluoride,andfluorinatedhydrocarbons[10].However,carbondioxide(CO2)isthemostpopular
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306 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
SFEsolventbecauseitissafe,isreadilyavailable,andhasalowcost.Itallowssupercriticaloperationsatrelativelylowpressuresandatnearroomtemperatures.
TheonlyseriousdrawbackofSFEisthatinvestmentcostsarehigherthanthosefortraditionalatmosphericpressureextractiontechniques.However,thebaseprocessscheme(extractionplusseparation)isrelativelycheapandverysimpletobescaleduptoindustrialscale.
Inthischapter,wefocusouranalysisontheextraction,isolation,andfractionationof essentialoils.EarlyworksonSFEof essentialoils frequentlyusedhighpressures (>350bar),evenwhensupercriticalCO2 (SCCO2)–solublecompoundshadtobeextracted(forexample,terpenes,sesquiterpenes,fattyacids).Operatinginthismanner,thesolventpoweroftheSCFwasenhanced,butitsselectivitywasverylow.Sincethen,theconceptoftheoptimizationbetweensolventpowerandselectivityhasbeenappliedandSFEoperatingconditionshavebeenchosentoobtaintheselectiveextractionofthecompoundsofinterest,reducingtoaminimumthecoextractionofundesiredcompounds[1].Moreover,forsuccessfulextraction,notonlymustthesolubilityofthecompoundstobeextractedbetakenintoaccountbutalsothesolubilitiesoftheundesiredcompounds.Masstransferresistancesduetothestructureof therawmaterialand to thespecific locationof thecompounds tobeextractedcanalsoplayarelevantrole.Microscopicanalysisofthenaturalstructurecanhelpinunderstandingwheremasstransferresistancesarelocated.Specificexperimentsperformedvaryingparticlesizeandsupercriticalsolventresidencetimecanalsobehelpful in thissense.Thecomplex interplaybetween thermodynamics(solubility)andkinetics(masstransfer)hastobeunderstoodtoproperlyperformSFE.
Fractionalseparationoftheextractsisanotherwellknownconceptthatcanbeuseful in improvingSFEselectivity. Indeed, in severalcases, it isnotpossible toavoidthecoextractionofsomecompoundfamiliesthatshowdifferentsolubilities,buttherearealsodifferentmasstransferresistancesintherawmatter.Inthesecases,onecanperformanextractioninsuccessivestepsatincreasingpressurestoobtainthefractionalextractionofthesolublecompoundscontainedintheorganicmatrix.FractionalseparationallowsfractionationoftheSCFextracts,equippingtheplantwithsomeseparationvesselsoperatinginseriesatdifferentpressuresandtemperatures.ThescopeofthisoperationistoinducetheselectiveprecipitationofdifferentcompoundfamiliesbasedondifferentsaturationconditionsintheSCF.
Inseveralothercases,thefeedisaliquidmixture.Therefore,theprocesstobeappliedisthecontinuousliquidextractioninapackedtower.Notethat,althoughtheextractionfromsolidsisadiscontinuousoperation,thepackedtoweriscapableofcontinuoussteadystateoperation thatallows theprocessingof largequantitiesofliquidmixturesinarelativelysmallapparatusandinashorttime.
Insomeothercases,thematerialtobetreatedisaliquidmixturethatcontainssolidcompoundsdissolvedinit.Theextractionofthesecompoundsfromtheliquidsolutioncannotbeperformedinapackedtowersincethesolidmatterprecipitatesonthepackingsofthebed.Inthiscase,supercriticalantisolventextraction(SAE)canbeadopted.ThepreconditionstoapplySAEaresimilartotheonescharacteristicof supercritical antisolventmicronization (SAS): the liquid solventhas tobeverysoluble inSCCO2,whereas the solidshave tobe insoluble in theSCF.Thescope of SAE is not the micronization but the purification of the liquid solution
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Essential Oils Extraction and Fractionation Using Supercritical Fluids 307
fromundesiredsubstances.Theseconditionscanbefrequentlyobtainedsincemanyorganic solvents are readily soluble inSCCO2evenatmildoperatingconditionsand many highmolecularweight solids show negligible solubilities in SCCO2,especiallyatlowCO2densities.
Duetothestructuralcomplexityandvariability(withseason,kind,crop,etc.)of the materials to be treated and to the large variety of compounds that can beextracted (different molecular weights, polarities, links with the structure, etc.),theseprocessesarefar frombeingexhaustivelystudied,althoughsomeindustrialapplicationshavealreadybeendeveloped.Moreover,anincreasinginteresthasbeenregisteredintheextractionofhighaddedvalueessentialoils,suchasoilsthatshowantioxidantandpharmaceuticalproperties.
Therefore,inthischapter,weanalyzeSFE,SAE,andliquidfractionationstudiesperformedonessentialoils and relatedmaterials andconsider the evolutionof theextractionprocesses,products,andmaterialstreated.Acriticalanalysisisperformed.
10.2 solIds ProCessIng
SolidsprocessingisthemoststudiedSCFapplicationbecausethemostfrequentlyrequiredseparationprocessistheextractionofoneormorecompoundfamiliesfromasolidnaturalmatrix.Thebasicextractionschemeconsistsofanextractionvesselchargedwiththerawmattertobeextracted.SCFattheexitoftheextractorflowsthroughadepressurizationvalvetoaseparatorinwhich,duetothelowerpressure,the extracts are released from the gaseous medium and collected. As a rule, thestartingmaterialisdriedandgrindedtofavortheextractionprocessandisloadedin a basket located inside the extractor to allow fast charge anddischargeof theextractionvessel.
Moresophisticatedextractionschemes,suchastheonereportedinFigure10.1,containtwoormoreseparators.Inthiscase,itispossibletofractionatetheextractintwoormorefractionsofdifferentcompositionsbysettingopportunetemperaturesandpressuresintheseparators[11–28].Solidspreprocessingisalsoaparameterthatcanlargelyinfluenceseparationperformance.Forexample,soliddryingandparticlesizeoptimization,asarule,havetobetakenintoaccount.
Dryingof thesolidmaterials is frequentlyrequiredbeforeextractionbecauserawvegetablemattercancontainupto90%water.Waterisonlyslightlysolublein
2
1
3 4 OutSCF IN
FIgure 10.1 1)CO2pump;2)extractor;3)firstseparator;4)secondseparator.
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308 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
SCCO2atthecommonextractionconditions;but,duringthepressurizationoftheextractorcanbepartlyexpressedfromthevegetablematerialandtravelsalongtheplanttogetherwiththesupercriticalsolvent.Moreover,waterinthesolidstructurecan obstacle SCCO2 penetration (diffusion), lowering extraction efficiency. As arule,watercontentsbetween5%and10%w/warerequiredtoperformSFEproperly.Fortunately,thesewaterpercentagesarealsotheonesusuallypresentindriedmaterials.Attention shouldbepaid to the selecteddryingprocessbecause it canlargelyinfluencethefinalcontentofvolatilecompoundsinthetreatedmaterial.Insomeparticularcases,thepresenceofwaterisnotdetrimentalforSFE,forexample,inthecaseofcaffeineextractionfromcoffeebeans,becauseinthatcasewaterunhooksthecaffeinesodiumsaltfromthevegetablematrix.
Other possible variations of the SFE solid processing scheme are multistageextractionandcosolventsaddition.Multistepoperationinvolvesvaryingpressureortemperatureineachprocessstep[29,30].ThisstrategycanbeusedwhentheextractionofseveralcompoundfamiliesfromthesamematrixthatshowdifferentsolubilitiesinSCCO2isrequired.IttakesadvantageofthefactthatSCCO2solventpowercanbecontinuouslyvariedwithpressureandtemperature.Forexample,itispossibletoperformafirstextractionoperatingatlowCO2density(e.g.,0.29g/cm3,90bar,50°C) followedbya secondextraction stepathighCO2density (e.g.,0.87g/cm3,300bar,50°C).Themostsolublecompounds(suchastheessentialoils)areextractedduringthefirststep,whereasthelesssolublecompounds(forexample,antioxidantsandcoloringmatter)areextractedinthesecondone[31–34].
AliquidcosolventcanbeaddedtoSCCO2toincreaseitssolventpowertowardpolarmolecules. Indeed,SCCO2 isagoodsolvent for lipophilic(nonpolar)compounds,whereasithasalowaffinityforpolarcompounds.Variousauthorsaddedsmall quantities of liquid solvents (for example, ethyl alcohol) that are readilysolubilizedbySCCO2andmodifyitssolventpower[22,26,34–69].Thisstrategyhasthedrawbackthatalargersolventpoweralsoimpliesalowerprocessselectivityand because, as a rule, the cosolvent is liquid at atmospheric pressure, itwill becollectedintheseparatortogetherwiththeextractedcompounds.Subsequentprocessingforsolventeliminationisrequired;therefore,oneoftheadvantagesofSFE(i.e.,solventlessoperation)islost.
Anotherpossibleprocessarrangementisthecontinuousfeedinganddischargingofthesolidtoobtaincontinuousprocessingofthesolidmatter[70].Thisoperationismadepossiblebyaddingtwosolidextrudersatthetopandbottomoftheextractorandcanavoidtheuseoftwoormoreextractorstosimulatecontinuoussolidprocessing.Designandoperationof the twoextruders isnotcheapandsimple.Apatentexistsonthisoperationmode,butithasnotyetbeenindustriallyapplied.
10.2.1 Selection of the operating parameterS
Selection of the operating conditions depends on the specific compound or compoundfamilytobeextracted;molecularweightandpolarityhavetobetakenintoaccount case by case. However, some general rules can be applied. First of all,processingtemperatureforthermolabilecompoundshastobefixedbetween35°Cand60°C (specifically, in thevicinityof thecriticalpoint andas lowaspossible
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Essential Oils Extraction and Fractionation Using Supercritical Fluids 309
toavoiddegradation).TheincreaseoftemperaturereducesthedensityofSCCO2(forafixedpressure), thusreducingthesolventpowerofthesupercriticalsolvent.However,italsoincreasesthevaporpressureofthecompoundstobeextracted;thus,thetendencyofthesecompoundstopassinthefluidphaseisincreased.Themostrelevantprocessparameter is theextractionpressure thatcanbeused to tune theselectivityoftheSCF.Thegeneralruleisthis:thehigherthepressure,thelargerthesolventpowerandthesmallertheextractionselectivity.Frequently,solventpowerisdescribedintermsoftheSCCO2densityatthegivenoperatingconditions.CO2densitycanvaryfromabout0.15to1.0g/cm3andisconnectedtobothpressureandtemperature.Itsvariationisstronglynonlinear;therefore,properselectionrequirestheuseoftablesofCO2properties[71,72].
The other crucial parameters in SFE are CO2 flow rate, particle size of thematrix,anddurationoftheprocess(extractiontime).Theproperselectionoftheseparametershasthescopeofproducingthecompleteextractionofthedesiredcompoundsintheshortesttime.Theyareconnectedtothethermodynamics(solubility)andkineticsoftheextractionprocessinthespecificrawmatter(masstransferresistances).Theproperselectiondependsonthemechanismthatcontrolstheprocess;theslowestonedeterminestheoverallprocessvelocity.CO2flowrateisarelevantparameteriftheprocessiscontrolledbyexternalmasstransferresistanceorbyequilibrium;theamountofsupercriticalsolventfeedtotheextractionvessel,inthiscase,influencestheextractionrate.Particlesizeplaysadeterminingroleinextractionprocessescontrolledbyinternalmasstransferresistances;asmallermeanparticlesizereducesthelengthofdiffusionofthesolvent.However,ifparticlesaretoosmall,theycancausechannelingproblems inside theextractionbed.Partof thesolventflowsthroughchannelsformedinsidetheextractionbedanddoesnotcontact thematerialtobeextracted,thuscausingalossofefficiencyandyieldoftheprocess.Asarule,particleswithmeandiametersrangingbetweenapproximately0.25and2.0mmareused.Theoptimumdimensioncanbechosencasebycaseconsideringthewatercontent in thematrixand thequantityofextractable liquidcompoundsthatcanproducecoalescenceamongtheparticles,thusfavoringirregularextractionalongtheextractionbed.Moreover,theproductionofverysmallparticlesbygrindingcouldproducethelossofvolatilecompounds.ProcessdurationisinterconnectedwithCO2flowrateandparticlesizeandhastobeproperlyselectedtomaximizetheyieldoftheextractionprocess.
Essentialoilsaremainlyformedbyhydrocarbonandoxygenatedterpenesandbyhydrocarbonandoxygenatedsesquiterpenes.Theycanbeextractedfromseeds,roots, flowers, herbs, and leaves using the process of hydrodistillation (HD). HDis a very simple process but suffers from many drawbacks: thermal degradation(forexample,ofcissabinenehydrateandcissabinenehydrateacetateinmarjoramessentialoil),hydrolysis(forexample,oflinalylacetateinlavenderessentialoil),andsolubilizationinwaterofsomecompoundsthataltertheflavorandfragranceprofileofmanyessentialoilsextractedbythesetechniques.Insomecases,acomparisonbetweenthecompositionsoftheessentialoilsobtainedbySFEandthoseobtainedbyHDhasbeenmade.Forexample,inthecaseofrosemaryoil[1],theSFEessentialoilcontainedhigherpercentagesoflinalool,verbenone,andisobornylacetate;theircontentwasalmostdouble that in thehydrodistilledoil.Thedifferencewas
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310 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
moreevidentintermsofthetotalpercentageofoxygenatedmonoterpenes(whichstronglycontribute to thefragrance):73.7%for theSFEoilagainst59.4%for thehydrodistilledoil.Organoleptictestsconfirmedthatthehydrodistilledoilpossessedalessintenserosemaryaroma.
Insomeothercases,liquidsolventextractionisalsoperformed;theresultsarethesocalledoleoresinsorconcretesthataresolidorsemisolidandcontainessentialoilcompoundstogetherwithwaxes,fattyacids,coloringmatter,antioxidants,andothercompounds.Volatileoilorabsoluteisalsosometimesthetargetoftheprocessthatisroughlyspeakingthevolatilefractionoftheoleoresin.
Essential oil isolation is an example of extraction plus fractional separation.Indeed,thisprocesscanbeoptimallyperformedoperatingatmildpressures(from90to100bar)andtemperatures(from40°Cto50°C)because,attheseprocessconditions,alltheessentialoilcomponentsarelargelysolubleinSCCO2[73–76].Forexample,at40°C,linalooliscompletelymisciblewithSCCO2atpressuresgreaterthanabout85bar[73],andlimonene[74–76],αpinene[76],andfenchone[76]arecompletelysolubleatabout80bar.
However,essentialoilcompoundsareatleastpartlylocatedinsidethevegetablestructure;therefore,masstransferresistanceshavetobeconsidered,too.Atthepreviously discussed operating conditions, essential oil components are extractedtogetherwithcuticularwaxes(i.e.,paraffiniccompoundslocatedonthesurfaceofvegetablematterwiththescopeofcontrollingitsperspiration).Paraffinsexhibitarelativelylowsolubilityattheseoperatingconditions[77].Whenextractionpressureisincreased,theircontributionintheextractismoreprevalentandothercompounds(suchasfattyacids)canalsobeincreasinglyextracted.Sincewaxesareonthestructuresurface,theirextractioniscontrolledbytheirsolubility,whereasessentialoilextractioniscontrolled,atleastinpart,byinternalmasstransferresistancesinthevegetable structure. As a result of these interactions, essential oil and waxes arecoextractedatalloperatingconditions.Toisolate theessentialoil, it isnecessarytotakeadvantageofthefactthat,atlowtemperatures(from–5°Cto+5°C),waxesarepracticallyinsolubleinCO2,whereasterpeniccompoundsmaintainverylargesolubilities (they are completely miscible in liquid CO2). Therefore, it is possibletoobtainafractionationoperating,forexample,theextractionat90barand40°Cand,then,performingafirstseparation,forexampleat0°C,90bar,andasecondseparationat15°C,20bar. In thismanner, theselectiveprecipitationofwaxes isobtainedinthefirstseparatorandnoprecipitationoftheotherextractedcompoundsoccurs,whereas, in the secondseparator, essentialoil is recovered.An industrialplant(V=1200dm3)thatusesthisprocessarrangementhasbeenconstructedandsuccessfully operated since 1996 (Essences, Italy). One must take into account,however,thatitisnotpossibletoperformSFEdirectlyat0°Cand90barbecausethevegetablemattercontainsmanyothercompoundfamilies(antioxidants,colors,etc.)thataresolubleattheseprocessconditionsand,therefore,acomplexmixtureofessentialoilplustheseothercompoundsisobtained.
Dataonessentialoils,volatileoils,andoleoresinsobtainedbySFEareshowninTable10.1,alphabeticallyorganizedbythecommonname(rawmaterial),thebotanicalname,and the targetcomponents (theextract). InTable10.1, laboratory,pilotplant, and analytical studies performed using very small extractors are included.
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table 10.1sFe of oleoresins (or), essential oils (eo), and Volatile oils (Vo)
raw Material botanical name extract references
leaves
Basil Ocimum basilicum EO [73,78]
Eucalyptus Eucalyptus globulus L. EO [11]
Laurel Laurus nobilis EO [14]
Lemonbalm Melissa officinalis EO [79]
Lemonbergamot Monarda citriodora EO [79]
Lemoneucalyptus Eucalyptus citriodora EO [79]
Lemongrass Cymbopogon citratus EO [79,80]
Lovage Levisticum officinale Koch. EO [22,63,81]
Marjoram Origanum majorana L. EO [82,83]
Mint Mentha spicata insularis EO [15,78]
Oregano Origanum vulgare L. EO [84]
Sage Salvia desoleana EO [15,23,63,85]
Spikedthyme Thymbra spicata EO [86]
Thyme Thyme zygis sylvestris EO [87]
Flowers
Chamomile Chamomilla recutita L.R. EOandOR [88,89]
Lavender Lavandula angustifolia EO [90]
seeds
Aniseed Pimpinella anisum L. EO [91]
Fennel Foeniculum vulgare Mill. EO [17]
Lovage Levisticum officinale Koch. EO [22,63,81]
roots
Celery Apium graveolens L. EO [22]
Lovage Levisticum officinale Koch. EO [22,63,92]
other Matrices
Bacurifruitshells Platonia insignis Mart. EO [93]
Blackpepperfruits Piper nigrum L. EO [94,95]
Cashewnutshell Anacardium occidentale VO [30,96]
Clovebud Eugenia caryophyllata EO [13]
Lemonbalmherb Melissa officinalis EO [16,21]
Oreganoherb Origanum vulgare L. EO [61,63,78,97]
Pennyroyalplant Mentha pulegium L. EO [50]
continued
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312 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
Inonlysomecasestheoperatingconditionshaveoptimizedtomaximizeboththeyieldandtheselectivityoftheprocess.Therefore,theyieldandtheoperatingconditionsindicatedbytheauthorsarelargelyinfluencedbythefinalscopeofthepaper:toisolatetheessentialoilortoextrapolateitscompositionfromtheunfractionatedextract(“concretelike”).Ananalysisontheinfluenceofsomeprocessparameters,suchaspressure,temperature,extractiontime,percentageofcosolvents,andsolventflowrates,isavailableinsomeofthepapersconsideredinTable10.1.
Inseveralcases, thesamematrixcontainsessentialoils(orvolatileoils)withknownbiologicalactivityandhighmolecularweightcompoundsthatexhibitnutraceutical or pharmaceutical activity (see Table10.2 for several examples). A largespectrumofcompoundscanbeinsertedinthesecategoriesbecausefoodadditiveswithnutritionalandpharmaceuticalproperties(nutraceuticals)rangefromtocopherolstocarotenoidstoalkaloidstounsaturatedfattyacids.Pharmaceuticalcompoundslike Artemisinin (an antimalaria drug), Hyperforin (an antidepressant drug), andsterolscanbeextractedfromvariousmatters.Inthesepapers,adifferentemphasisisgiventothesecharacteristicsandtheextractischaracterizedmoreforitsfunctionalitythanwithrespecttoessentialoilcomposition.
10.2.2 exampleS
Aspreviouslystated,essentialoilscanbeextractedfromdifferentmatrices,includingleaves,flowers,andseeds.Inthissection,weillustratesomeexamplesofextraction.
10.2.2.1 leaves
Essentialoilisolationhasbeenperformedasaruleinplantsoperatedwithatleasttwoseparatorsinseries.AnexampleofSFEfromleavesissage(Salvia officinalis)essentialoilextraction[85].Thebestisolationconditionshavebeenfoundat90barand50°C.Sagewaxeseliminationhasbeendemandedtothefirstseparator,fixingthe conditions at 85bar and–12°C. In the second separator, operating at 17bar,
table 10.1 (continued)sFe of oleoresins (or), essential oils (eo), and Volatile oils (Vo)
raw Material botanical name extract references
other Matrices (continued)
Redpepperfruits Capsicum frutescens L. OR [98]
Staranise Illicium anisatum EO [13]
Juniperfruits Juniperus communis L. VO [99]
Concretes
Jasmine Jasminum grandiflorum L. VO [100]
Rose Rosa damascena Mill. VO [101]
Tuberose Nepeta tuberosa L. EO [12]
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–6°C,onlyessentialoilisfound.Theessentialoilobtainediswaxfreeandcontainsonly traces of highmolecularweight compounds. These highmolecularweightcompounds were identified as two flavones. The main compounds extracted are1,8cineole,camphor,andcaryophyllene.Theasymptoticvalueofthesageessentialoilyield,expressedasweightofextractdividedbytheweightofthestartingmaterial,is1.35wt%ofthechargedmaterial.
table 10.2essential oil–related and biologically active Compounds
raw Material botanical name extract references
leaves
Aloevera Aloe barbadensis Miller αtocopherol [45]
Eucalyptus Eucalyptus camaldulensis var. brevirostris
Gallicandellagicacids [39]
Hawthorn Crataegus sp. Flavonoidsandterpenoids [48]
Marjoram Origanum majorana L. Carotenoidsandchlorophylls [19]
Marjoram Origanum majorana L. Phenolicandtriterpenoidantioxidants
[102,103]
Rosemary Rosmarinus officinalis L. Rosmanol,carnosicacid,andcarnosol
[31–33,36,41,104–107]
Sage Salvia officinalis L. Carnosolicacid [23]
Savory Satureja hortensis L. Oil [20]
Flowers
Chamomile Matricaria recutita Flavonoidsandterpenoids [48]
Hawthorn Crataegus sp. Flavonoidsandterpenoids [48]
Marigold Calendula officinalis Flavonoidsandterpenoids [48]
seeds
Coriander Coriandrum sativum Tocopherols,flavonoids,andterpenoids
[108]
other Matrices
Aniseverbena Lippia alba Limoneneandcarvone [109,110]
Coffeepowder Coffea arabica Aroma [111]
Gingerrhyzomes Zingiber officinale Roscoe Gingerolsandshogaols [40,41]
Horsetailplant Equisetum giganteum L. Oleoresin [112]
Mosobambooplant Phyllostachys heterocycla EthoxyquinA,sesquiterpeneA,andcyclohexanoneA
[46]
Paprikaflake Capsicum annuum L. Carotenoids,tocopherols,andcapsaicinoids
[113,114]
Sawpalmettoberries Serenoa repens Fattyacidsandβsitosterol [56]
Spearmintplant Mentha spicata Tocopherol [115]
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314 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
10.2.2.2 Flowers
As examples of SFE of essential oils from flowers, we consider lavender [90]andchamomile[89]essentialoilextractionandisolation.Inthecaseoflavenderflowers[90],theoperatingconditions,intermsofpressureandtemperature,havebeenfixedasfollows:intheextractor,90barand48°C;inthefirstseparator,80barand–10°C;inthesecondseparator,25barand0°C.Waxesprecipitateinthefirstseparator,andthelavenderessentialoilprecipitatesinthesecondseparatorandmainlyconsistsof1,8cineole,linalool,camphor,4terpineol,αterpineol,andlinalylacetate.Theasymptoticvalueofthelavenderessentialoilyieldis4.9wt%ofthechargedmaterial.
In the case of chamomile flower [89], the operating conditions are similar.Themajor constituentsof this essential oil areoxygenated sesquiterpenes,whichrepresent 78.48% of the oil composition. In Table10.3, the compositions of thechamomileessentialoilandthechamomilecuticularwaxeshavebeenreportedtogiveanexampleofdetailedidentificationofessentialoilcomponents.Oxygenatedsesquiterpenescontainthemostcharacteristicchamomileessentialoilcompounds,namelybisabololoxideB(16.88%),αbisabolol(0.35%),bisaboloneoxide(7.76%),andbisabololoxideA(50.42%).Matricine(evaluatedaschamazulene)represents,inthiscase,3.52%anddicycloetherscontributemorethan12.97%tothetotalextract.Theyieldsobtainedare1.18%foressentialoiland0.8%forcuticularwaxes.
10.2.2.3 seeds
Inthecaseofseedoils,atleasttwoSCCO2extractablecompoundfamiliesarecontainedinthevegetablematrix:essentialoilandseedoil;therefore,extractionconditionshavetobesettoavoidtheircoextraction.Anexampleisgivenbyfennelessentialoilisolation[116].Thefirststepoftheextractionprocessisperformedat90barand50°C,withtheaimofselectivelyextractingfennelessentialoil.Waxeseliminationisdemandedtothefirstseparator.Theextractionandsimultaneousisolationoffennelessentialoilhasbeensuccessful.Inthefirstseparator,paraffinicwaxesarecollectedwithcarbonatomnumbersbetween25and37; thiswaxcompositionagreeswellwiththatofvariousothervegetablematterextractedbySCCO2[1].Inthesecondseparator,fennelessentialoil iscollected.It ismainlyformedbyestragole(about80%), anethole, fenchone, and limoneneand isnot contaminatedbywaxesorbyhighermolecularweightcompounds.Anessentialoilasymptoticyieldof1.8wt%oftheloadedmaterialhasbeenobtained.
Thesecondstepoftheextractionprocess,performedat40°Cand200bar,producestheextractionoffennelvegetableoil.Also,inthiscase,thefirstseparatorisusedtoprecipitatecoextractedwaxes.Thewhitemasscollectedinthefirstseparatorisagainformedbyparaffins,althoughtheirmolecularweightisslightlylarger(carbonatomsfrom25to41).Fenneloiliscollectedinthesecondseparator.However,higherpressuresarecommonlyusedinthisstepoftheprocesstoacceleratetheextractionofthevegetableoil.
10.2.2.4 other Matrices
Asexamplesofextractionfromdifferentmatrices,weconsiderclovebudandstaraniseessentialoils.Inbothcases,thebestessentialoilprocessconditionsare90bar
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Essential Oils Extraction and Fractionation Using Supercritical Fluids 315
table 10.3Composition of the Chamomile essential oil and of Cuticular Waxes (adapted from [89])
Compoundretention time
(min)area (%)
essential oil
6methyl5hepten2one 20.02 0.07
Ocimene 25.33 0.11
Linalool 28.26 0.57
Isoborneol 33.07 0.10
Menthol 33.43 <0.05
4terpineol 34.02 0.07
αterpineol 35.00 0.09
nid.C10H16O 38.25 0.11
Nerol 39.58 0.65
Geraniol 41.07 0.24
Menthylacetate 45.13 0.17
n.id.C12H22O2 48.10 0.17
βelemene 49.17 <0.05
βcaryophyllene 51.01 0.13
βfarnesene 53.33 1.53
trans-nerolidol 60.07 0.42
Spathulenol 60.53 0.65
Caryophylleneoxide 63.18 0.17
n.id.C15H26O 64.03 0.39
Tcadinol 64.39 0.36
BisabololoxideB 65.38 16.88
αbisabolol 66.14 0.35
Bisaboloneoxide 67.09 7.76
Matricine(chamazulene) 69.39 3.52
BisabololoxideA 70.53 50.42
n.id.C15H26O 71.43 0.34
n.id. 72.09 0.56
n.id.C15H26O 74.07 0.18
cis-dicycloetherMW200 77.46 9.64
trans-dicycloetherMW200 78.23 3.33
trans-farnesol 79.02 0.32
cis,trans-farnesol 79.55 0.42
cis-dicycloetherMW214 79.92 <0.05
trans-dicycloetherMW214 81.18 <0.05
continued
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316 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
and50°Cforextraction,90barand–10°Cinthefirstseparator,and15barand10°Cinthesecondseparator.
Theobtainedclovebudessentialoilcomprisesmainlyeugenol(65.9%),caryophyllene(11.1%),andeugenylacetate(19.0%).Theyieldis20.7%byweightofthematerialchargedintheextractor.
Theobtainedstaraniseessentialoilcontains94.2%anethole,1.4%estragole,1.7%limonene,and0.3%linalool.Theasymptoticyield is7.3%byweightof thechargedmaterial.
10.2.2.5 Flower Concretes Fractionation
Whenvegetablematerialscharacterizedbyaveryshortlifehavetobeprocessed,asinthecaseofmanyflowers,thefragranceproductionisperformedintwosteps.Thefirststepconsistsofsolventextraction,usuallybyhexane,whichyieldsanintermediateproductcalled“concrete.”Itismainlycomposedoffragrancerelatedcompoundsbutalsocontainslargequantitiesofparaffins,fattyacids,fattyacidsmethylesters, diterpenic and triterpenic compounds, pigments, and other substances. In
table 10.3 (continued)Composition of the Chamomile essential oil and of Cuticular Waxes (adapted from [89])
Compoundretention time
(min)area (%)
Waxes
Hexadecane 13.12 0.44
Octadecene 17.43 2.17
Docosene 26.82 0.83
Tricosane 29.59 1.64
Tetracosane 32.09 0.31
Pentacosane 34.65 10.50
Hexacosane 36.98 1.52
Methylhexacosane 38.73 0.26
Heptacosane 39.74 17.56
Octacosane 41.78 2.74
Methylheptacosane 43.07 0.85
Nonacosane 43.94 24.12
Triacontane 45.54 2.86
Entriacontane 47.73 19.71
Methyltriacontane 49.24 1.26
Dotriacontane 49.80 1.54
Methylentriacontane 51.39 1.16
Tritriacontane 52.57 9.46
Methyldotriacontane 54.70 1.13
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the second step, theconcrete ispostprocessedby steamdistillationor solubilization ina largeexcessofalcohol toobtainavolatileoil containing the fragrance.Theseconventionalpostprocessing techniquesare subject to somedisadvantages,suchasthermaldegradation,incompleteeliminationofnonvolatilecompounds,andfractionationofthecompoundsthatformthefragrance.Becausefloweressentialoilshavealargecommercialvalue(thousandsofdollarsperliter),exploringtheuseofSCCO2extractionasanewwaytofractionatetheconcretecouldbebeneficial.
In thecaseof roseconcrete [101],apreliminarystudyshowed that themajorvolatilecompoundscontainedinthestartingmaterialwere2phenylethanol(25.1%),citronellol(4.7%),and2phenylethylacetate(2.7%).However,italsocontainedmanyothercompoundsthatdonotcontributetorosefragranceformation,likeparaffins.Amongthese,twolongchainparaffinicalcoholshavebeendetected.Theyarecommonly called “steroptens” and are characteristic of rose concrete and adverselycontributetorosefragrance.
Roseconcretehasbeenwarmedupto35°C,mixedwith2mmdiameterglassbeads to perform SCCO2 processing, and then charged into the extractor. Themixinghasbeenperformedtoobtainathinlayerofconcretearoundtheglassbeads.Thisprocedurehasbeenusedtomaximizethecontactsurfacebetweentheconcreteandthesupercriticalsolventandtoavoidchanneling.Thesolutionattheexitoftheextractor,asforsolidextraction,flowedintothetwoseparatorsoperatedinseries,inordertofractionatetheextract.Thefirstseparatorwassetat80barand–16°C,whereasthesecondseparatorwassetat15barand0°CtominimizethelossofvolatilecompoundsinthegaseousCO2streamattheexitoftheapparatus.Attheendoftheextractionprocess,glassbeadswererecoveredbywashingwithwarmethanol.
TheoptimizedSFEisperformedat80barand40°C,andamaximumyieldofvolatilecompoundsof49%byweightofthechargedmaterialhasbeenrecoveredinthesecondseparatorusingfractionalseparation.Theprocesswasextremelyselective:nounwantedcompoundsweredetected.Thewaxesyieldinthefirstseparatorwasabout2%byweightoperatingat80barand40°Cbutcanreachupto10.4%byweightiftheextractionisperformedat120barand40°C.
At the endof theSFEprocess at80bar and40°C,on the exhaustedcharge,a further extraction step has been performed operating at 120 bar and 40°C for100min.Thehigherpressurestepyieldsafurther14.9%byweightofthecharge.The extract is still liquid but contains only 1.0% 2phenylethanol, whereas thepercentage of steroptens is 21.3%. The second step was performed to evaluatewhetherothervaluablecompoundswerestillcontainedinthestartingmaterialandwerenotpreviouslyextractedat80barand40°C.
Inthecaseoftuberoseflowers,concretefractionationisalsorequiredbecauseSCCO2 extraction performeddirectly on the tuberose flower is not applicable atanindustrialscalesincetheyieldinessentialoilfromflowersislessthan0.1%byweight.TuberoseconcretefractionationusingSCCO2hasbeenperformed[12]withtheaimofseparatingvolatileoilfromthehighermolecularweightcompounds.Theextractionprocesshasbeencoupledagainwiththefractionalseparationtechniquethatusestwoseparationstagesoperatinginseries.
SystematicSFEtestsontuberoseconcretehavebeenperformedintherangeof80to100bar,operatingatatemperatureof40°Candanalyzingtheproductcollected
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318 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
in thetwoseparatorsbygaschromatography–massspectrometry.Byoperatingat80bar,theresearchersobtainedthemaximumcontentoffragrancecompoundsintheextractcollectedinthesecondseparator.
Since the different compound families that constitute the tuberose volatile oil(hydrocarbonterpenes,oxygenatedterpenes,andbenzenederivatives)showdifferentextraction rates, thecompositionsof the tuberoseoilchangesduring theextractionprocess.Theextractrecoveredafter20minutesofextractioncontainsahighpercentage(>83%)ofoxygenatedcompounds(monoterpenesandbenzenederivatives).Thevolatilefractionrecoveredintheextractiontimeintervalbetween360and480minutesconsistsofalowerpercentage(<79%)ofoxygenatedcompoundswithanincrementofthepercentageofthetransmethylisoeugenolandoftheeugenylacetate,whereasthemostvolatilecompounds,suchasmethylbenzoateandmethylsalicylate,arereduced.
The fraction recovered in the extraction time interval between 690 and 750minutescontainsverylowquantitiesofaromacompoundsbutstillcontainslargequantitiesoftransmethylisoeugenol,eugenylacetate,andlactones.
Bydividingthetuberoseoilcompoundsintothreefamilies,hydrocarboncompounds(monoterpenesandsesquiterpenes),oxygenatedcompoundswith10orlesscarbonatoms(monoterpenesandbenzenederivatives),andoxygenatedcompoundswith 15 or more carbon atoms (sesquiterpenes and lactones), the contribution ofeach compound family can be calculated as the sum of the area contribution ofall compounds belonging to that family. Tuberose oil contains a low percentageofhydrocarboncompounds(monoterpenesandsesquiterpenes)andtheirpercentagedecreasebyincreasingtheextractiontime.Thepercentageofoxygenatedcompoundswith 10 or less carbon atoms also decreases during the extraction, whereas thepercentage of oxygenated compounds with 15 or more carbon atoms increases,especially at extraction times longer than450minutes.Thismeans thatdifferentsolubilitiesandperhapsmasstransferresistancescharacterizethevariouscompoundfamiliesduringtheextractionprocessandtheextractiontimeplaysarelevantroleinthefinalcompositionoftuberoseoil.Moreover,byinterruptingtheextractionoftheoilatdifferenttimes,itispossibletofractionatethefragranceandtoobtainanextractinwhichtopnotesorbottomnotesprevail.
It is also possible to divide the tuberose oil compounds in two groups: thefragrance compounds (oxygenated compounds) and the nonfragrance compounds(hydrocarboncompounds).Theyieldcurvesshowanexponentialtrendagainsttheextractiontime;thehydrocarboncompoundscurvegetsflatafterthefirst300minutesofextraction,whereas theyieldcurveoffragrancecompoundsasymptotizesonlywhencompleteextractionisperformed(after750minutes).
Asconfirmedbythetwoexamples,SFEisgenerallyapplicabletoflowerconcretesandcanbeveryselective.Thus,itcanbeusedtorecoverinarelativelycheap,singlestepSFEoperationessentialoilswidelyusedintheperfumeindustry.AbetterproductisalsoobtainedwithSFEthanwithtraditionaltechniques.
10.3 lIquId Feed ProCessIng
Thefractionationofliquidmixturesintotwoormorefractionsisanotherrelevantprocess.Inatypicalapparatus,twopumpsdelivertheliquidsolutionandSCCO2to
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Essential Oils Extraction and Fractionation Using Supercritical Fluids 319
thepackedcolumn.ThepackingisaninertmaterialcharacterizedbyalargespecificsurfacewhosescopeistofavorthecontactbetweentheliquidandtheSCF.SCCO2generallyflowsalong the column from thebottom to the top,whereas the liquidsolutionisusuallyaddedtothetop.However,itisalsopossibletofeedtheliquidatanintermediatepositionalongthecolumnandtoaddarecycleofpartofthefluidphaseexitingatthetop.AschemeoftheapparatusisshowninFigure10.2.
10.3.1 Selection of the operating parameterS
Selectionof theoperatingparameters is basedon thedifferent solubilitiesof theliquidstobeseparatedinSCCO2.TheidealcaseisobtainedwhenonlythecompoundstobeextractedaresolubleinSCCO2andalltheotherliquidcomponentsarecompletelyinsoluble.However,thiscaseisrareandalimitedsolubilityoftheotherliquidcompoundsformingthemixturehastobetakenintoaccount.Forthisreason, pressure and temperature of the process have to be accurately chosen toselect theconditionsatwhichthemaximumdifferenceinsolubilityexistsamongthecompounds tobeextractedandall theothercompounds in themixture.Alsointhiscase,CO2densityisfrequentlyusedasacriteriontofindtheconditionsofmaximumselectivity.ThedifferenceindensitybetweentheliquidandSCCO2isanotherparametertobetakenintoaccount;toallowthecountercurrentoperation,SCFdensityhastobelowerthanthedensityoftheliquidmixture.
The traditionaloperationofpackedcolumns requires that liquidflow ratebelargerthantheminimumamountthatassuresthecompletewettingofthepacking.Thefeedratioisalsoselectedtoavoidthemassiveentrainmentoftheliquidinthefluidphase(flooding).TheseconditionshavetoberespectedalsowhenaSCFisusedasthefluidprocessingmedium.Theclassicalcalculationintermsofthenumberoftheoreticalequilibriumstagesrequiredforseparationcanalsobeapplied.Apossiblevariation of this processing scheme can consist of the adoption of a temperature
Liquid IN
2
3 4 5
Out
SCF IN
1
FIgure 10.2 1)CO2pump;2)liquidpump;3)packedtower;4)firstseparator;5)secondseparator.
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320 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
profilealongthecolumn,withtheaimofoptimizingtheseparationtemperaturewithrespecttothecompositionofthemixturesatdifferentlevelsinsidethecolumn.
The extraction of liquid mixtures is controlled by the relative solubilities inSCCO2ofthevariouscompoundsformingthemixture—thatis,thethermodynamiclimitationoftheprocess.Masstransferbetweenthetwophasesrepresentsthekineticlimitation.Thedistancefromtheequilibriumconditionisthedrivingforcefortheseparationalongthecolumn.
10.3.2 exampleS
ThefractionationofliquidmixturesbySFEhasbeenproposedforvariousapplications, including some essential oils, with the aim of improving their fragranceandeliminatinghydrocarbonterpeniccompoundsthatcanrapidlydecomposeandtherefore can shorten the shelf life of the product. The problem does not have asimple solution. Selective elimination of hydrocarbon terpenes (deterpenation) isrequiredincitruspeeloilsbecausethesecompoundscontributetoasmallextenttothecitruspeelfragranceandarerapidlyoxidizedbyairandcanundergostructuralrearrangements. These essential oils are mainly formed by hydrocarbon andoxygenatedterpenes,butalsocontainsmallquantitiesofsesquiterpenesandhighmolecularweightcompounds likecoumarins,psoralens,andwaxes.Hydrocarbonterpenepercentagecanrangefrom60%to99%.InTable10.4,someexamplesoffractionationsofliquidmixturesarereported.
The fractionation of a peel oil with SCCO2 was studied using a mixture offour key compounds [117]. The composition of this mixture was determined bythefollowingconsiderations.Limoneneisahydrocarbonterpeneanditisthepredominant compound inallpeeloils,withconcentrationsbetween30%and80%.Therefore, it canbechosenas themost abundantkeycompound,withaconcentration of 60% by weight. Linalool is one of the most representative among theoxygenatedcompoundsand,therefore,itsconcentrationwassetat20%byweight,representingthesecondmostabundantcomponentinthekeymixture.γTerpinenehasamolecularweightsimilartolimonenebutisahydrocarbonterpenethathasavolatilityveryneartooxygenatedcompounds.Therefore,itspresenceinthemixture(10%)allowsevaluationoftheeffectivenessofseparationforthosecompoundsthat
table 10.4Fractionation of liquid Mixtures by sFe in Continuous (C) and semicontinuous (sC) Plants
Initial mixture objective Process references
Citruspeeloilkeymixture Separationoflimonenefromlinalool C [117]
Citrusoil Deterpenation C [118]
Citrusoil Deterpenation SC [119]
Orangepeeloil Deterpenation C [120]
Oreganumoil Deterpenation SC [121]
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Essential Oils Extraction and Fractionation Using Supercritical Fluids 321
aremoredifficulttoseparatethanlimonene.Linalylacetate(10%)isrepresentativeofoxygenatedcompoundswithamolecularweighthigherthanlinalool.
Systematicexperiments[117]havebeenperformedona2mcolumnoperatedincountercurrentandequivalenttoabout2.5theoreticalequilibriumstages.Theoperatingpressureandtemperaturerangedbetween75and90barandbetween40°Cand80°C,respectively.Solventtofeedratiosof60,80,and120wereused.Theeffectsofdifferentfeedinsertionpointsandcolumnpackingswerealsotested.Experimentalresultsindicatedthatfractionationcouldbesuccessfullyobtainedbetween75and80barandbetween50°Cand80°C.Ingeneral,anincreaseinsolubilitycorrespondsto a decrease in selectivity and, thus, optimization of the separation is required.Experiments also indicated that temperature helps separation and, furthermore,increasestherecoveryofoxygenatedcompounds.Theupperlimittotheoperatingtemperature is given, however, by the thermal stability of the product. The totalandpartialrefluxesoftheextractatthecolumntopshowadefinitelypositiveeffecton theseparation.However,onlywhen theoperation ina twocolumnserieswasperformed(i.e.,usingalengthofcolumnequivalenttoabout5equivalentstages),researchersobserved theelimination in the topproductof linalylacetateand thereductionoflinaloolcontenttoabout0.8%.
Liquid feed can be alternatively fractionated by adsorption/desorption of theliquidmixtureonanadsorbent.Forexample,SCCO2hasbeenusedtoselectivelydesorbbergamotpeeloilcomponentsfromsilicagel[122].Themaximumdesorptionselectivityhasbeenobtainedoperatingat40°C, in twosuccessivepressuresteps.Thefirststep,performedat75bar,producestheselectivedesorptionofhydrocarbonterpenes;thesecondone,performedat200bar,assuresthefastdesorptionofalltheoxygenatedcompounds.
Inanotherwork[123],thirteenterpenesformingamixturecharacteristicofpeelessentialoilswereselectivelyadsorbedonsilicagelusingSCCO2.Thesecompoundsareαpinene,βpinene,myrcene,limonene,γterpinene,βcaryophyllen,citronellylacetate,geranylacetate,linalylacetate+geraniol,linalool,citronellal,andcitral.Theycanbegroupedinfourfamiliesofpseudocomponents:hydrocarbonterpenes(likelimonene),asesquiterpene(βcaryophyllene),terpeneacetates(likegeraniol),andoxygenatedcompounds(likelinalool).Theexperimentswereperformedatdifferentpressures(130to210bar),temperatures(37°Cto57°C),andconcentrations(0.9 to 7.6 g/kgsolvent). The different pseudocomponents show a different adsorptionbehaviorthatcanbejustifiedlookingattheinteractionswiththeactivesitesof the adsorbent. Hydrocarbon terpenes and sesquiterpenes can be adsorbed onCH3groupsofsilicagelandarerapidlyshiftedbythemorepolarcompounds(displacement).OxygenatedcompoundscanalsobondwithOHgroups(silanol).Compoundswithhighermolecularweightsorhigherpolaritiescanmove(displace)theotherspeciesfromtheadsorptionsiteinwhichtheywerelocated.Thedisplacementof the lessstronglyadsorbedcompoundsismoreevidentat lowerconcentrationsandallowstheselectiverecoveryofthevariousfractionsattheexitoftheadsorption/desorptioncolumn.
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322 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
10.4 antIsolVent extraCtIon
Therecoveryofsolidcompoundsfromaliquidmixturerequiresdifferentprocessapproachessincethefixedbedextractordoesnotadapttoprocessliquidmixtures.Inaddition,thepackedtowercannotbeusedinthesecasesbecausethesolidcompoundsprecipitateontheinternalpackings.
The Supercritical antisolvent extraction (SAE) process is conceptually verysimilartoSupercriticalantisolventmicronization(SAS),butthescopeoftheprocessistherecoveryofoneormoresolidcompoundsfromaliquidmixture.ItconsistsofthecontinuousflowofSCCO2andoftheliquidmixtureinapressurizedprecipitationvessel.Iftheprocessconditionshavebeenproperlyselected,theliquidisrapidlydissolvedintheSCF,whereasthesolidprecipitatesatthebottomoftheprecipitationvessel.Therefore,inapossiblerepresentationoftheprocess,twopumpsdelivertheliquidsolutionandtheSCF,respectively.Theprecipitationvesselisusedtocollectthe solid and a vessel located downstream the precipitator and operated at lowerpressure(forexample,30barand25°C)isusedtorecovertheliquid.AschematicoftheapparatusisshowninFigure10.3.
10.4.1 Selection of the operating parameterS
Thefirststepofthisprocessistheformationofasprayoftheliquidsolution.Theintentofthisoperationistoproduceaverylargeliquidsurfaceduetotheformationofsmallliquiddropletstostronglyenhancetherateofsolubilizationoftheliquid
Liquid IN
2
3
41
Liquid Recovery
Out
SCF IN
FIgure 10.3 1)CO2pump;2)liquidpump;3)precipitator;4)separator.
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Essential Oils Extraction and Fractionation Using Supercritical Fluids 323
phaseinthesupercriticalmedium.Forthesamereason,theprocessisperformedatoperatingconditionsatwhichtheliquidsolventiscompletelysolubleinSCCO2.TheknowledgeofsolubilitydataontheliquidsolventsandofthesolidsinSCCO2ismandatoryfortheproperselectionofprocesstemperatureandpressure.
InthecaseofSAE,theinteractionsbetweenthermodynamicsconstraintsandmass transfer mechanisms also control the process performance. The enhancedmasstransferthatcharacterizesSCFsisagainadistinctiveadvantageoftheiruseasextractionmedia,togetherwiththefastandcompleteseparationbysimpledepressurizationbetweenthesupercriticalsolventandtheliquid.
Alimitationofthisprocessisthepossibleformationofaternarymixtureliquid/solid/SCCO2.Indeed,thepresenceoftheliquidcaninduceanincreaseofthesolubilityofthesolidcompoundsinSCCO2.Inthiscase,theliquidcanactasacosolventfromthepointofviewofsolidsolubilization.Whenthisphenomenonoccurs,thepart of the solid retained in thefluidphaseobviouslydoesnot precipitate and islostintheliquidrecoveredintheseparationvessel.Thelimitcaseisthecompletesolubilizationofthesolidinthefluidphasethatproducestheprocessfailure.
10.4.2 exampleS
Untilnow,SAEhasbeenusedinalimitednumberofprocesses(forexample,therecoveryofessentialoilfromamixtureessentialoil+triglycerideoil[124,125])butithasalargepotentialforfutureapplications,someofwhicharediscussedhere.
10.4.2.1 Proteins and aroma extraction from tobacco
Aprocesshasbeendeveloped for the recoveryof tobaccoproteins, aminoacids,andaromausingethylalcohol[126].Afractionationprocessisrequiredtoseparateproteinrelated(solid)compoundsfromtobaccoaroma,obtainingthesimultaneouseliminationoftheliquidsolvent.Theethanolicextractispreparedusinganethanolsolution 1% potassium hydroxide using a solution containing a tobacco blend ofapproximately10to1(v/w),for3hoursat40°C.Potassiumhydroxideisaddedtothesolutionforitsstabilization.
EthanolisreadilysolubleinSCCO2;therefore,thecouplesolventantisolventshouldnotdeserveproblemsinSAEprocessing.Proteinsandtheirderivativeaminoacidsshouldbevirtually insoluble inSCCO2dueto theircompositionandtheirmolecularweights.Flavoringcompoundscanalsobereadilyextracted.Therefore,basedontheseconsiderations,afractionationof theethanolicextract is, inprinciple, possible if processing conditions are selected to induce proteic compoundprecipitationandthetransferinthefluidphaseoftheliquidsolventtogetherwithflavoringcomponents.
Experimentshavebeenperformedatdifferentpressuresandsolidconcentrations.Goodresultshavebeenobtainedat150bar,40°C,and100mg/mL.Theethanolicextracthasbeenefficientlyfractionated,andtheyieldofprecipitatedmaterialwasabout40%(w/wofextract).
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324 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
10.5 MatheMatICal ModellIng
Mathematical modelling gives the possibility to generalize experimental resultsand,ifsuccessful,toobtainindicationsaboutsystemsdifferentfromthosestudied(simulation).Moreover,itisusefulinthedevelopmentofscalingupproceduresfromlaboratorytopilotandindustrialscales.Forthesereasons,severalattemptsatmathematicalmodellingofSFEhavebeenpresentedin the literature[1,127,128]andsomeofthesemodelsarerelatedtoSFEofessentialoils.
Amodelshouldnotbeameremathematicalinstrument,butitshouldreflectthephysicalknowledgeofthesolidstructureandtheexperimentalobservations.Therefore,mathematicalmodels that havenophysical correspondence to thematerialsandtheprocessstudiedareoflimitedvalidity,althoughtheycanbeusedtofitsomeexperimentaldata.
ThreedifferentapproacheshavebeenproposedforthemathematicalmodellingofSFE:(1)empirical[129,130],(2)basedonheatandmasstransferanalogy[131,132],and(3)differentialmassbalancesintegration[127,133,134].Themostproperanalysis is obtained from the integration of the differential mass balances: timedependentconcentrationprofilesareobtainedforfluidandsolidphases.
In facingmathematicalmodellingofSFE, severalgeneral aspectshave tobetakenintoaccount:
1.Solid material structure:Knowledgeofthebotanicalaspectsorscanningelectronmicroscope(SEM)analysisofthematerialisnecessarytovisualizethestructure.Forexample,seedsareessentiallyformedbyspecializedstructuresthatoperateassmallrecipientscontainingtheoil.Theirshapeandstructurechangeseedbyseed,butthegeneralorganizationisalwaysthesame.
2.Location of the compounds to be extracted: Thedistributionofthesolutewithinthesolidsubstratemaybeverydifferent.Theextractablesubstancesmaybefreeonthesurfaceofthesolidmaterialorinsidethestructureofthematerialitself.Essentialoilcanbelocatedneartheleafsurfaceinglandulartrichomes or in vacuoles (intracellular structures located well inside theleaf)[29,135].
3. Interactions of solutes with the solid matrix: Dependingontheinteractionsbetween thecompoundsand the solid structure,different equilibriamaybe involved. Indeed, if the material has no interactions with the matrix,equilibriumsolubilityhas tobe taken intoaccount.Thematerial canbeadsorbedontheoutersurfaceorinsidethesolidstructure;inthiscase,apartitioningequilibriumbetweensolidandfluidphaseoccurs.
4.Broken-intact cell structures: Partofthecompoundstobeextractedmaybenearthesurfaceofthestructureduetocellbreakingduringgrinding.Moreover,membranesmodificationsmayoccurduetodrying,freeingpartofthesolublematerial.
5.Shape of particles:Particlesmaybespherical,platelike,orothershapesasaresultoftheoriginalshapeofthematerial(forexample,leaves)andofthegrindingprocess.Theirshapecaninfluencethediffusionpathof thesupercriticalsolvent[29,127].
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Essential Oils Extraction and Fractionation Using Supercritical Fluids 325
Fromthepointofviewoftheextractionmechanisms,otherconsiderationsarenecessary.Theequilibriummayexistif:
1. thematerialislargelyavailable 2. itisdistributedonornearthesurface 3. thekindofequilibriumdependsontheinteractions(ifany)withthesolid
structure
Masstransferresistances,ingeneral,maybeoftwotypes:externalorinternal(and,inthiscase,variouspossibilitieshavetobeconsidered).Totakeintoaccountmasstransferresistances,differentialmassbalancesareapplied.
Essential oils forwhichmathematicalmodellingofSFEhasbeenattemptedarereportedinTable10.5.Wehavealsoindicatedifthemodelisbasedonempiricalkineticequations,on theanalogybetweenheatandmass transfer (HMT),orondifferentialmassbalancesequations(DMBE)alongtheextractionbedoronasingleparticle.
A brokenintact cell model of the SFE of essential oils can be based on thefollowinghypotheses:
1.Thebehaviorofallcompoundsextractedissimilarandcanbedescribedbyasinglepseudocomponentwithrespecttothemasstransferphenomena.
2.Concentrationgradientsinthefluidphasedevelopatlargerscalesthantheparticlesize(i.e.,concentrationvariationsinthefluidphasehaveacharacteristiclengthscalelargerthanthediameterofparticles).
3.Thesolventflowrate,withsuperficialvelocityu,isuniformlydistributedinallthesectionsoftheextractor.
table 10.5Mathematical Modelling of sFe of essential oils
raw Material extract type of Model references
Basilleaves Essentialoil DMBE [136]
Carawayseeds Essentialoil DMBE [136]
Clovebud Essentialoil DMBE [137]
Fennelseeds Essentialoil DMBE [116,138]
Gingerrhizomes Oleoresin DMBE [139]
Jalapenopepperflakes Oleoresin DMBE [140]
Lavenderflower Essentialoil DMBE(shrinkingcore) [141]
Marigold Oleoresin Variousmodelsproposed [142]
Marjoram Essentialoil DMBE [136]
Orangeflowerconcrete Volatileoil DMBE [143]
Oreganobracts Essentialoil HMT(singleplatemodel) [29]
Pennyroyal Essentialoil DMBE [135]
Pepper,black Essentialoil DMBE [144]
Rosemaryleaves Essentialoil DMBE [136]
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326 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
4.Thevolumefractionofthefluid,ε,isnotaffectedbythereductionofthesolidmassduringextraction.
5.Thesoluteinthesolidispresentintwoseparatephases.Onephaseincludesthesolutecontainedinsidetheinternalstructureoftheparticles.Itfillsafractionφtoftheoverallvolumeoccupiedbytheseedparticles.Theotherphase ismade of the solute freely available on the particle surface.Theconcentrationhereisalwaysthesameand,accordingtothehypotheses,itisequaltothepuresolutedensityρ0(thesoluteisfreelyavailableonthesurfaceand,therefore,itsconcentrationisconstant).
6.The fraction of the volume filled by the free solute before extraction isφf=1–φt.
7.Thefractionofthevolumeoccupiedbythefreesoluteduringtheextractionisψφf,whereisψ≤1.
8.Alinearequilibriumrelationshipappliesbetweenphases.
According to the above hypotheses, the mass balance on the solute in theextractoris:
ε ρ ε ρ ρ ε φ⋅ ⋅ ⋅ ∂∂
+ ⋅ ⋅ ∂∂
+ ⋅ ⋅ ∂∂
+ −( ) ⋅f L f f fDC
z
Ct
uCz
2
21 ρρ
ε φ ρ ψ
s
f
Pt
t
⋅ ∂∂
+ −( ) ⋅ ⋅ ∂∂
=1 00 (10.1)
whereDListheaxialdispersion;uthesuperficialvelocity;ρfthefluiddensity,whichissupposedlynotaffectedbythepresenceofthesolute;ρs thebulkdensityofthenonsolublesolidthatisthemassofnonsolublesolidsinthevegetablematerialsperunitoffilledparticlevolume,thatisthetotalvolumeoftheparticleminusthevolumeofbrokencells.
Thegeneralmassbalanceonthephaseofthefreesolutealoneis:
ρψ ρ
ε φψ
00
1∂∂=−
−( )−( )t
k a K Cf
funtil ψ > 0 , (10.2)
otherwise,
∂∂
=ψt
0 (10.3)
whereaisthespecificsurfaceoftheparticlesandkfistheexternalmasstransferresistance.
Themassbalanceontiedsoluteis:
∂∂
= −⋅ −( )
−( )Pt
k a P K Ci p
t1 ε φ (10.4)
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Essential Oils Extraction and Fractionation Using Supercritical Fluids 327
wherekiistheinternalmasstransferresistance.Thissystemofequationshasauniquesolutionwhentheinitialconditions(i.c.)
onC,P,andψandtheboundarycondition(b.c.)onCaregiven:
(i.c.) Att=0: C=C0; P=P0; ψ=ψ0; foreachz (10.5)
(b.c.) Atz=0:u
C DCzLε
⋅ − ⋅ ∂∂
= 0 foreacht (10.6)
(b.c.) Atz=L:∂∂
=Cz
0 foreacht. (10.7)
Thesetofdifferentialequationscanbenumericallyintegratedusingafinitedifferencemethod.
Reverchonetal.[145–148]usedSEManalysistoconfirmthepresenceofbrokencellsonparticlesurfaces.Theconceptofbrokenandintactcellswascombinedwithequilibriumrelationshipsforeitherfreesolute[149,150]orsoluteinteractingwithmatrix[133,135].Bothtypesofequilibriumwerealsoassumedtooccursimultaneouslybyvariousauthors,thefreesoluteinbrokencellsandtheinteractingsoluteinintactcells[135,137,146,147,151].
Sovovà[128]proposedthatageneralmodelapproachcanbeappliedtoseedoilandessentialoilextraction.Themodelisbasedonthedivisionoftheprocessintotwoextractionperiods:thefirstonegovernedbyphaseequilibriumandthesecondonebyinternaldiffusioninparticles,takingintoaccounttheconceptofbrokenandintactcellstoexplainthesuddenreductionoftheextractionrateafterthefirstextractionstep.Thiseffectisparticularlyevidentinthecaseofseedoilextraction.Thenewfeatureofthemodelisthedescriptionofthefirstextractionperiodconsideringdifferenttypesofphaseequilibria:independentonmatrix(solubilityequilibrium),adsorbedonmatrix(partitionbetweenthetwophases),anddifferentflowpatterns,mainlydispersion.Themodelhasbeenverifiedondatasetsfromliteraturerelatedtoseeds(almond)andessentialoils(orangepeels,pennyroyal).Thismodelpresentsalimitinthecaseofessentialoilextractionwhentheextractablematerialislocatedonlyinsidethematrix;theconceptofbroken(inthesurface)andintactcells(insidethe particle) is no longer applicable and the first part of extraction controlled byequilibriumdoesnotapply.
Gaspar et al. [29]modeled theextractionoforeganoessentialoil.Themodelisbasedontheprevalentgeometryofparticles:thoseobtainedfromleavestendtomaintainaplatelikegeometry.Massbalancesontheparticlehavebeenproposed.
Adsorptiondesorption processes can also be treated as extraction processes:adsorptiondesorptionisothermsbeingtheequilibriumcurvesduetointeractionsofthesolutesbetweenthesolidmatrixandthefluidphase.Differentialmassbalancesinthiscasecanalsodescribetheextractionprocess.Thisapproachhasbeenusedby Reverchon [152] to model the selective desorption from silica gel of two keycompoundsofessentialoils:limonene(representativeofthehydrocarbonterpenesfraction) and linalool (representative of the oxygenated terpenes fraction). The
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328 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
modelhasalsobeenextendedtothefractionaldesorptionofbergamotpeeloil[122],describingatwostepdesorptionprocess,withthefirststepperformedat40°C,75bartodesorbhydrocarbonterpenesandthesecondstepat40°C,200bartodesorbtheoxygenatedcompounds.Later,Reverchonetal.[123]modeledtheselectiveadsorption on silica gel of a complex terpenic mixture formed by 13 components. Themixturewasdividedintofourfamiliesconsideredasfourpseudo–keycomponents.Theintegrationofdifferentialmassbalancesgaveaccountofthecompetitionamongthedifferentcompoundsfortheoccupationoftheadsorptionsitesandofdisplacementeffectsobservedattheexitoftheadsorptionbed.
Mathematicalmodelling has alsobeenperformed in this case [143, 153]; thevolatileoilhasbeenconsideredasamixtureoffourcompoundfamilies(pseudocomponents)extractedfromanactivelayerofconcreteputonasphericalinertcore.Successful modelling of terpenes oxygenated terpenes and oxygenated sesquiterpenesextractionwasobtained.
Mathematicalmodellingofcountercurrentpackedcolumnhasbeenstudiedbyonlyafewauthors[154–158]andonlyinsomecaseswithreferencetonaturalmatterfractionation[154,157].ThemostinterestingworkistheoneproposedbyRuivoetal.[154],whichperformedthedynamicmodellingandsimulationofapackedcolumn.Theyusedexperimentaldatafromamodelbinarymixtureformedbysqualeneandmethyloleate,fractionatedusingSCCO2.Themodelwasformedbyasetofpartialdifferentialequationsthatcorrespondtothedifferentialmassbalancesonthepackedcolumnandalgebraicequationsthatdescribethemasstransfer,thehydrodynamicsofthetwophaseflowthroughthepackingsandtheternarythermodynamicequilibriumforthestudiedsystem.Thecolumnwasconsideredatconstanttemperature.Afairlygoodagreementwasobtainedbetweenmeasuredandpredictedcompositionprofilesoftheoutletstreamsovertime.
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337
11 Processing of Spices Using Supercritical Fluids
Mamata Mukhopadhyay
Contents
11.1 Introduction................................................................................................. 33711.2 ImportanceofSpices.................................................................................. 33811.3 BeneficialAttributesofSpices.................................................................... 33911.4 BioactiveIngredientsinSpices................................................................... 34111.5 SaleableSpiceProducts.............................................................................. 34311.6 ConventionalExtractionMethods..............................................................34611.7 SupercriticalCarbonDioxideastheExtractant......................................... 34711.8 CommercialSCFEProcess......................................................................... 34911.9 ComparisonofSpiceExtractsbyConventionalandSCFEProcesses....... 35111.10 ProcessAnalysisofSCFEfromSelectedSpices........................................ 354
11.10.1 CelerySeed(Apium graveolen).................................................... 35411.10.2 RedChili....................................................................................... 35611.10.3 Paprika.......................................................................................... 35711.10.4 Ginger........................................................................................... 35811.10.5 Nutmeg.......................................................................................... 35811.10.6 BlackPepper................................................................................. 35911.10.7 Vanilla........................................................................................... 35911.10.8 Cardamom.................................................................................... 35911.10.9 Fennel,Caraway,andCoriander...................................................36011.9.10 Garlic............................................................................................ 36111.10.11 Cinnamon...................................................................................... 362
11.11 CorrelationforSpiceOilSolubilityinSC-CO2.......................................... 36211.12 Conclusions.................................................................................................364References.............................................................................................................. 365
11.1 IntroduCtIon
In recent years, increasing demand for superior quality and safety of foods andmedicines, as well as concern for environmental pollution during their commer-cialproduction,havetriggeredstringentregulationsonthetoxinlevelsinfoodsandmedicinesaswellasonthedischargeofpollutantstotheenvironment.Inaddition,there has been increasing consumer preference for natural substances. All thesefactorshavegiven strong impetus todevelopmentofcost-effectivenew technolo-gies,suchastheoneforeco-friendlyextractionfromnaturalsubstancesemploying
7089_C011.indd 337 10/8/07 12:19:04 PM
338 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
greenandsafesolvents. In recentyears, supercriticalfluidextraction (SCFE)hasemergedasahighlypromisingenvironmentallybenigntechnologyforproductionofnaturalextracts,suchasflavors,fragrances,spiceoils,andoleoresins;naturalanti-oxidants;naturalcolors;nutraceuticals;andbiologicallyactiveprinciples.Thestateofasubstance iscalledsupercriticalwhenboth temperatureandpressureexceedtheircriticalpointvalues.Asupercriticalfluid(SCF)combinesthetwinbeneficialproperties,namelyhighdensity(whichimpartshighsolventpower)andhighcom-pressibility(whichpermitshighselectivityduetolargevariabilityofsolventpowerbysmallchangesintemperatureandpressure).Inaddition,itoffersveryattractiveextractioncharacteristics,owingtoitsfavorablediffusivity,viscosity,surfacetension,andotherthermo-physicalproperties.
Since the1980s,severalpotentialapplicationsofSCFEtechniqueshavebeenreported.Sofar,themostpopularSCFhasbeencarbondioxide(CO2),owingtoitseasyavailability,lowcost,nonflammability,nontoxicity,andaspectrumofsolventproperties in a single substance. Its critical temperature is 31.1°C and its criticalpressure is 73.8 bar. Dense or supercritical carbon dioxide (SC-CO2) could verywell be the most commonly used solvent in this century due to its wide-rangingapplications. Its near-ambient critical temperature makes it ideally suitable forprocessingof thermally labilenatural substances. It isgenerally regardedas safe(GRAS),andityieldsmicrobial-inactivated,contaminant-free,tailor-madeextractsof superior organoleptic profile and longer shelf life,withhighpotencyof activeingredients. The SCFE technique ensures high consistency and reliability in thequalityandsafetyofthebioactiveheat-sensitivebotanicalproducts,asitdoesnotalter thedelicatebalanceofbioactivityofnaturalmolecules.Allof theseadvan-tagesarealmostimpossibleinconventionalprocesses.Therefore,SCFEtechnologyusingSC-CO2asthesolventisanidealalternativetotheconventionaltechniquesforextractionofbioactive ingredients fromspices.Thischapterpresentssomeof theprinciplesandmethodsofSCFEtechnologyforprocessingofspicesforuseinfoodproducts,medicines,anddietarysupplements.
11.2 ImportanCe of spICes
Bydefinition,aspiceisan“aromatic,pungentvegetablesubstanceusedtoflowerfood,”asoriginatedfromtheLatinname“speciesaromatacea.”Anherbisdefinedasa“plantwithoutwoodytissuethatwithersanddiesafterflowering.”Spicesandherbsbothfunctionasflavoringagentsforfoodand,accordingly,theU.S.FoodandDrugAssociationincludesspicesandherbstogetherinaclassofaromaticvegetablesthatimpartflavorandseasoningtofoodratherthannutritionalvalue.Thus,spiceimplies a tropical herbal plant or some part of it that is used in cooking and incondiments aswellasincandies,cosmetics,fragrances,andmedicationsinordertoprovidearoma,flavor,andcolor,alongwithstimulatingpungencyandtaste[1].
In ancient ages, spices were employed for embalming, preserving foods, andmaskingbadodors.TheEbersPapyrus,writteninEgyptinabout1500B.C.,evenmentions some common spices, such as coriander, cumin, fenugreek, and mint,and describes how these spices were used in foods and medicines. In MedievalandRenaissance times,GreeksandRomansused tospendvast fortuneson trade
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Processing of Spices Using Supercritical Fluids 339
with Arabia, which was then the center of the spice trade. Exotic spices used tobeexhibitedasa symbolofwealthandpower, somuchso that thereweremanylongexplorationsinsearchofsourcesofspices.TheextraordinaryvoyagesmadeforthesespicesresultedinthediscoveryoftheNewWorldand,inturn,demonstratedthat theglobecouldbecircumnavigatedbysea.Thestrongmotivation tocontrolspice sources lured the British to India, the Portuguese to Brazil, the Spanish toCentralandSouthAmericaandthePhilippines,theFrenchtoAfrica,andtheDutchtoIndonesia.However,countriesexploringthesenewregionshadtodealwiththenatives toestablishmonopolisticcontrolofpowerover thespice-growing regionsandmajorspicetraderoutes.Overthislonghistoricaljourney,manyoftheexoticspices of earlier attraction, such as nutmeg and saffron, lost their pride of place,whilelessvaluedspiceshavingsomemedicinalvalues,suchasgarlic,peppers,andothercommonherbs,havenowbecomeincreasinglypopular[2].
11.3 BenefICIal attrIButes of spICes
Inpresentdays,mostpeopleaspireforgoodhealthandthereisagrowingfascina-tion in theuseofnaturalhealth careproducts. It is interesting tonote thatmorethan80%oftheworld’spopulationbelievesthat“preventionisbetterthancure,”forwhichtheypreferbotanicalproductshavinghealthpromoting,diseasepreventing,medicinal properties. Hardly any spices have no medicinal effects [2]. The mostcommonlyusedspicesarewellproventobemedicinal;forexample,blackpepper,cayenne,cinnamon,garlic,ginger,licorice,onion,andchivesallcontainavarietyofbiologicallyactivecompounds[3]thatareGRAS.Table11.1listssomespicesandtheirsynergistictherapeuticbenefitsbasedonthenumberofbioactivecomponentspresentforaspecificbiologicalaction.Itisnowbelievedthatthenaturallyoccur-ringsynergisticeffectofthetotalextractrendersbettereffectivenessforaspecificbiologicalactionthantheisolatedactiveingredient[2].
Spicescanbeusedtopromotehealth,curedisorders,andpreventdiseases,fromcancer anddiabetes to liver andheartproblems toobesity.Diverseagro-climaticzonesprevalentinsomegeographicallocationsareresponsibleforproducingbio-diversefloraandfauna.Thesezonesledtothedevelopmentoftheancientmedicinalsciences,likeAyurveda,Sidda,andUnani,basedontheregionalnaturalresourcesavailableinIndiaandChina.Naturalflavorsandfragrancesobtainedfromspicesand herbs are often used to relieve stress by aromatherapy. Spices and herbs arealsoused for gastrointestinal therapies, aphrodisiacs, and nonspecific tonics. Themore pungent ones are counterirritants and can be used for pain relief and anti-inflammatoryeffects.Manyhaveantibacterialorantifungalproperties.Spicesareclaimedtopreventcancersduetotheirstrongantioxidantproperties,thoughmostofthemorepotentmedicalbenefitshavenotbeenvalidatedowingtothedifficultyofidentifyingtherelevantbioactivecompounds.
For years, researchers have recognized that garlic reduces hypertension,cholesterol, respiratory and urinary tract infections, and digestive and liverdisorders.Italsocuresdiphtheria,hepatitis,ringworm,typhoid,andbronchitisandinhibitspathogenicbacteria,amoebae,fungi,andyeast,evenatlevelsof10ppm.Itisbothanantioxidantandanantiseptic.Itisthuspopularlybelievedthat“agarlic
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340 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
taBle 11.1number of Bioactive Components responsible for specific therapeutic Benefits of Common spicesspice (part) therapeutic Benefits (Correlated to number of Bioactive Compounds)*
Allspice P(8),AC(5),AI(3),AB(2),AU(2),AA(2),H(3),AD(2),AAM(2)
(Fruit) AG(2),AM(2)
Blackmustard P(8),AC(10),AS(3),FC(3),AB(6),AO(4),AV(4),AG(3),L(2)
Blackpepper P(31),AC(21),AS(16),AI(8),FC(10),AB(14),AO(4),AV(6),
(Fruit) ST(7),AA(4),AN(8),H(8),AG(5)
Cardamon P(2),AC(8),AI(2),HG(2),AM(2),HP(3)
Cassia P(10),AC(7),AS(5),ADB(3),AO(3),AU(4),AV(3),AA(3),
(Bark) AD(3),AM(3),I(4)
Cinnamon P(29),AC(14),AS(10),AI(7),FC(10),AB(11),AU(5),AV(6),
(Bark) ST(8),AA(4),H(8),AG(4),ADB(3)
Clove P(11),AC(10),AS(4),AI(6),FC(5),AO(3),AU(5),ST(3),
(Bud) AN(2),AA(3),AG(2)
Coriander P(40),AC(27),AS(9),AI(8),FC(11),AB(20),AO(7),AV(12),
(Fruit) ST(8),H(7),AG(6),AAM(5)
Cumin P(27),AC(11),AS(5),AI(7),FC(6),AB(11),AO(5),AU(5),
(Fruit) AV(7),ST(6),H(6),AG(3),AAM(3)
Garlic P(23),AC(21),AS(6),FC(8),AB(13),AO(9),AU(6),AV(5),
(Bulb) ST(5),AA(9),AD(5),HG(6),HP(5)
Ginger P(43),AC(25),AS(11),FC(18),AB(17),AO(6),AU(13),AV(6)
(Rhizome) ST(11),H(7),HP(8)
Licorice P(45),AC(26),AS(23),AI(12),FC(21),AB(20),AO(10),AU(6),
(Root) AV(8),ST(6),AN(9),AA(5),E(8)
Nutmeg P(32),AC(15),AS(11),FC(14),AB(15),AU(4),AV(4),ST(6),
(Seed) AN(5),AA(6),E(4),H(6),AG(3)
Poppy P(5),AO(3),AU(6),HPT(4),AD(5),AM(4)
Sesame ADB(4),P(7),AC(17),AB(5),AO(7),AD(7)
Turmeric P(15),AC(9),AS(4),AI(5),FC(7),AB(8),AO(3),AU(6),AV(3)
(Rhizome) AN(3),H(4),AG(3),I(4)
Vanilla P(20),AC(7),AS(9),FC(9),AB(7),AO(7),AU(3),AV(3),AN(5)
(Fruit) E(4)
* P:Pesticidal E:Expectorant
AC:Anticancerous H:Herbicidal
AS:Antiseptic AG:Analgesic
AI:Anti-inflammatory AD:Antidepressant
FC:Fungicidal HG:Hypoglycermic
AB:Antibacterial AM:Antimigraine
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Processing of Spices Using Supercritical Fluids 341
clove a day keeps the doctor away.” Ginger is often used to cure colds, cough,asthma, tuberculosis, joint pain, high cholesterol, low blood pressure, and evenmotion sickness. It is a heart stimulant, bactericide, and antidepressant. Onion isknown tobe abloodcleanser,weight regulator, andantidiabetic. It alsopreventscoldsandinfections. Cloveandcinnamonareknownpainkillers,antifungals,andantidiabeticsandcanbeusedforsparinginsulin.Theygenerallycureindigestion,nausea,andhyperacidity;inhibittuberculosis;relievefever,insomnia,andallergies;andevenlowerbloodpressure.Cumincurespiles,hoarsenessofvoice,dyspepsia,jaundice,insomnia,colds,andfever.Italsocureshook-worminfection. Turmericiswellknownforitsanti-inflammatory,antiseptic,andanticarcinogenicproperties.Itcuresarthritis,respiratorytractinfection,skinallergies,bronchialasthma,andviralhepatitis.Essenceofjojobaisconsideredauniqueproductofnatureasitisalmostidenticaltonaturalskinoilandisusedforherbalskincare.Moisturizersandbeautyoilsarealsomadefromnaturaloils(e.g.,olive,almond,wheatgrass,andaloevera).Teatreeoilisanuniqueherbaloilthatisanantibacterial,antifungal,anti-infective,and antiseptic. It is obtained fromneedle-like leavesof a small herb, a nativeofAustraliathatisnowextensivelygrownintheU.S.
11.4 BIoaCtIve IngredIents In spICes
Spicesmaybeclassifiedaccordingtotheirtherapeuticbenefitsorbioactiveingredi-ents,inadditiontotheiraroma,taste,color,andconsistencyimpartedtofoodsandmedicines.Table11.2listsgroupsofactiveingredientspresentinspicesandtheirtherapeuticvalues.Thevolatilefractionofaspiceisknownasitsessential oilandisresponsiblefortheessenceorflavorofthespice.Essentialoilsarefoundinvariouspartsofaplant(e.g.,sandalwood,clovebud,cinnamonbark,orangepeel,roseandjasmine flowers). The essential oil constituents may be classified into four majorgroups:monoterpenes,diterpenes,sesquiterpenes,andoxygenatedcompounds.Thecompoundsbelongingtothelastgroup,namelyesters,ketones,alcohols,andethers,areveryspecifictothespeciesorthegenusofthespiceplant.Eventhoughthesecompoundsarepresentinverysmallquantities,theyarethesubstancesresponsibleforthecharacteristicflavorofthespice,theabsenceofwhichsometimeschangesthearomacompletely.
taBle 11.1 (continued)number of Bioactive Components responsible for specific therapeutic Benefits of Common spices
AO:Antioxidant ADB:Antidiabetic
AU:Antiulcerous AAM:Antiasthmatic
AV:Antiviral L:Laxative
ST:Sedative I:Immunostimulant
AN:Anaesthetic HP:Hepatoprotective
AA:Antiaggregant
Source: Mukhopadhyay,M.,Natural Extracts Using Supercritical Carbon Dioxide,CRCPress,BocaRaton,FL,2000.Withpermission.
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342 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
The relatively nonvolatile fraction of a spice extract is viscous and resinous,andsoitiscalledoleoresin. Itisresponsibleforthetasteorpungencyofthespice.Thiscomprisesnonvolatileconstituentsorlarge-molecular-weightcompounds,suchas fatty acids, resins, paraffin waxes, and alkaloids. Most of the ingredients thatare responsible for themedicinalattributesofaspicearepresent in this fraction.Forexample,thecompoundresponsibleforthemedicinalvalueinblackpepperispiperine, an alkaloid having bitter taste that is present in the oleoresin. It is notpresent in theessentialoilanddoesnotcontribute to thearomaofblackpepper.Among the bioactive compounds present in oleoresin, another important groupdeters the formation and propagation of free radicals and is called antioxidants.Theypreventdiseasescausedbyoxidativedamage(e.g.,aging,cataracts,coronaryheartdisease,cancer,memoryloss,Alzheimer’sdisease,andkidney-failure).Thesenaturalbioactivechemicalcompoundsareoftencommerciallycallednutraceuticals,inlinewiththetermpharmaceuticals, andarealsotermedphytochemicals iftheyarederivedfromleaves,roots,stems,seeds,orfruits.Thephytochemicalspresentin functional foods include phenolics and polyphenolics, and some are commer-ciallyusedasnaturalantioxidants.VitaminsA,C,andEandflavonoidsaresomenaturalantioxidantsthatareaddedinsmallconcentrationstofoodsassupplementsforpreservation.TheprovitaminAactivityinspicesisduetothecarotenoidspresentinspicesandplaysanimportantroleasanantioxidant.Spicesandherbsthatpossessantioxidant properties include clove, turmeric, allspice, rosemary, mace, sage,oregano,thyme,nutmeg,ginger,cassia,cinnamon,savory,blackandwhitepepper,aniseed,andbasil.Inthefoodindustry, thesenaturalantioxidantsareusedalongwith syntheticantioxidants, suchasbutylatedhydroxyanisole,butylatedhydroxytoluene,tertiarybutylhydroquinone,andpropylgallate.
taBle 11.2Classification of Bioactive Constituents in spices
group examplespice: active Ingredient therapeutic value
Alkaloids Bitteramines Chili:Capsaicin Counter-irritantforpain
Bioflavonoids Phenolicpigments Rosemary:Luteolin Antioxidant
Essentialoils Mixturesofvolatiles Clove:Various Aphrodisiac,perfume
Glycosides Carbohydratederivatives Garlic:Alliin Expectorant
Phenylpropanoids Cinnamicacidderivatives Cinnamon:Eugenol Topicalanesthetic
Resins Terpeneoxidants Myrrh:Resinacids Antibacterial
Saponins Soapyhemolysants Licorice:Glycyrrhizin Anti-inflammatory
Sterols Steroidprecursors Sesame:Linoleicacid Antioxidant
Tannins Polyphenolics Tea:Catechin Antioxidant
Terpenes Isoprenederivatives Ginger:Zingiberene Antinauseant
Carotenoids Carotenes Paprika,redchili:β-carotene
Antioxidant,color
Anthocyanins Curcuminderivatives Turmeric:Kokum Naturalcolor
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Processing of Spices Using Supercritical Fluids 343
In addition to the antioxidants, some spices (e.g., turmeric, paprika, saffron,mustard,andblackpepper)havenatural colorasanimportantingredient,whichisusedforfoodcolor.Occurrenceofthecomponentcurcumininturmeric(Curcuma longa)hasmadeitbeneficialnotonlyasanaturalcolortofoodprocessingindustriesbutalsoasanantioxidant,anti-inflammatory,antimutagenic,andantivenomagentforhumanhealth.Alargenumberofbioactivecomponentsarepresentintheessentialoilobtainedfromturmeric,suchas,curcumin,ar-turmeric,andturmerone.SomeofthebioactivecompoundspresentinothercommonlyuseddomesticspicesarelistedinTable11.3.
11.5 saleaBle spICe produCts
Spicesarenotnecessarilysoldaspurespicesinwholeorgroundformbutarepre-ferredintheformofblendsandformulationsfortheeaseofusage.Inmanycases,additivesareaddedtoimprovethequalityorshelflife.Manypurespicessoldintheformofpowderundergocaking.Accordingly,anticakingadditivesareoftenaddedtomaintainthespicedryandfreeflowing.Forexample,asilicagel(sodiumsilicate)isoftenaddedasananticakingagent.Calciumstearate,magnesiumstearate,andpotassiumstearatearealsousedaseffectiveanticakingagents.Somespicesaresoldasblendsofspices,suchascurrypowder.Theingredientsandtheircompositionsinaspiceblendmaychangewiththefoodapplications,asinthecaseofchilipowder.
Alternatively, spice extracts can replace spice powder in food and flavorformulations.Spiceextractscanprovidethetrueessenceofspiceintheformofthevolatile essential oil, the taste components in the formof thenonvolatile resinousfraction, and the food colors in the form of the pigments. The formulations withflavors,spiceoils,andoleoresinareverymuchanart rather thanastandardtech-nology and vary depending on the buyer’s preferences for food habits and healthcareproducts.Liquidspiceflavorsareaddedtotheediblegum,powderedstarch,orcellulosesubstanceandcareistakensothattheblendcanretainitspowderynaturebytheadditionofanticakingagents.Furthermore,flavordehydrates,suchasdehydratedchicken,meat,andcheesepowders,areaddedtotheblend.Eachspiceformulationisdevisedsothatitiseffectiveduetoitsowncharacteristics,andeachformulahastobewithinthelimitsspecifiedbytheregulatoryboards.Spiceblendingequipment,suchasblendersandfilters,shouldbecleanedonaregularbasistoensurethatthereisnocontaminationwithresidualspiceblendfromthepreviousoperation.
Liquidspiceextracts,suchasessentialoilsandoleoresins,providefoodtech-nologists with many advantages, as food manufacturers can select the specificflavorprofileswithmuchgreaterprecisionthanif theyweretosimplyuseblendsofwholespices.Inaddition,hygienicconcernsaswellastransportationcostsaregreatlyreducediftheoilsandoleoresinsareextractedclosetotheareaswherethespicesaregrown.Spiceoilsandoleoresinsprimarilyfindusesinprocessedmeat,fishandvegetables;soups,sauces,chutneysanddressings;cheesesandotherdairyproducts;bakedfoods,confectionery,snacks,andbeverages.Thedemandforspiceoilsandoleoresinsisincreasinggloballydaybyday,duetotheincreasingdemandforspicyfast-foodsnackstobeintroducedintothemarketandwithaneyetowarddevelopingacharacteristictasteinthesnacksforthefuture.Spiceoilsandoleoresins
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344 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
taB
le 1
1.3
Bio
logi
cally
act
ive
Con
stit
uent
s in
Com
mon
spi
ces
spic
epl
ant
part
Bio
-act
ive
Con
stit
uent
s (p
pm)
Gar
lic
(All
ium
sat
ivum
)B
ulb
Ajo
ene,
Alli
cin,
Alli
in,A
llist
atin
-I,A
llist
atin
-II,
Arg
inin
e(6
000–
1500
0),A
scor
bic
Aci
d(1
00–8
00),
Cho
line,
Citr
al,D
ially
ldi
sulfi
de,G
eran
iol,
Glu
tam
ica
cid
(805
0–19
320)
Lin
aloo
l,N
iaci
n(4
–17)
,Sco
rodi
n-A
,Try
ptop
han
(660
–158
4)
Gin
ger
(Zin
gibe
r of
ficin
ale)
Roo
tA
ceta
ldeh
yde,
Asc
orbi
cA
cid
(0–3
10),
Asp
arag
ine
Bor
neol
(55
–110
0),B
orny
lace
tate
(2–
50),
Cam
phen
e(2
5–55
0)C
havi
col,
1-8
Cin
eole
(30
–650
)C
itral
(0–
1350
0),D
ehyd
rogi
nger
dion
e,G
eran
iol(
2–5
0),G
inge
rdio
nes,
Gin
gero
ls(
1820
0),
Hex
ahyd
rocu
rcum
in,L
imon
ene,
Lin
aloo
l(30
–650
),M
ethi
onin
e(6
70–7
35),
Myr
cene
(2–
50),
Pin
ene
(5–2
00),
Se
linen
e(3
5–70
0),S
hoga
ols
(180
0),Z
inge
rone
,Zin
giba
in,T
rypt
opha
n(6
30–6
90).
Clo
ve
(Syz
ygiu
m a
rom
atic
um)
Bud
Ane
thol
e,B
enza
ldeh
yde,
Car
vone
,Car
yo-p
hyle
ne(
7400
–816
0),C
havi
col(
465–
510)
,Cin
nam
alde
hyde
,Ela
gic-
Aci
d,
Eug
enol
(10
8000
–120
000)
,Eug
enol
ace
tate
(36
000–
4000
0),F
urfu
ral,
Gal
lica
cid,
Kae
mfe
rol,
Lin
aloo
l(1)
,M
ethy
lEug
enol
(31
0–34
0)
Cas
sia
and
cinn
amon
(C
inna
mon
um, c
assi
a &
ve
rum
)
Bar
kB
enza
ldeh
yde
(25–
100)
,Cam
phen
e,C
amph
or,C
aryo
phyl
lene
(13
5–13
15),
1,8
Cin
eole
(16
5–18
00),
Cin
nam
alde
hyde
(6
000–
3000
0),C
umin
alde
hyde
(5–
100)
,p-c
ymen
e(5
5–44
5),E
ugen
ol(
220–
3520
),F
arne
sol(
3–10
),F
urfu
ral(
3–10
),
Lim
onen
e(4
5–18
0),L
inal
ool(
230–
950)
,Met
hylE
ugen
ol,M
yrce
ne(
5–20
),N
iaci
n(8
),P
inen
e(2
0–23
5),P
iper
itone
(7–
25),
Sa
frol
e,T
erpi
neol
(1–
260)
Cum
in
(Cum
inum
cym
inum
)Fr
uit
Ani
sald
ehyd
e(8
35),
Asc
orbi
cac
id(
0–75
),B
orny
lace
tate
(35
),d
elta
-3-c
aren
e(2
70),
bet
a-ca
rote
ne(
5),C
arve
ol(
435)
,C
aryo
phyl
lene
(14
0–32
0),1
,8C
ineo
l(40
–135
),C
opae
ne(
30),
p-C
ymen
e(81
0–12
600)
,Far
neso
l(83
0),L
imon
ene
(60–
695)
,L
inal
ool(
30–3
15),
Met
hylC
havi
col(
30),
Myr
cene
(35
–120
),N
iaci
n(4
5),P
inen
e(1
0–66
00),
Pip
erito
ne(
170)
,Te
rpin
ene
(25–
1180
0),T
erpi
nen-
4-0l
(30
),T
erpi
neol
(30
–275
)
Cor
iand
er
(Cor
iand
er s
ativ
um)
Frui
tA
neth
ole
(1–2
),A
scor
bic
acid
(18
0–62
90),
Bor
neol
(2–
50),
Cam
phor
(10
0–13
00),
Car
vone
(20
–25)
,Car
yoph
ylle
ne(
1–8)
,1-
8C
ineo
le,p
cym
ene
(70–
725)
,Fer
rulic
acid
(46
0–13
60),
Ger
anio
l(30
–440
),L
imon
ene
(30–
1230
),L
inal
ool(
4060
–169
00),
Pi
nene
(50
–137
50),
Ter
pine
ol(
30–4
0),V
anill
ica
cid
(220
–960
)
Car
dam
on
(Ele
ttar
ia c
arda
mom
um)
Frui
tB
orne
ol(
30–8
000)
,Cam
phen
e(1
0–30
),C
amph
or(
5–20
),1
,8C
ineo
le(
525–
5600
0),C
itron
ella
l,C
itron
ello
l(10
–40)
,p-
cym
ene
(130
–280
00),
Ger
anio
l(45
–140
),L
imon
ene
(595
–948
0),L
inal
ool(
1285
–800
0),M
yrce
ne(
335–
3000
),
Ner
ol1
0–30
),N
eryl
acet
ate,
Pin
ene
(70–
3000
),T
erpi
nen-
4-ol
(25
0–23
200)
,Ter
pine
ne(
20–1
40)
7089_C011.indd 344 10/8/07 12:19:07 PM
Processing of Spices Using Supercritical Fluids 345
Tur
mer
ic
(Cur
cum
a do
mes
tica
)R
hizo
me
Asc
orbi
cA
cid
(0–2
90),
Bis
desm
etho
xyc
urcu
rmin
(60
–270
00),
Bor
neol
(15
–350
),C
amph
or(
100–
720)
,1,8
,Cin
eole
(30
–720
)C
inna
mic
aci
d,c
urum
in(
10–3
8500
),p
-cym
ene,
Nia
cin
(5–6
0),p
-Tol
met
hyl-
carb
inol
(50
0–17
50),
Tur
mer
one
(180
0–4
3200
)
Bla
ckp
eppe
r(P
iper
nig
rum
)Fr
uit
Asc
orbi
cac
id(
10),
Ben
zoic
Aci
d,B
orne
olC
amph
or,C
arva
crol
,Car
veol
,Car
yoph
ylle
ne,1
,8C
ineo
le,C
inna
mic
aci
d,C
itral
,C
itron
ella
l,p-
cym
ene,
Eug
enol
,Lim
onen
e,L
inal
ool,
Myr
cene
,Myr
istic
in,P
inen
e,P
iper
idin
e,P
iper
ine,
Saf
role
,Te
rpin
en-4
-ol
Bla
ckm
usta
rd
(Bra
ssic
a ni
gra)
Seed
/Lea
fA
llyli
soth
iocy
anat
e(6
510–
1176
0,s
eed)
,Arg
inin
e(1
810–
2665
7,L
F),A
scor
bic
acid
(23
5–40
00,L
F),β
-car
oten
e(3
0–47
5,L
F),
Eru
cic
acid
(77
0–11
340,
LF)
,Met
hion
ine
(230
–339
0,L
F),N
iaci
ne(
3–48
,LF)
,Try
ptop
han
(270
–397
5,L
F)
Saff
ron
(Cro
cus
sati
vus)
Flow
erβ-
caro
tene
,1-8
Cin
eole
,Cro
cetin
,Cro
cin
(200
00),
Del
phin
idin
,Hen
tria
Con
tane
,Kae
mfe
rol,
Lyco
pene
,Myr
icet
in,
Nap
htha
lene
,Pin
ene,
Que
rcet
in
Lic
oric
e(G
lycr
yrrh
iz g
labr
a)R
oot
Ace
tica
cid
(2),
Ane
thol
e (1
),B
etai
ne,C
holin
e,O
-Cre
sol,
Est
rago
le,E
ugen
ol(
1),F
erul
ica
cid,
Gly
cryr
hizi
cA
cid
(100
000–
2400
00),
Gua
iaco
l,K
aem
fero
l,L
inal
ool,
Man
nito
l,N
iaci
n(7
0)
Mac
ean
dnu
tmeg
(M
yris
tica
frag
ans)
Seed
Bor
neol
(42
00–2
5600
),1
,8-C
ineo
le(
440–
3500
),p
-cym
ene
(120
–960
),E
lem
icin
(20
–350
0),E
ugen
ol(
40–3
20),
Fu
rfur
al(
1500
0),G
eran
iol,
Lim
onen
e(7
20–5
760)
,Lin
aloo
l,M
ethy
lEug
enol
(20
–900
),M
yrce
ne(
740–
5920
),
Myr
istic
in(
800–
1280
0),P
inen
e(3
000–
4000
),S
afro
le(
120–
2720
),T
erpi
nen-
4-ol
(60
0–48
00),
Ter
pine
ol(
120–
9600
)
Thy
me
(Thy
mus
vul
gari
s)Pl
ant
Bor
neol
(15
–146
0),B
orny
lace
tate
(15
–540
),C
affe
icA
cid,
Cam
phen
e(1
5–27
0),d
elta
-3-
Car
nene
(51
0),B
eta-
caro
tene
(2
0–25
),C
arva
rol(
15–1
8720
),C
hlor
ogen
ica
cid,
1,8
Cin
eole
(80
–459
0),p
-cym
ene
(145
–208
00),
Ger
anio
l(0–
1066
0),
Lim
onen
e(1
5–52
00),
Lin
aloo
l(18
0–17
420)
,Met
hion
ine
(137
0–19
80),
Myr
cene
(35
–675
),N
iaci
n(5
0),P
inen
e(1
5–16
00),
R
osm
arin
icA
cid
(500
0–60
00),
Ter
pine
n-4-
ol(
70–8
320)
,Ter
pine
ol(
35–6
500)
,Thy
mol
(15
–240
00),
Try
ptop
han
(186
0–20
00),
Urs
olic
aci
d(1
5000
–188
00)
Red
Pep
per/
Chi
lli
(Cap
sicu
m c
orra
ls)
Frui
tA
rgin
ine
(400
–800
0),A
scor
bic
Aci
d(3
50–2
0000
),A
spar
agin
e,B
etai
ne,C
apsa
icin
(10
0–22
00),
bet
a-ca
rote
ne(
0–46
0),
Chl
orog
enic
aci
d,H
espe
ridi
n,M
ethi
onin
e(1
00–1
900)
,Nia
cin
(5–1
70),
Oxa
lica
cid,
Try
ptop
han
(100
–200
0)
Van
illa
(Van
illa
pla
ntif
olia
)Fr
uit
Ace
tald
ehyd
e,A
cetic
aci
d,A
nisa
ldeh
yde,
Ben
zald
ehyd
e,B
enzo
icA
cid,
Cre
osol
,Eug
enol
,Fur
fura
l,G
uaia
col,
Van
illic
aci
d,
Van
illin
(13
000–
3000
0),V
anill
ylA
lcoh
ol.
Sour
ce:
Muk
hopa
dhya
y,M
.,N
atur
al E
xtra
cts
Usi
ng S
uper
crit
ical
Car
bon
Dio
xide
,CR
CP
ress
,Boc
aR
aton
,FL
,200
0.W
ithp
erm
issi
on.
7089_C011.indd 345 10/8/07 12:19:08 PM
346 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
areparticularlysuitableforsuchsnacksbecausetheycanbeusedveryconveniently(withouthavingtohandleinbulkrawspicessuchasginger,garlic,chili,onion,car-damom,andcinnamon).Forexample,approximately7000kgofonionsareneededtoproduce1kgofhighlyconcentratedonionoil.
Mostspiceextractscanbesuppliedinbothoil-andwater-solubleforms.Asaresult,spiceoleoresinsandessentialoilsarenowshippedindispersionsofedibleoilorotherliquids.Furthermore,dispersionscanbestandardizedwithotheringredients,such asmono-, di-, or triglyceridesor polysorbates.Another technique for easierspice application is to make a liquid emulsion of spice oleoresins, essential oils,andastarchorspraydrytheessentialoiltoapowder.Inthiscase,thespray-dryingprocesshastomakesurethatotherproductsarenotspray-driedonthesameequip-menttoavoidcontamination.Oleoresins,spiceoils,substrates,anddiluentallshouldindividuallymeetreliablestandardization.
Theblendformulationsforspiceseasoningsuseflavorenhancersandotherflavoringredients,suchasmonosodiumglutamate,sodiumerythorbate(usedindelimeats),dextrose,maltodextrin,andhydrolyzedvegetableproteins.Manyoftheseflavoringsandingredientsarecornorsoybased.Otherprocessedingredientsarederivedfromproductsthatgothroughamultistageconversionprocessofenzymolysis,fermenta-tion,andregenerationuntilthefinalproductisachieved.Thequalityandsafetyoftheformulationareassuredbeforemarketing[4].Moreinformationmaybegatheredfromthereferencesprovided[5,6].
11.6 ConventIonal extraCtIon methods
Figure11.1 outlines the various alternative steps involved in the conventionalmethodsforproductionofspiceextracts.Thespicearomaoressentialoilistradi-tionallyproducedbysteamdistillation(SD)ofthegroundspiceorSDoftheextractsobtainedbysolventextraction(SE)oraqueousalkalineextraction(AE)ofthegroundspice.Avarietyofsolvents,suchasalcohols,acetone,andhexane,canbeusedforextractionofspices.However,removaloftheseorganicsolventsleavessomeresidualsolventbehind,whichrequiresthedesolventizationatelevatedtemperatures.Thiscancausechemicalmodificationsoftheoleoresins.
Table11.4listsafewexamplesofspiceextractsthatareproducedcommerciallywiththeirpercentageyieldsofessentialoilsandoleoresinsfromgroundspice,asreportedbyMarionetal.[7].Variationsinyieldandqualityofspiceextractsmayoccurduetovariationsintheoriginandharvestingtimeofspices.
Theyieldandqualityofextractsalsodependonpreprocessingoperations,suchasgrinding,thetechniqueofextraction,andthenatureofthesolventwhich,inturn,are decided based on the desired specification of the end product in terms of itsaroma,flavor,andsolubility.Eachextractplaysaspecificroleintheformulationanditsselectionisthekeytotheproductdevelopment,asperthespecificrequirementofvalueaddition.
Conventionalextractionmethods,suchasSE,AE,anddirectorindirectSD(i.e.,hydrodistillation[HD]),arenotselective.Asaresult,theextractsoftencon-tain color (e.g., chlorophyll) or some other undesirable components. Therefore,further purification is imperative using a number of techniques, such as color
7089_C011.indd 346 10/8/07 12:19:08 PM
Processing of Spices Using Supercritical Fluids 347
adsorptionbyactivatedcharcoal,dryingusingsilicagel,chromatographicsepa-ration,vacuumfractionation,ormoleculardistillation.Owing to thebanon theusageofthechlorinatedsolvents,themostcommonlyusedsolventforSEtodayishexane.Formostspiceoilsandoleoresinsintheinternationalmarket,theresidualhexanecontent inproductshas tobereduced to less than25ppm.This limit isexpected to go down further. Hence, the SE process may be phased out in thenearfuture.Thefoodindustryneedstocombatstrictregulationsandcomplywithmeasuresforsafety,reliability,andstandardizationofnaturalproductstobecon-sumedasnutrientsandfoodadditives.ThismaybeachievedbyadoptingSCFE techniques,asSC-CO2canrecovertheactiveingredientsinnaturalformwithoutdegradationorcontamination.Over thelast twodecades,SCFEhasemergedasasuperioralternative toconventionalprocessessuchasSDandSE in the food,pharmaceutical,andcosmeticsindustries.
11.7 superCrItICal CarBon dIoxIde as the extraCtant
Several spice extracts—such as those from basil, black pepper, cardamom, chili,cinnamon,clove,cumin,fennel,fenugreek,ginger,garlic,nutmeg,paprika,savory,turmeric,andvanilla—arenowcommerciallyproducedusingSC-CO2,asitiscurrentlythemostdesirableSCFsolventforextractionofnaturalproducts.ThesolubilityoftheextractinSC-CO2increaseswithpressureordensityofSC-CO2anddecreaseswith
Spice EmulsionSeasonings
Steam Distillation Solvent/Alkali Extraction SC CO2 Extraction
Essential Oil Steam Distillation Essential Oil Oleoresin
Dispersed in Carrier
Dispersed inVegetable Oil
Spray Dried in EdibleGum or Starch Solution
Dispersed in StarchSolution
Blended with OtherFlavors
Commercial SpiceOil
Liquid Oleoresin EncapsulatedSeasonings
Ground Spice/Herb
fIgure 11.1 Various alternative steps for spice extraction. (From Mukhopadhyay, M.,Natural Extracts Using Supercritical Carbon Dioxide,CRCPress,BocaRaton,FL,2000.Withpermission.)
7089_C011.indd 347 10/8/07 12:19:09 PM
348 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
temperatureuptoalimitingpressure(termedcross-over pressure),beyondwhichthesolubilityincreaseswithbothpressureandtemperature.ThisphenomenonisutilizedforrecoveringandrecyclingCO2afterextractionbysimplyloweringthepressure,increasingthetemperature,orbothintheseparators.ThereisnosolventresidueintheextractasCO2isinagaseousstateattheambientcondition.
AbroadrangeofselectivityandextractabilitycanbeachievedusingSC-CO2justbymanipulatingtheoperatingconditions,suchaspressureandtemperature,therebytargetingthespecificcompoundsofinterest.BecauseSCFEishighlyselective,theconcentrationofthedesiredactivecompoundinthetotalextractishigherandtheyieldofthedesiredactivecompoundisclosertothetotalyield.ThereisrarelyanyneedforadditionalprocessingstepsforSCFextracts,whereasorganicsolvent–extracted
taBle 11.4Commercial spice extracts [2]
spiceessential oils, min-max (%)
oleoresins (%)
Anise 1.0–4.0
Caraway 3.0–6.0
Cardamon 4.0–10.0
Carrot 0.5–0.8
Cassia 1.0–3.8
Celeryseed 1.5–2.5
Cinnamon 1.6–3.5
Clovebud 14.0–21.0
Coriander 0.1–1.0
Cumin 2.5–5.0
Curcuma 2.0–7.2 7.9–10.4
Dill(seeds) 2.5–4.0
Fennel 4.0–6.0
Garlic 0.1–0.25
Ginger 0.3–3.5 3.5–10.3
Marjoram 0.2–0.3 —
Mace 8.0–13 22.0–32.0
Nutmeg 2.6–12 18.0–37.0
Pepper 1.0–3.5 5.0–15.0
Pimentoberry
3.3–4.5 6.0
Saffron 0.5–1.0
Savory 0.5–1.2 14.0–16.0
Vanilla 29.9–47.0
Source: Mukhopadhyay,M.,Natural Extracts Using Supercritical Carbon Dioxide,CRCPress,BocaRaton,FL,2000.Withpermission.
7089_C011.indd 348 10/8/07 12:19:10 PM
Processing of Spices Using Supercritical Fluids 349
oleoresinsincludeundesirableresinsthatprecipitateandmakethesolutioncloudy,requiringanadditionalstepoffiltration.Evensteam-distilledoil formsan immis-ciblelayerduetothepresenceofmonoterpenehydrocarbons,toalargeextent,whichinhibitthesolubilityofoilinsoftdrinksandbeverages.TheremarkableselectivityofSC-CO2overorganicsolventsfacilitatestherecoveryofspiceextractswithdesirableconstituents and superior blending characteristics. Studies on SCFE with SC-CO2andSEwithalcoholindicatethat,althoughtheoverallyieldobtainedusingalcoholasthesolventishigherduetocoextractionofundesirablecomponents(whichyieldsanaccordinglyhigherquantityoftotalextract),thepercentageofthedesiredactivecompoundinthatextractislower[2].
Inadditiontoselectiveextractionandtheabsenceoforganicsolventresidues,SCFE offers another unique advantage; namely, simultaneous fractionation ofdifferentcompoundsispossibleusingthesamesolvent,SC-CO2.Forexample,theactivecomponentsinblackpeppercanbeextractedwithSC-CO2andseparatedintotwofractionsbychangingpressureand temperature; thefirst fraction isenrichedinoleoresinandthesecondfractioninessentialoil.Accordingly,SC-CO2can,inasingleprocessofSCFE,selectivelyextracttheoleoresinandessentialoilfractions(asopposedtoprocessingofspicesbySE,AE,orSD)andthenseparatethembysequentialdepressurization.Furthermore,mostraffinate(thematerialleftoverafterextraction)isuncontaminatedandhasahighmarketvalueduetothecontentoffiberandprotein,whichremaininsolubleinSC-CO2.
However,inviewofthefactthatSC-CO2isessentiallynonpolar,itisunsuitableforextractingwater-solubleconstituents.Thisseemingdisadvantagemaybeeasilyovercomebyaddingafood-gradepolarcosolvent(typicallyinverysmallquantities,say3to5mole%)toSC-CO2.Thebinaryhomogeneousmixture is thencapableof extractingwater-solubleorhigh-molecular-weight compounds.Thebest candi-datesforsuchcosolvents,especiallyforfoodsandnutraceuticals,areethanol,ethylacetate,andinsomecaseswater.Theremarkablevalue-additionthat theSC-CO2extractsofferasnaturalconcentrates,inadditiontotheiradvantagesfromthestand-pointofenvironmentandhealth,hasgeneratedagreatdealofcommercialinterestsforusingSC-CO2astheextractantinthefoodindustry.
11.8 CommerCIal sCfe proCess
Forsolidfeeds,SCFEisusuallyasemi-batchprocessinwhichCO2flowsinacontin-uousmode,whereasthefeedischargedintheextractorbasketinbatches.However,for better viability on the commercial scale, theprocess ismade semicontinuoususingmultipleextractionandseparationvessels,asschematicallydescribedintheflowdiagramshowninFigure11.2.Extractionandseparationoftheextractareoftencarriedoutinstages,bymaintainingdifferentconditionsofpressureandtempera-tureintheextractorsandseparators.Thisallowseasyfractionationoftheextractforenrichmentofthespecificactivecomponents,whicharesubsequentlyfractionatedineachoftheseparators.Itisthuspossibletoproduceavarietyofproductsusingthesamehardwarebymerelychangingpressure,temperature,andcosolventconcen-trationandmakeaplantformultipleproducts[2].ThecommercialSCFEprocessworksinaclosedloopwithconstantcirculationofCO2inthesystem,withatypical
7089_C011.indd 349 10/8/07 12:19:10 PM
350 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
batchtimeof2to4hours.TypicaloperatingconditionsforSCFEareintherangeof100to500barand40°Cto80°C.
ThefeedfortheSCFEprocessneedstohavelowmoisturelevel(lessthan10%)andbe in theformofgroundpowder(100 to300mesh).However,preprocessingshouldbedone in such away that there areminimal lossesof essential oils andactiveingredientsandnegligiblethermalandchemicaldegradationduetotheriseintemperatureorexposuretoatmosphere,respectively.Accordingly,dryingcouldbecarriedoutinafluidizedbeddrierinaninertenvironment,suchasinflowingnitrogenorcarbondioxide.Similarly,grindingcouldbeachievedinaliquidCO2ordry-ice,precooledgrinderwhilecontrollingthehumidityoftheincomingairtoavoidcondensationofmoistureinthefeed.
ItisgenerallybelievedthatSCFEiscapital-intensive,duetotherequirementsoftheprocesstobeoperatedathighpressureswithverypreciseprocesscontrol.ThereisalsoageneralconcernthatSCFEtechnologyisenergy-intensive.However,itisinterestingtonotethattheenergyneededtoattainasupercriticalstate(P>73.8bar,T > 31.1°C) is more than compensated for by the negligible energy required forsolventrecoveryfromtheextractbyasimplestepofdepressurization.Asaresult,theoverallenergyconsumptionofSCFEusingCO2islowerthanthatfortraditionalSDorSE,duetosteamgenerationinSDandduetosolventevaporationandblowingoffofsteamforremovalofresidualsolventfromtheresiduesinSE.Forexample,theremovalofresidualsolventfromanextractbySErequiresabout8kWhofenergyperkilogramofplantextract[8],whereasextractionwithSC-CO2requiresone-tenthofthisenergy.Also,thesolventlossinthebatchSEprocessisuptoone-thirdofthefeedsolvent(thoughitissomewhatlower[10%to15%]inthecontinuousSEpro-cess),whereasthelossofCO2intheSCFEprocessisnegligiblebecauseCO2canbe
E1 E2 E3
S1 S2 T
Condenser
Sub-cooler Pre-heater Co2 Pump
CO2 Supply
Depressurisation Line
HE
E1, E2, E3 : Extractors S1, S2 : Separators T : CO2 Day Tank HE : Heat exchanger
I.I.T., Bombay
Entrainer Pump
fIgure 11.2 ProcessflowsheetofSCFEofspices.
7089_C011.indd 350 10/8/07 12:19:12 PM
Processing of Spices Using Supercritical Fluids 351
easilyregeneratedandrecycled.Thesolventrequirementinthebatchprocessis10to20timesthatofthefeedchargedandthatinthecontinuousSEprocessismuchlower,namelyabout3to4timesthefeed,whereasanhourlycirculationrateofSC-CO2isintherangeof16to24timestheamountoffeedcharged.
TherelativelyhigherinvestmentrequiredintheSCFEprocessiswellbalancedbyotherbenefitsofSCFE,suchaslowsolvent(CO2)cost,lowerbatchtimes,higherconcentrations of active desirable components in the extract, and no additionalpurification-andpollution-abatement-relatedcosts.SCFEalsogeneratespracticallynoeffluent.Inaddition,theextractedresidue(cake)doesnotundergoanydegrada-tionorcontamination,unlike inSEandSD.ResiduefromtheSCFEprocesshasamarketvalueas it retainsall theuseful ingredients,suchasedibleproteinsandfibers.Thiscanbesoldasahighvalueby-producttoyieldadditionalrevenue.
ThenormalSCFEprocesssimultaneouslyandseparatelyyieldsbothliquidandsolidproducts,startingwiththesamefeedofspicesinasinglestep,unlikeSDandSE.InSD,thesteamvolatileessentialoil,whichisaliquidproduct,isdistilledout,whereasinSE,theliquid(essentialoil)andsolid(oleoresin)productsareobtainedtogether.Subsequently,SDorSEwithanothersolvent isemployed torecover theessentialoilfromthemixedproduct.Liquidspiceproductsaremorestable,haveamorereproduciblequalitythantheirconventionalforms,andcontainthecharacter-isticaroma,taste,andodor.Duringtheirutilization,asmallerquantityisrequiredforobtainingthesameeffect.Thestandardizationofthesenewliquidspiceproductsimpliescontrollingtheircomposition.Adetailedfeasibilitystudyshowsthatevenattheexistingprice(ofextractsfromSEandSD)ofoilandoleoresins,theinvestmentinSCFEisprofitable,whichjustifiesittobethepreferredroutefromalong-termperspective.TheinstrumentationandcontrolsystemnecessaryfortheSCFEprocessisdesignedtoprovideaccuratecontroloftheparameters,ensuringhighconsistency,reliability,andstandardizationofthefinalproduct.
11.9 ComparIson of spICe extraCts By ConventIonal and sCfe proCesses
AlthoughrecoveryofessentialoilsfromspicesbySDandAEhasbeenpracticedforcenturies,theoilsproducedbytheseprocessesmaycontainartifactsformedduringtheprocessing,inadditiontothefactthattherecoveryoftheoilsisquitelow,assomeofthecomponentsarenotsteamvolatile.WhenSEinvolvestheuseoforganicsol-ventstoextractessentialoilsfromgroundspices,thequalityoftheextractisdecidedbythepresenceofresidualsolvent,artifactsformedduetothermaldegradationdur-ingtherecoveryofthesolvents,orbycoextractionofundesirablecomponentsduetopolarityofthesolvent.Apolarsolventislikelytoextractmostpolarcomponentsfromspices,someofwhichmayevenbeundesirable.ExtractionofspiceswithSC-CO2orsubcriticalliq-CO2ismostfavorableforcommercialproductionofessentialoilsandoleoresins,asCO2isanaturalsolventandisideallysuitableforthermallylabile natural products. The oleoresins are extracted at relatively high pressures,whereastheessentialoilsarerecoveredatrelativelylowpressures,whichcanformaclearsolutionwhenaddeddirectlytosoftdrinks.Butsimultaneousextractionofoleoresinsandessentialoilsataveryhighpressureintherangeof250to350bar,
7089_C011.indd 351 10/8/07 12:19:13 PM
352 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
followedbystage-wiseselectivefractionationatsupercriticalandsubcriticalcondi-tions,ispreferredforcommercialproduction.Theoperatingconditionsofpressure,temperature,andcosolventareappropriatelyselectedinordertoobtainaspecificproductprofile.Thecosolventisoftenselectedontheconsiderationthatitcanbeleftbehindintheextractedproductorusedformakingtheformulationwithallow-ancemadefordilutionlevel.Thecosolventselectedisafood-gradeGRASorganicsolvent,suchasethanol,ethylacetate,aceticacid,orwater.TheadvantageofSCFEwithcosolvent-mixedSC-CO2overSEisthattheformerisselectiveandretainsallotheradvantagesoftheSCFEprocessbecausethecosolventaddedtoSC-CO2isinverysmallamount(3to5mole%).MostofthecosolventaftertheSCFEprocessescapeswithCO2intheseparator,ensuringmarginalcontentofresidualsolventinthefinalextract.TheextractionyieldsobtainedbyCalameandSteiner[9]usingSDandSC-CO2aregiveninTable11.5.
ItisnowanestablishedfactthatSC-CO2extractionatoptimizedconditionsyieldsmuchmoreactiveingredientsthanSEorSD.Butotherfactors,suchasparticlesize,preprocessingmethods (e.g.,dryingandgrinding), timeofextractionandstorageafterharvesting,andevengeographicaloriginoftherawspice,responsiblefortherecoveryoftheextractarenotincludedinTable11.5.Accordingtotheexperienceoftheauthor,higheryieldsmaybeobtainedfromsomeofthesespiceswithSC-CO2,evenwithoutacosolvent,ascanbeseenlaterinTable11.7.
Notonlytheyieldsoftheextractsbutalsotheirorganoleptic(sensory)charac-teristicsmaybedifferentforextractsobtainedbydifferentmethods.Accordingly,the criteria for selection of the best process condition are based on the desired
taBle 11.5yields by sd and sC-Co2 extraction with a Cosolvent
steam distillation sC-Co2 extraction
spice yield (%) Cosolventextractor
p/t (bar/°C)separator
p/t (bar/°C) yield (%)
Allspice 2.5 Ethanol 300/40 55/37 5.3
Basil 0.5 Ethanol 200/40 56/15 1.3
Cardamom 4.0 Methylacetate 150/60 50/9 5.8
Coriander 0.6 Ethanol 300/40 54/13 1.3
Ginger 1.1 Ethanol 300/40 52/11 4.6
Juniperberry 1.5 Hexane 300/60 52/11 7.2
Marjoram 2.06 Ethanol 250/40 50/35 1.7
Oregano 3.0 Ethanol 150/40 55/14 5.4
Rosemary 1.44 Ethanol 250/60 53/12 7.5
AU 1.34 Hexane 250/60 53/12 7.5
Sage 1.1 Methylacetate 200/40 53/12 4.3
Thyme 1.85 Hexane 150/46 50/9 2.1
Source: Mukhopadhyay,M.,Natural Extracts Using Supercritical Carbon Dioxide,CRCPress,BocaRaton,FL,2000.Withpermission.
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Processing of Spices Using Supercritical Fluids 353
quality. Inanycase,CO2extractshavemore topnotes,morebacknotes,nooffnotes,nodegradation,moreshelf life,andbetteraromaandblendingcharacter-istics than steamdistilled andhexane extracts, as canbe seen inTable11.6. Ingeneral, theextractproducedbySEcontainsall the ingredients thataresolublein theorganic solvent, including thevolatileoils and resins.Some triglycerides(lipids)presentinspicesarecoextractedandactasnature’sownfixativeresultingeasyandproperblending.
Theyieldof essentialoilbySDof cumin (2.5%) is less than thatbySC-CO2
extraction(3.5%)at120barand40°C[10].Acomparisonofthecompositionofthe
taBle 11.6spice Constituents (area %) by various methods
Constituents distillation (%) l Co2 (%) sC-Co2 (%) hexane (%)
GingerExtract(byGC)
α-Curcumene 10.0 3.7 2.3
α-Zingiberene 44.0 19.6 12.1
β-Zingiberene 8.0 3.4 2.0
β-Bisabolene 8.3 3.7 2.4
β-Sesquiphellandrene 17.8 7.9 4.9
Zingerone 0.8 0.7 0.3
GingerExtract(byHPLC)
6-gingerol 0.2 16.4 0.9
8-gingerol 0.3 3.1 0.7
10-gingerol — 3.8 0.8
6-shogaol 0.3 2.8 6.3
8-shogaol — — 1.6
CuminExtract2(byGC)
α-pinene — 1.1 —
(EthylEther)-pinene 13.0 21.0
p-cymene 13.0 9.4
γ-terpinene 24.8 20.0
Cuminaldehyde 16.0 20.3[19] 21.0 11.4
Cymol 33.4[20] 26.7[19] 15.2 13.5
CloveExtract(byGC)
(EthylEther)
Eugenol 76.4 77.1 71.8* 73.3
Eugenolacetate 5.6 4.9 11.1* 4.6
β-caryophyllene 5.8 8.5 9.3* 10.4
* PilotplantexperimentatIITBombaySource: Mukhopadhyay, M., Natural Extracts Using Supercritical Carbon Dioxide, CRC Press,
BocaRaton,FL,2000.Withpermission.
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354 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
cloveextractsobtainedbyliquidCO2,SEwithethylether,andSDindicatesthatliquidCO2andethyletherextractsaresimilar,thoughliquidCO2extracthasthecharacter-isticsofbothessentialoilandoleoresin[11].LiquidandSC-CO2extractsarealwaystransparentandcontainmoreactivecomponentsthatareclosertothoseinthefreshornaturalspice,duetolowoperatingtemperatureandinertenvironment.SC-CO2extractionfollowedbyfractionalseparationwascarriedoutforavarietyofspicesusinga10-Lextractorcapacitypilotplantat Indian InstituteofTechnology (IIT),Bombay.Thecompositionoftheactiveingredientsoftheextractsseparatedinthetwoseparators,asanalyzedbyeithergaschromatography(GC)orgaschromatography-massspectrometry(GC-MS),are indicated inTable11.7(except forpepper,wherepiperinewasquantifiedbyultraviolet[UV]method).TheyieldsandthecompositionsoftheSC-CO2extractsarecomparedwiththoseofhexane-extractedproductsfromthesamespices(Table11.7).SC-CO2extractionyieldsarebetterforclove,cumin,andblackpepper.Inmostcases,theconcentrationsoftheactiveingredientswerehigherintheSC-CO2extractedproduct[2].
11.10 proCess analysIs of sCfe from seleCted spICes
Spiceextractsareusuallyacomplexmixtureofvolatileessentialoils,waxes,tri-glycerides,andresinousandothermiscellaneousmaterials,withthecompositionoftheconstituentscontributingtoaroma,flavor,andpungencyselecteddependingonthespecificapplication.Accordingly,forcustomizedapplications,SC-CO2extractionofspicesrequiresfractionalseparationofselectedgroupsofconstituents.Thiscanbeachievedintwoways:bystage-wiseextractionfollowedbydepressurizationoftheextract-ladenSC-CO2orbysingle-stageextractionataveryhighpressurefollowedbystage-wisedepressurizationforfractionalseparation.Intheformermethod,thevolatileoil isfirst extractedat relativelymilder conditions and, subsequently, thenonvolatileoleoresinsareextractedatrelativelymore-severeconditions.Inthelattermethod,thefinelygroundspiceismoreorlesscompletelyextractedatarelativelymore-severeconditiontorecoverbotholeoresinsandvolatileoilsimultaneouslyandefficientlysothatthetimeofextractionisgreatlyreduced.Theextract-ladenSC-CO2is subsequentlydepressurized in twoor three separators at predeterminedcondi-tionssothatspecificproductsareselectivelyfractionatedandcollected.Thesecondmethodofferssignificantadvantages,asthequalityoftheproductisimprovedandthebatchtimeforextractionisreduced,resultinginhigherproductioncapacityandcosteffectivenessoftheSC-CO2extractionprocess.Simultaneousfractionationatpreciselyselectedconditionsallowsproductionofcustomizedqualityfractionsandeliminationofundesirablecontaminantsfromthem.ThespecificadvantagesofthisSC-CO2extractionand fractionationprocessarementioned in the following sub-sectionswithrespecttoafewcommonspices.
11.10.1 Celery Seed (Apium grAveolen)
AcomparisonofchemicalcompositionofceleryseedoilbyHDandSC-CO2extrac-tion [12] at 100bar and40°C indicated that theHDoil containedmostlymono-terpenes,whereastheSC-CO2extractedoilcontainedmostlyphthalides(Table11.8).
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Processing of Spices Using Supercritical Fluids 355
The SC-CO2 extract contained some additional components, such as fatty acids,whichwerenotpresentintheHDoil.Monoterpenesconstituted57.6%oftheHDoil,whereastheSC-CO2oilcontained56.8%phthalides[12].Thelowlevelofphthalides(15.2%)intheHDoilwasattributedtotheirhighboilingpointsandlowvolatilityinsteam.Morethan10hrofHDwasnecessaryfortheircompleterecovery.Phthalidesare cyclic esters or lactones with outstanding odor characteristics of celery. TheodoroftheSC-CO2-extractedoilismoreintenseandlessterpenic.Therefore,theSC-CO2-extractedoil ispreferred to theHDoil to impart theceleryflavor.With
taBle 11.7yields and Concentrations of active Ingredients in extracts with sC-Co2 and hexane
spices (active Ingredient)
sC-Co2 extraction (200 bar, 40°C) (by wt.)
solvent (hexane) extraction (by wt.)
yield (%) % ess. oil % oleoresin yield (%) % extract
Clove 23.8 16.8
Eugenol 71.8 — 70.7
Eugenolacetate 11.1 — 11.3
(byGC,10%FFAP)
Cumin 21.0 12.2
Cymol 15.2 — 13.5
Cuminaldehyde 15.3 — 11.4
(byGC-MS,DB5)
Coriander 3.6 20.0
Linalylacetate 7.8 — 5.8
D-linalool 13.0 — —
(byGC-MS,DB-5)
Ginger 4.6 4.9
Zingiberene 26.7 1.6 31.6
Gingerol 5.65 10.1 5.4
(byGC-MS,DB-5)
Cinnamon 3.0 5.11
Cinnamicaldehyde 77.5 45.0
(byGC,SPB-1)
Pepper 4.6 5.0
Piperine — 53.0 46.4
(byUVmethod)
Ajwain 4.5 5.18
Thymol 63.6 — 24.6
(byGCOV-101)
Source: Mukhopadhyay, M., Natural Extracts Using Supercritical Carbon Dioxide, CRC Press,BocaRaton,FL,2000.Withpermission.
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356 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
SC-CO2extractioncarriedoutfromceleryseedsatarelativelymoderatepressureof100barand40°C,theyieldofessentialoilwasmerely2.03%ofthechargedmate-rial[13].TheyieldofessentialoilfromceleryleavesbySC-CO2extractionat90barand40°Cwasevenlower(0.04%).ThecompositionsoftheessentialoilsfromceleryseedsandceleryleavesbySC-CO2extractionwerefoundtobesignificantlydiffer-ent,ascanbeseeninTable11.9.Theceleryseedextractscontainedmoreparaffinandfattyacidmethylestersthantheceleryleafextract.
11.10.2 red Chili
SC-CO2extractionofredchiliiscarriedoutinthepressurerangeof300to500barand80°C to100°C,withsimultaneousfractionationof theextracts into lightandheavyfractions.Thelightfractioncontainsmostofthecapsaicin(i.e.,thecompoundresponsible for the hotness of the spice), in addition to the essential oil, whereastheheavy fractioncontains triglyceridesand thecolorcompounds, inaddition to
taBle 11.8Composition (%) of Celery seed essential oil by sC-Co2 extraction and hydrodistillation
Class of Compounds sC-Co2 extraction hd
Monoterpenes 16.1 57.6
Oxygenatedmonoterpenes 0.2 0.6
Sesquiterpenes 19.7 23.3
Phthalides 56.8 15.2
Others 4.8 0.3
Source: Mukhopadhyay, M., Natural Extracts Using Supercritical Carbon Dioxide,CRCPress,BocaRaton,FL,2000.Withpermission.
taBle 11.9Composition of essential oils from Celery seeds and leaves by sC-Co2
ComponentCelery seeds
(100 bar, 40°C)Chinese Celery seed
(100 bar, 40°C)Celery leaves (90 bar, 40°C)
Limonene 3.7 14.9 33.4
β-Selinene 33.8 17.6 3.0
α-Selinene 5.3 1.8 0.5
Butylphthalide 19.8 5.5 2.8
Sedanenclide — 22.4 —
Bedanolid — 28.8 —
Germacrone 21.0 — 45.4
Source: Mukhopadhyay, M., Natural Extracts Using Supercritical Carbon Dioxide,CRCPress,BocaRaton,FL,2000.Withpermission.
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Processing of Spices Using Supercritical Fluids 357
a small quantity of capsaicin [14], as shown in Table11.10a. The distribution ofcapsaicininthetwofractionsisadjustedbyselectingtheconditionsinthesepara-torsforfractionation.SCFEofamorepungentvarietywasperformed[28]overalowertemperaturerange(35°Cto70°C)andawiderpressurerange(100to550bar).Itwasshownthatthecapsaicinoid(capsaicin,dihydrocapsaicinandthelike)couldberaisedto75%inthe0.2%to0.3%oleoresinifextractionwascarriedoutfromthedriedmaterialandupto99%fromfreshmaterial.Itwasobservedthecapsaicinoidcontent increasedwithtimeofextractionandadditionofacosolvent,suchas5%aceticacid,anditincreasedfurtherbysuccessivelyincreasingthetemperature,aspresentedinTable11.10b.
11.10.3 PaPrika
Paprikaisusefulinindustryforitsnaturalcolor.ForSC-CO2extractionofpaprika,mostof the color compounds are collected in theheavy fraction,while aroma iscollectedinthelightfraction.Researchindicates[14]thatthecolorvalueofSC-CO2-extractedproductcouldreachashighas7200ASTA,whereasanormalcommercialproductischaracterizedtohaveacolorvalueintherangeof1000to2000ASTA.
taBle 11.10(a)Composition in light and heavy fractions of Chili extract
products % Capsaicin% dihydro- Capsaicin
total % Capsaicinoid
Rawmaterial 0.21 0.14 0.39
Lightfraction 8.10 4.05 13.50
Commercialproduct 1.83 1.52 3.93
Heavyfraction 0.57 0.31 0.95
RatioL/H 14.2 13.1 14.2
taBle 11.10(B)sCfe from fresh Chili with successive Increase in temperature and pressure with 5% acetic acid (by wt.) of feed Chargedpressure
(bar)temperature
(°C) time (min)yield
(% wt. of feed)Capsaicinoid (% of extract)
100 40–50 60 0.065 47.0
130 50–60 45 0.040 57.5
150 60–70 45 0.010 87.4
175 70–75 45 0.003 98.8
250 75 45 0.106 0.8
Source: Mukhopadhyay, M., Natural Extracts Using Supercritical Carbon Dioxide,CRCPress,BocaRaton,FL,2000.Withpermission.
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358 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
11.10.4 GinGer
Ginger extract usingSC-CO2 is fractionated into two fractions: the essential oil–enrichedlightfractionandtheoleoresin-enrichedheavyfraction,thecompositionsofwhicharecomparedinTable11.11aandTable11.11b.Gingerols(G)andshogaols(S)arethecompoundsresponsibleforthepungencyofginger,andtheyaremostlycollectedintheheavyfractionoftheSC-CO2extract,ascanbeseeninTable11.11a.Shogaols,beingtheoxidationproductsofgingerols,arepresentinverylessquanti-ties in theSC-CO2-extracted fractions.Aproduct of desired specification canbeformulatedbycombiningthetwofractionsinasuitableproportion[14].
11.10.5 nutmeG
SC-CO2extractionandfractionationofnutmegcanyieldgoodqualitynutmegbutterastheheavyfractionwithverylittlevolatileoilandnutmegoilasthelightfraction,
taBle 11.11(a)light and heavy fractions of sC-Co2-extracted ginger oleoresin
product % 6-g% 8-g+ 6-s
% 10-g+ 8-s
(8g+6s) % total
total % extract
Rawmaterial 0.87 0.14 0.27 0.11 1.28
Heavyfraction 13.95 2.58 4.37 0.12 20.90
Commercialproduct 2.81 5.83 1.19 0.52 11.12
Lightfraction 1.43 0.61 0.36 0.25 2.40
RatioH/L 9.8 4.2 12.1 0.5 8.7
G:Gingerol;S:Shogaol
taBle 11.11(B)Compositions (%) light and heavy fractions of sC-Co2-extracted ginger essential oil
productraw
materialheavy
fractionlight
fractionratio l/h
Essentialoil(ml/100g)
2.0 4.4 98.8 22.5
β-pinene 2.5 0.5 2.6 5.2
Camphene 7.0 1.6 7.3 4.6
Cineole 8.4 2.3 8.6 3.7
Limonene 1.2 0.3 1.2 4.0
Zingiberene 21.8 17.4 22.7 1.3
Bisabolene 8.7 8.6 8.8 1.0
Sesquiphellandrene 11.9 12.7 11.9 0.9
Source: Mukhopadhyay,M.,Natural Extracts Using Supercritical Carbon Dioxide,CRCPress,BocaRaton,FL,2000.Withpermission.
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Processing of Spices Using Supercritical Fluids 359
inwhichtheundesirablehallucinatorycompoundmyristicinispresentinnegligibleconcentration[14].ItispossibletouseSC-CO2toproducenutmegoildevoidofthiscompound.ThisisanimportantadvantageoftheSCFEprocess,asthepresenceofthiscompoundinnutmegoilisbannedinsomecountries.
11.10.6 BlaCk PePPer
WhenblackpepperisextractedandfractionatedintotwofractionsusingSC-CO2,thelightfractionmaybecompletelyfreefrompiperine,theactiveingredientofpepper,whereastheheavyfractionmaybeenrichedwithupto60%piperine[14].Besidestheconcentrationofthespecificcomponent,allSC-CO2-fractionatedproductsareofsuperiorquality.Therateofextractionat500barisalmostdoubletherateat300barand60°C.Theproductioncapacityofthefractionsmaybeenhancedfourtimesat500barusingacascadeoffourextractors.Thus,theoperatingcostofextractioncanbereducedtoone-fourthofthatobtainedbythetraditionalSCFEplant.ThecurrentcommercialpracticeistofollowthistechniquetoimprovetheefficiencyandcosteffectivenessofSC-CO2extractionofmajorspices.
11.10.7 Vanilla
Naturalvanillafragranceisextractedfromcuredvanillabeans.Greenvanillabeansarecuredtobringabouthydrolysisoftheglucosidespresentinthebeanstogeneratevanillinandotherflavorandfragrancecomponents.Thecuringprocesschangesthegreenvanillabeansintodark,brownish,softbeans.Thecurrentcommercialextrac-tionmethodusesaqueousalcoholof35to40vol.%inconcentrationatatemperatureashighas87°Cinanumberofsteps,makingtheextractthermallydegraded.SC-CO2extractionofcryogenicallyground,driedbeansresulted10.6%yieldofoleoresinat110barand36°C,whichisevenhigherthan5.3–8.4%yieldsbyalcoholextraction[15].Thevanillaoleoresincontainedashighas16%to36%vanillinbySC-CO2extraction,whichamountedto74%to97%recoveryof the totalvanillincontent,respectively.Otherflavorand fragranceconstituents in thenaturalvanillaextractarep-hydroxybenzaldehyde,vanillicacid,andp-hydroxybenzoicacid.Thequalityof theextract is,however,characterizedbyitsvanillincontent.ThecompositionsofthenaturalvanillaextractsbytraditionalalcoholextractionandSC-CO2extrac-tionarecomparedinTable11.12.ThehighestpurityvanillincouldbeobtainedbySC-CO2extractionofwater-presoakedbeans,thoughtheyieldwasonly3%.Ontheotherhand, cryo-grinding apparently releasesmore compounds and, accordingly,theyieldwasalsohigh(10.6%).Thepurityofthealcoholextractaswellaspercentrecoveryofvanillin(61%)islowerthanthatoftheSC-CO2extract.Eventhecoloroftheextract,whichisyellowcomparedtothedarkbrowncolorofthealcoholicextract,issuperiorinthecaseofSC-CO2extraction.
11.10.8 Cardamom
SC-CO2 extraction of cardamom requires much higher pressure (100 bar) thansubcriticalpropane,whichrequiresas lowas20bar toyield thesameamountof
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360 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
essentialoil.AdditionofethanoltoSC-CO2doesnotgreatlyincreasetheyield,butincreasesthecoextractionofpigments,ascanbeseeninTable11.13.Reductioninpressure of SC-CO2 usually reduces the contents ofβ-carotene, chlorophyll, andpheophytinintheextract.
Theamountofpigmentextractedissignificantlymorewhensubcriticalpropaneisusedastheextractant.However,betterrecoveryofaroma(Table11.14)ispossiblewithSC-CO2at100barand35°C,asreportedbyIllesetal.[16].
11.10.9 Fennel, Caraway, and Coriander
Recovery of active components from fennel, caraway, and coriander by differentmethodsofextractioniscomparedinTable11.15.ItisclearthatSC-CO2extractsarericherinactivecomponents,owingtobetterselectivityoftheextractant[17].
taBle 11.12Comparison of Compositions of vanilla extracts
solvent(Beans)
sC-Co2 (120 bar, 33°C) ethanol + h2o(Water soaked)(dry) (ground) (Water soaked)
p-hydroxybenzoicacid(area%) 0.2 0.1 0.1 1.1
Vanillicacid(area%) 0.1 1.3 0.1 1.1
p-hydroxybenzaldehyde(area%) 0.6 1.9 0.9 2.7
Vanillin(mass%) 21.0 16.1 36.3 20.0a
Unknown 0.0 2.4 0.0 8.0a: Water-freebasis.
Source: Mukhopadhyay, M., Natural Extracts Using Supercritical Carbon Dioxide, CRC Press,BocaRaton,FL,2000.Withpermission.
taBle 11.13yield of Cardamom oil and pigment by sC-Co2 and propane
process Conditionsyield % (g/g oil)
β-Carotene (g/g oil)
Chlorophyll (g/g oil) pheophytin
SC-CO2(80bar,25°C) 5.65 0.8 0.65 —
SC-CO2(100bar,35°C) 5.45 2.1 0.30 —
SC-CO2(200bar,35°C) 5.95 3.9 0.36 0.33
SC-CO2(300bar,35°C) 6.65 5.8 4.53 2.36
CO2+ethanol(100bar,25°C) 5.28 1.64 9.65 2.10
Ethanol — 0.80 11.95 2.60
Propane(50bar,25°C) 7.24 18.6 10.80 4.80
Propane(20bar,25°C) 6.85 16.2 3.40 2.10
Source: Mukhopadhyay, M., Natural Extracts Using Supercritical Carbon Dioxide,CRCPress,BocaRaton,FL,2000.Withpermission.
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Processing of Spices Using Supercritical Fluids 361
11.9.10 GarliC
SC-CO2extractionofvaluableingredientsfromgarliciscomparabletothatbyhexane[18].Themajorcomponentsofgarlicoilarediallyldisulfide(30%),diallyltrisulfide(30%),anddiallylsulfide(15%).Alliin,amajorgarlicactiveingredient,isknowntodegrade toallicinbyanenzymatic reaction,andothergarliccomponentsarealsosusceptibletooxidationwithtemperature.Acomparisonofhigh-performanceliquidchromatography(HPLC)andGCanalysisofextractsobtainedbySEwithavarietyofsolventswithvaryingpolaritywiththatbySC-CO2indicatedthattheformercontained
taBle 11.14peak area (× 103) of aroma Constituents of Cardamom oil by sCf
β-pinene Cineole linalool α-terpinol Borneole
CO2(80bar,25°C) 16.1 295 34.8 47.8 356
CO2(100bar,35°C) 27.6 450 73.5 91.2 579
CO2(300bar,35°C) 17.4 341 32.7 46.4 340
Propane(20bar,25°C) 15.5 286 25.6 36.9 304
Propane(50bar,25°C) 26.9 386 72.1 82.7 521
CO2+ethanol(100bar,25°C) 6.5 198 5.8 8.9 112
Source: Mukhopadhyay, M., Natural Extracts Using Supercritical Carbon Dioxide, CRC Press,BocaRaton,FL,2000.Withpermission.
taBle 11.15recovery of active Components from fennel, Caraway, and Coriander by various methods
active Component
sC-Co2
ultrasound water hexane steam
p (bar) 80 100 200–300
t (°C) 28 30 35
fennel
Fenchon 10.7 13.1 9.2 21.9 16.3 0.3
Estragol 1.6 0.5 1.5 6.6 3.1 1.7
Transanethole 68.2 50.8 72.5 70 70 77.6
Caraway
Limonene 33.5 32.0 33.3 30.1
D-carvone 56.9 54.0 54.3 50.2
Coriander
Linalool 20–30 15 80–85 67 80 79
Source: Mukhopadhyay, M., Natural Extracts Using Supercritical Carbon Dioxide, CRC Press,BocaRaton,FL,2000.Withpermission.
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362 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
morecomponents.ThisisattributedtodegradationofthecomponentsinSE.ClinicaltestsalsoindicatedthattheSC-CO2garlicextracthasmorepotentbioactivity,closetothatofrawgarlic[18].
11.10.11 Cinnamon
Twotypesofessentialoilnamely,leafandbarkoil,areproducedfromtwodifferentpartsofacinnamontree.CinnamonleafoilismainlyproducedinSriLanka.ItisalsoproducedinIndiaandSeychelles.Mostcinnamonoilisproducedfromleaves.Barkoilamountstoonly15%oftotalproduction.Leavesyield1%oil.However,therootbarkyields3%oil[19].ThecomparisonofthecompositionsofextractsfromSrilankancinnamonbarkand leaves isgiven in Table11.16.SDof thecinnamonbarkyields1.4%oil,whereasSC-CO2extractionat200barand60°Cresults1.5%yield.However,additionofethanolasacosolvent increasestheyieldto2.6%[9].Theleafoilrichineugenolmakesitasubstituteforcloveoilandmaybeusedforconversiontovanillin.Barkoilismorevaluablethantheleafoil,althoughbothfindwideusesinflavoringandpharmaceuticalindustries.
11.11 CorrelatIon for spICe oIl soluBIlIty In sC-Co2
Solubility of spice oils in SC-CO2 is an important process parameter needed fordesignandscale-upofthecommercialSCFEplant.SolubilitydepictsthemaximumpossiblesolventcapacityofSC-CO2atagiventemperature,pressure,orcosolventconcentration in SC-CO2, though the actual loading or dissolution of the soluteis much less than this solubility in the presence of the solid substrate. However,theneatsolubility(withoutthepresenceofthesubstrate)behaviorofspiceoilcansufficeforselectionoftheprocessconditionsforthemostefficientperformanceoftheSCFEprocess.Becauseexperimentalmeasurementofsolubilityistediousand
taBle 11.16Compositions of Cinnamon leaf oil and Cinnamon Bark oil
% leaf oila % Bark oil
Componentsteam
distillatesteam
distillatesC-Co2 extract (200 bar, 60°C)
sC-Co2 + ethanol extract (200 bar, 60°C)
Eugenol 85–95 3.3 2.0 2.8
Caryophyllene 6 Traces 2.1 1.6
Cinnamicaldehyde 38 7.88 1.98 6.8
Isoeugenol 21.9 1.2 0.4
Linalool 20.1 0.9 0.8
Cinnamylacetate 23.6 5.1 1.8
o-methroxycinnamic
aldehyde 1.3 1.6 2.6a: Fromreference(Wright,1994)Source: Mukhopadhyay, M., Natural Extracts Using Supercritical Carbon Dioxide, CRC Press,
BocaRaton,FL,2000.Withpermission.
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Processing of Spices Using Supercritical Fluids 363
timeconsumingatdifferentconditions,areliablecorrelationcanservethepurpose,as itcanbeutilizedforestimationofsolubilityspiceoil inSC-CO2.Chrastil [21]relatedtheequilibriumsolubilityofasoluteinSC-CO2byalinearrelationshipintermsofitsdensityas:
lny*=klnρ+aT
+b (11.1)
where,y*isthesolutesolubility(g/L),T(K)isthetemperature,ρisthedensityofSC-CO2(g/L),anda,b,andkareadjustableconstantsthatcanbeevaluatedfromthelimitedexperimentaldata.
DeValleandAguilera [22]modified theChrastil’scorrelationbyaddingonemoreregressableconstanttowidenitsvalidityforthetemperaturerangefrom20°Cto80°Candforpressuresvaryingfrom150to280baras:
lny*=klnρ+aT
bT
+2
+C (11.2)
Silva et al. [23] correlated the experimental data (as reported in Table11.17)intermsofthedensityofSC-CO2asreportedbyAngus[24].TheconstantsinthecorrelationsarepresentedinTable11.18.Ferreiraetal.[25]reportedSCFEofblackpepperessentialoilfromwhichthesolubilitydataweregeneratedandwerecorre-latedintermsofvaporpressures(Ps),consideringoilasapseudo-purecomponent:
y*=PP
S
exp[A+Bρ] (11.3)
taBle 11.17Black pepper oil solubility in sC-Co2
t (°C) p (bar)density of sC-Co2
(g/cm3)oil solubility (g/cm3 Co2)
30 150 0.8478 0.0755
40 150 0.7812 0.0728
50 150 0.7010 0.06015
30 200 0.8909 0.1006
40 200 0.8404 0.08774
50 200 0.7851 0.7812
30 300 0.9486 0.13698
40 300 0.9106 0.1243
55 300 0.8712 0.1093
Source: Silva,D.C.M.N.etal.,Correlatingsolubilityvaluesofblackpepperoilin supercritical CO2 using empirical models, in Proceedings of the Sixth International Symposium on Supercritical Fluids,Nice,France,2003,Tome1,279.Withpermission.
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364 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
where A and B are empirical constants evaluated from the experimental data.However,thisrequiresvaporpressureofspiceoilasafunctionoftemperature.
Essentialoilobtainedfromturmeric(Curcuma longa)containsalargenumberofcomponents,suchascurcumin,ar-turmeric(42%),turmerone(12%),andtherest(<5%).Consideringit tobeasinglecomponentbyresearchers[26]thesolubilityofturmericoilinSC-CO2wasmodeledusingthesteady-stateextractiondataattheinitialperiodaswellasusingNaik’scorrelation[27]as:
Y= Y t
B t∞
+ (11.4)
where Y=extractionyield(kgextract/kgcurcumin)×100 t=CO2mass(kgCO2/kgcurcumin) Y∞=extractionyieldatequilibrium B=CO2massneededtoreachthehalfofY∞.
AcomparisonofthepredictedsolubilitieswiththecorrespondingexperimentaldataisgiveninTable11.19.Itmaybenotedherethatthefraction[0.5Y∞/B]issimilartotheslopeoftheextractioncurveattheinitialstage(i.e.,whentheextractionofspiceoil is controlledby its solubility).Bothmethods result similaragreementwith theexperimentaldataandmaybeconsideredforascertainingthesolubilitybehavior.
11.12 ConClusIons
SCFEofspicesisconsideredasuperioralternativetotheconventionaltechniquesofSD,SE, andASE for simultaneousproductionof essential oils andoleoresinsinasinglestep.SCFEensureshighconsistencyandreliability in thequalityandsafetyofthebioactivenaturalmolecules.SC-CO2isGRASandyieldscontaminant-free, tailor-made extracts of superior organoleptic profile, with high potency ofactive ingredientswithoutanyresidualorganicsolventandartifacts.Theextractsareveryclosetothatinnatureinsmellandtasteandhavelongershelflivesduetocoextractionofantioxidantsandbetterblendingcharacteristicsduetocoextractionof triglycerides. SCFE is known to be commercially viable for high-value, low-volumeextracts,andifmultipleproductsareobtainedoperatingthesameplantattherespectiveoptimizedprocessconditions.
taBle 11.18parameters for the solubility Correlations of Black pepper oil
Correlation a b c k
Chrastil –14807.9 26.123 — 3.84
DeValle&Aguilera 70207.45 –13500128.15 –107.409 3.84
Source: Silva,D.C.M.N.etal.,CorrelatingsolubilityvaluesofblackpepperoilinsupercriticalCO2usingempiricalmodels,in Proceedings of the Sixth International Symposium on Supercritical Fluids,Nice,France,2003,Tome1,279.Withpermission.
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Processing of Spices Using Supercritical Fluids 365
referenCes
1. Darling,M.Louis,BiomedicalLibrary,UCLA,Spices: Exotic Flavors & Medicines, Availableathttp://unitproj.library.ucla.edu/biomed/spice/
2. Mukhopadhyay,M.,Natural Extracts Using Supercritical Carbon Dioxide,CRCPress,BocaRaton,FL,2000.
3. Duke, J.A., Biologically active compounds in important spices, in Spices, Herbs, and Edible Fungi, Charalambous,G.,Ed.,ElsevierSciencePublishers,Netherlands,225–250,1994.
4. Rosen,R.T.,Ta’am Tov B’Tuv Ta’am: A Flavorful Blend of Kashrus and Spices, Avail-ableathttp://www.kashrut.com/articles/spices/September12,2006.
5. Tainter,D.R.andGrenis,A.T.,Spices and Seasonings: A Food Technology Handbook,SecondEdition,CulinaryandHospitalityIndustryPublicationsService,1997.
6. Raghavan Uhl, S., Handbook of Spices, Seasonings, and Flavorings, Culinary andHospitalityIndustryPublicationsService,1996.
7. Marion, J.P., Audrin, A., Maignial, L. and Brevard, H., Spices and their extracts:Utilization, selection,quality control, andnewdevelopments, inSpices, Herbs, and Edible Fungi,Charalambous,G.,Ed.,71–95,1994.
8. Pellerin,P.,Comparingextractionbytraditionalsolventswithsupercriticalextractionfrom an economic point and environmental standpoint, in Proceedings of the Sixth International Symposium on SCFs, France,2003,Tome1,13.
taBle 11.19solubility of essential oils of Curcuma longa
t (°C) p (bar)solubility (g/100 g Co2)
By naik’s modelsolubility (g/100 g Co2)
from extraction data
30 100 0.39 0.67
150 0.82 1.00
200 0.87 1.12
250 0.95 1.20
280 1.59 —
40 100 0.17 0.20
150 0.58 0.63
200 1.24 1.34
250 1.67 1.51
280 1.88 1.74
50 100 0.19 0.31
155 0.77 0.89
200 1.51 1.53
250 2.54 1.96
280 2.80 2.16
35 280 1.59 1.54
Source: Blasco,M.etal.,SCFEofCuruma longa:Solubilityofessentialoil,in Proceedings of the Sixth International Symposium on Supercritical Fluids, Nice, France, 2003,Tome 1, 279.Withpermission.
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366 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
9. Calame,J.P.andSteiner,R.,Supercriticalextractionofflavors,inTheory and Practice of Supercritical Fluid Technology,Hirata,M.andIshikawa,T.,Eds.,TokyoMetropolitanUniv.,275–318,1987.
10. GangadharaRao,V.S.G.andMukhopadhyay,M.,Selectiveextractionofspiceoilcon-stituents by supercritical carbon dioxide, Proceedings of the Annual Convention of Indian Institute of Chemical Engineers, Baroda,India,1988.
11. Meireles, M.A.A. and Nikolov, Z.L., Extraction and fractionation of essential oilswithliquidCO2,inSpices, Herbs, and Edible Fungi,Charalambous,G.,Ed.,ElsevierSciencePublishers,Netherlands,171–199,1994.
12. Zhang, J. et al., Volatile compounds of a SCF extract of Chinese celery seed, inProceedings of the Fourth International Symposium on Supercritical Fluids,Sendai,Japan,1994,235–237.
13. DellaPorta,G.,Reverchon,E.andAmbrousi,A.,PilotplantforisolationofceleryandparsleyessentialoilbySC-CO2,inProceedings of the Fifth Meeting of Supercritical Fluids,Nice,France,1998,Tome2,613–618.
14. Nguyen,U.Y.,Anstee,M.andEvans,D.A.,ExtractionandfractionationofspicesusingSCFCO2,inProceedings of the Fifth Meeting of Supercritical Fluids,Nice,France,1998,Tome2,523–528.
15. Nguyen,K.,Barton,P. andSpencer, J.S.,Supercritical carbondioxide extractionofvanilla,J. Supercrit. Fluids,4,40–46,1991.
16. Illes,V.,Daood,H.,Karsai,E.andSzalai,O.,Oilextractionfromcardamomcropbysubandsupercriticalcarbondioxideandpropane, in Proceedings of the Fifth Meeting of Supercritical Fluids, Nice,France,1998,Tome2,533–538.
17. Then, M., Daood, H., Illes, V. and Bertalan, L., Investigation of biologically activecompoundsinplantoilsextractedbydifferentextractionmethods,inProceedings of the Fifth Meeting of Supercritical Fluids,Nice,France,1998,Tome2,555–560.
18. Nawrot, N. and Wenclawiak, B., Supercritical fluid extraction of garlic followed bychromatography, in Proceedings of the Second International Symposium on Super-critical Fluids,Boston,1991,451–455.
19. Mahindru,S.N.,Indian plant perfumes, Metropolitan,NewDelhi,India,1992. 20. GangadharaRao,V.S.G.,Studies on Supercritical Extraction of Spices, Ph.D.Disser-
tation,IndianInstituteofTechnology,Bombay,1990. 21. Chrastil,J.,Solubilityofsolidsandliquidsinsupercriticalgases,J. Phys. Chem,86,
3016–3021,1982. 22. DeValle,J.M.andAguilera,J.M.,Animprovedequationforpredictingthesolubility
ofvegetableoilsinsupercriticalCO2,Ind. Eng. Chem. Res.,27(8),1551–1553,1988. 23. Silva,D.C.M.N.,Ferreira,S.R.S.andMeireles,M.A.,Correlatingsolubilityvaluesof
blackpepperoilinsupercriticalCO2usingempiricalmodels,in Proceedings of the Sixth International Symposium on Supercritical Fluids,Nice,France,2003,Tome1,279.
24. Angus,S.,Ramstrong,B.andDeReuck,K.M.,International Thermodynamic Tables of the Fluid State:Carbon Dioxide, NewYork,PergamonPress,3,1976.
25. Ferreira,S.R.S.etal.,SCFEofblackpepperessentialoil,J. Supercrit. Fluids,14(3),235–245,1999.
26. Blasco,M.etal.,SCFEofCuruma longa:Solubilityofessentialoil,inProceedings of the Sixth International Symposium on Supercritical Fluids,Nice,France,2003,Tome1,341.
27. Naik,S.N.,Lentz,H.andMaheshawari,R.C.,Extractionofperfumesandflavourfromplant materials with liquid carbon dioxide at liquid–vapour equilibrium conditions,Fluid Phase Equilibria,49,115–126,1989.
28. McGaw,D.R.,Holder,R.,Commissiong,E.andMaxwell,A.,Extractionofvolatileand fixed oil products from hot pepper, in Proceedings of the Sixth International Symposium on Supercritical Fluids,Nice,France,2003,Tome1,111.
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367
12 Preparation and Processing of Micro- and Nano-Scale Materials by Supercritical Fluid Technology
Eckhard Weidner and Marcus Petermann
Contents
12.1 Introduction................................................................................................. 36712.2 ParticleGenerationbyHigh-PressureSprayProcesses.............................. 368
12.2.1 RapidExpansionofaSupercriticalSolution................................ 37012.2.2 AntisolventProcesses................................................................... 37212.2.3 SprayingofGasSaturatedLiquids............................................... 37312.2.4 Economics.................................................................................... 375
12.3 CompositeswithHigh-PressureSprayProcesses....................................... 37612.3.1 SprayAgglomerationwithaHigh-PressureSprayProcess......... 37612.3.2 Liquid-FilledCompositeswithaHigh-PressureSpray
Technology.................................................................................... 37812.4 ProcessingofNutraceuticalswithSupercriticalFluidTechnology............38012.5 Conclusions.................................................................................................384References..............................................................................................................384
12.1 IntroduCtIon
Thegenerationofnano-andmicro-particlesandtheformationofparticulatecom-positeshavebecomemoreandmore important inmany industrialareas. Infood,pharmaceutical,material,andlifescienceindustries,existingandnewproductsinnewapplicationformswith tailor-madepropertiesarebeingdevelopedfasterandfaster.Toformparticulateproducts,differentwell-establishedprocessesareavail-able.Powderscanbeobtainedbycrystallization,grinding,orspraydryingprocesses.However, all these techniques have drawbacks, especially if sensitive substancessuchasnutraceuticalsorbioactivesystemshavetobeprocessed.Inclassicalcrystal-lizationtechniques,solvents—inmanycasesorganicsolvents—havetobeusedasauxiliarymedia.Resultantresiduesofthesesolventsmaybefoundintheproductsand have to be removed by time-consuming and expensive technologies. Similar
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368 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
drawbacksappearinmostspraydryingprocesses.Inaddition,thehightemperaturesnecessarytoevaporatesolventsmaycauseproductdegradation.Grindingprocesses,whicharetypicallysolventfree,canonlybeappliedforbrittlesubstances.Toachievesufficient brittleness, deep-freeze conditions are sometimes required. But even ifmillingispossible,onlyparticleswithsharpedgesareavailable(Figure12.1).
Theincreasingdemandfornewproductpropertiesandthedrawbacksofexist-ingprocessesarecausingasteadysearchfornewtechnologicalpossibilitiesfortheformationofparticulatesystems.Somepromisingtechniquesincludeusingsuper-criticalfluids(SCFs)togeneratenano-andmicro-scaledparticlesystemswithwell-definedmorphologiesand,therefore,productbehavior.Inadditiontobeingusedforpureparticleformation,thesetechniquesarebeingusedmoreandmoretogeneratecompositesconsistingoftwoormoresubstances,eventhoseindifferentstatesofaggregate(liquid/solid).Thisallowsmanufacturingofhigh-qualityproductsoffer-ingtailor-madeproperties,suchascontrolledreleaseofactivesubstances[1–5].
12.2 PartICle GeneratIon by HIGH-Pressure sPray ProCesses
Generatingparticlesfrompuresubstancesorcompositesbyhigh-pressuretechnolo-giesrequiresunitoperationssimilartothoseusedforclassicallow-pressureprocesses.Thoseunitoperationscomprise,forinstance,melting,dissolving,mixing,spraying,separating,andpumping.PerformingsuchunitoperationsunderhighpressuresandinthepresenceofSCFsrequiresspecificadaptationsinplantdesignandprocess-ing. Due to extensive R&D work and industrial experience, those adaptations ofmachinesandapparatusesaremeanwhileknownquitewell.Inspiteofthefactthatmajortechnicalproblemsaresolvedforhigh-pressureapplications,itisundisputedthatthoseprocessesaremorechallengingfromthetechnicalandeconomicalpointsof view than are low-pressure processes. New possibilities to create value-addedproductsthatarenotaccessiblewithclassicaltechnologiesareastrongdrivingforcefor the industrial use of high-pressure processes. A series of such processes andprocessmodificationsthatallowsgeneratingnewproductformshasbeendeveloped
FIGure 12.1 Morphologyofgrindedparticles.
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Preparation and Processing of Micro- and Nano-Scale Materials 369
inthepastyears.Anextensivenumberofacronymsareusedtocharacterizethoseprocesses.Thoseacronymswillnotbe listedhere,but theprocessesaregroupedaccordingtothermophysicalprinciplestheyuse.
Inrapidexpansionofsupercriticalsolution(RESS)processes,thesubstancetobepowderizedisdissolvedintheSCF. Intheparticlesfromgas-saturatedsolutions(PGSS)processes,thesubcriticalfluidorSCFisadmixed,dispersed,anddissolvedin the substance to be powderized. In both types of processes, the particles aregeneratedbyexpansionincapillariesornozzles[6–9].Intheso-called“antisolventprocesses,”suchasthegasanti-solvent(GAS)process,theproducttobepowderizedisfirstdissolvedinaclassicalorganicsolvent.Afterward,thissolutionisadmixedwith aSCF.This causes adecreaseoffluiddensity and leads to reduced solventpowerof theorganic solvent.Resultantparticles areprecipitatedwithin themix-ture of organic solvent and SCF [10–13]. Compared to traditional crystallizationprocesses, in which organic solvents are evaporated at high temperatures or in avacuum,GASprocessesworkatlowtemperaturesandachievesupersaturationmuchfaster.Therefore,GASprocessesareadvantageouscomparedtotraditionalcrystal-lizationprocesses,eveniftheresidualsolventproblemhastobesolved.
Allprocesseshave incommonthat thesubstance tobepowderizedhas tobebroughtintoaliquidordispersedform.Thisisachievedbymeltingthesubstance,bydissolvingtheproductinclassicalsolvents,orbydispersingtheproductinaliquid.Inmanycases,thisstepisperformedatambientorslightlyelevatedpressures.Thentheproduct is compressedbycontinuouslyoperatedplungerpumps,gearpumps,orextruders.Thetypeofdosingsystemuseddependsmainlyonthepropertiesofthe product (e.g., melting point and viscosity�). Dosing of substances with highviscositiesathighpressuresrequiresspecialtechnicalsolutions,someofwhichhavebeenelaboratedinthepastyears[14].Manyresearchactivitiesdemonstratedthathighpressure,inconnectionwiththeuniquepropertiesofSCFs,opensthechancetogeneratepowdersfromhighlyviscousliquids,whichcannotbesprayedbyclassicaltechnologiessuchasspraydrying.Sprayabilityisachievedbyaconsiderablereduc-tionofviscosityandsurface tension ifsuchhighlyviscous liquidsareeffectivelyadmixedwithaSCFthatissufficientlysoluble[15,16].Admixingisachievedviastirrers,dispersers, impinging jets, staticmixers,ormembranes thatareoperatedunderhighpressure.
ImprovementofsprayabilityisnottheonlyadvantageofSCFs.AfteraSCFisadmixed,thephysicalpropertiesofthesubstancesarechangeddramatically[17–19].Themostimportanteffectsarereductionofviscosityandmeltingpointdepressionduetodissolvedgas.Botheffectsallowhandlingsubstancesnearorevenbelowtheirmeltingpointunderambientpressure.DuetocoolingoftheSCFthatoccursdur-ingexpansion,thetemperaturesareeven(much)loweraftertheparticleshavebeenformedviaexpansion.Astheheatofsolidificationisremovedbydirectheattransferfromtheparticlestothecoexpandedgas(inthecaseofRESSandPGSS),solidifica-tionoccursmuchfaster(some10milliseconds)[20–22]andthetemperaturestressontheparticlesislowerthanthatduringclassicalairdryingprocesses,wherebythe
�In discontinuous processes, products are dosed either as liquid or solid into an autoclave, whereadditionallycompressedgasisaddedandadmixed.Theelevatedpressureintheautoclaveisusedtotransporttheproductsintothenextprocesssteps.
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370 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
solventisevaporatedbyheattransferbetweenliquiddropletsandthesurroundinghotairorgas[23].Furthermore,unlikemanytraditionalspraydryingprocesses,thesupercriticaltechnologiesareintrinsicallyfreefromcontactingtheproductswithairoroxygen.Sothesetechnologiesaresuitableforsensitivesubstances[24].Anotherpositiveeffectisthataslongasthecontactbetweenthegeneratedpowdersandairiscarefullyavoided,dustexplosionsmaynotoccur.
Animportant—andsometimesunderestimated—stepofallhigh-pressurepro-cessesisthecollectionoftheparticles.UsingsprayprocesseslikeRESS,PGSS,andconcentratedpowderform(CPF),theparticleshavetobeseparatedfromagasstream.Dependingonparticlesizeandparticleconcentrationinthegasstream,differentseparation techniquesmightbesuitable.Coarse fractionscanbesepa-ratedjustbysettlingtheparticlesinspraytowersorbyenlargingtheseparationforcesincyclones.Forfinerparticlesandlowerparticleconcentrationfilters,mem-branesor sinter plates canbeused to collect themanufacturedproduct.For theantisolventprocesses,inwhichparticlesareformedbyprecipitationinaliquid,asolid-liquidseparationmustbeused.Thisseparationisachievedbysettlingorbydifferentfiltersystems.
After the particles are separated from the SCF or the solvent, postprocessingmightbenecessary.Inthecaseofantisolventprecipitation,solvent-wetparticlesareobtained,whichhavetobedried.Thiscouldbedone,forexample,byflushingwithheated gas or by additional SCF extraction steps [25]. Depending on the applica-tionoftheproduct,awholerangeoffurtherposttreatmentstepsmightbeconsidered(coating, sieving, agglomeration, sifting, size fractionation, dispersion, and soon),whichcouldeitherbeappliedaloneorusedincombinationwiththeupstreamparticlegenerationprocess.Someofthesecombinationshavealreadybeenstudied[26–28].
Anoften-discussed issue forallprocesses isgas recycling. If liquidsolventsareinthesystem,solventremovalfromthegasisrequiredinordertoavoidenrich-mentintherecyclegas.Insolvent-freeprocesses,theparticleshavetoberemovedcarefullybeforerecompressingthegasinordertoavoidpluggingoftherecyclingsystem.Bothpurificationmethodscantechnicallybeapplied,buteachisconnectedwith additional costs for equipment andoperation.On a case-by-casebasis, onemustconsiderwhetherrecyclingofthegasisfeasibleandreasonableaccordingtoeconomic and environmental aspects. Recycling might be a disadvantage of theuseofSCFforpowdergeneration,asthepressuredifferencesbetweenpreexpan-sionandpostexpansionrequirehighenergiesforrecompressingthegas.Therefore,reducingthegasdemandforparticlegenerationasfaraspossibleisrecommended.Someprocesses(e.g.,thePGSS-orCPF-method)allowgenerating1kgofpowderwith0.1to1kgofgas.IfacheapSCF,suchascarbondioxide(CO2),isused,gasrecyclingforsmall-andmedium-sizedplantsmightbemoreexpensivethanusingfreshgas.
12.2.1 Rapid Expansion of a supERcRitical solution
Oneoftheoldestprocessesthatusesthespecialpropertiesofcompressedgasesistheso-calledRESSprocess [29–37].Aflowscheme for thisprocess ispresentedinFigure12.2.The substance tobepowderized is stored in an extraction vessel.
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Preparation and Processing of Micro- and Nano-Scale Materials 371
Compressedgas(inmostcasesCO2)isledthroughthevessel.Underhighpressures,sometimesupto800bar,theproductis(partly)solubleinthegas.Theso-formedsolution is dosed through a heat exchanger and finally is expanded via a nozzle.Causedbytherapiddepressurization,thedissolutionpowerofthegasisreduced,supersaturationoccurs,andaprecipitationoffineparticlesisinduced.Particlefor-mationcanbeinfluencedbythepressureduringextraction,theconcentrationofthedissolvedsubstance, the temperaturebeforedepressurization, thegeometryof thenozzle,andconditionsinthespraychamberaftertheexpansion.Veryfinepowdersintherangeof0.1to10µmwithnarrowparticledistributionsareobtainedbyprop-erlyadjustingtheprocessparameters.
The RESS process is characterized by a rather simple setup in laboratory orsmallproductionscale,butitislimitedbythepoorsolubilityofmanysubstancesinCO2.Sometimesmorethan100kgofCO2wouldbenecessarytomanufacture1kgoftheparticulateproduct.Subsequently,theparticleshavetobeseparatedfromveryhighlydilutedgasstreams,whichisaproceduralchallenge.
Therefore, the RESS process offers a high potential for high value-addedproductssuchaspharmaceuticalsandcosmetics.Investigationswiththemodelsub-stancegriseofulvinshowedthat,comparedwithproductsmicronizedwithclassicalprocesses,anaccelerateddissolvingbehaviorcanbeachievedwiththeRESSproduct.Inaddition,researchersobservedbetterabsorptionbehaviorinanin-vitrotestsystem[38].Inallnano-scaledprocesses,theposttreatmentoftheparticlesafterparticlefor-mation isachallenging task.Nano-particles tend toagglomerate,andredispersingsuch systems is very difficult or sometimes nearly impossible. To stabilize RESSparticlesintheirnano-scale,researchersproposedtocollecttheminliquid-containingemulsifiers[39].Newlypublishedpapersshowedthat,usingthistechnique,along-termstabilityofnanosuspensionsisobtainedwithparticlessmallerthan100nmandconcentrationsofup to11g/dm³ [40].Other researchers sprayed ternarymixturesconsisting of a SCF, the active substance, and a polymer; they obtained resultantpowderswithencapsulatedactivesubstancesindifferentconcentrations[41–43].
Powder
Gas
Powder
FIGure 12.2 RESSprocessscheme.
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372 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
12.2.2 antisolvEnt pRocEssEs
Anotherimportantgroupofhigh-pressureprocessesaretheso-called“antisolventprocesses.”SimilartotheRESSprocess,thesetechniquesaresuitableformanufac-turingpowdersofpuresubstancesandcompositesfromnanotomicroscale.Alotofdifferentprocessmodificationsaredescribed[44–51],forexample:
GAS: gasantisolventSAS: supercriticalfluidantisolventPCA: precipitationwithacompressedantisolventASES: aerosolsolventextractionsystemSEDS: solution-enhanceddispersionbysupercriticalfluids.
Alltheprocessesbehindthesedifferentacronymsmakeuseofaneffectwellknowninclassicalcrystallizationtechniques.Byaddingathirdcomponent(antisolvent)toasolution,thesolubilityofthedissolvedcomponentisreducedandfinallythesub-stanceprecipitates.Inhigh-pressuretechnology,theantisolventisasupercriticalornear-criticalfluid.Theuseofcompressedgasesinsteadofotherantisolventsopensthewaytonewparticlemorphologiesandnewcomposites[52,53].
Thefirstthreeprocesses(GAS,SAS,andPCA)aretypicallyoperateddiscon-tinuously. A simplified process scheme is shown in Figure12.3. The substanceormixture tobepowderizedisdissolvedina liquid(mostlyorganic)solvent.Toprecipitateparticles,thesolutionhastobecontactedwithaSCF.Thisisachievedindifferentways:
IftheliquidsolutionisprovidedinavesselandafterwardtheSCFisdosedintothatvessel,theprocessiscalledtheGAS process.In the SAS and PCA processes, the SCF is provided in a high-pressurevesselandthesolutionissprayedintothatsupercriticalsolution.IntheSEDSandASESprocesses,bothfluids(thesolutionandtheSCF)aremixedinnozzlesandsprayedintoautoclaves.
•••••
•
•
•
Precipitation
Filtration Drying
FIGure 12.3 Antisolvent-process(GAS)processscheme.
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Preparation and Processing of Micro- and Nano-Scale Materials 373
Inalloftheseprocesses,admixingofthegascausesaprecipitationofthedissolvedproductintheliquidsolution.Inthenextprocessstep,theso-formedparticleshavetobeseparatedfromtheliquid.Inlaboratoryscale,filtersintegratedintothehigh-pressurevessels typicallyperform this step. Ina thirdprocess step, thecollectedparticleshavetobedried.Inanelegantway,thisisachievedbyaddingfreshSCFtothepressurevessel.Theliquidisextractedfromtheprecipitatedpowderbythegas.Oneadvantageofthisprocessingisthetemperaturerequiredinthesupercriticaldryingstep,whichismoderatecomparedwiththetemperaturerequiredforconven-tionaldryingprocedures.
The antisolvent processes have been successfully tested with many differentproducts. Beside the particle generation from explosives likeβ-HMX and nitro-guanidin,polymers(polyacrylnitril,polycaprolacton)andotherorganicsubstances(hydroquinoneandphenanthrene)havebeenpowderized.AsintheRESSprocess,themainfocusofresearchisaddressingpharmaceuticalslikeascorbicacid,insulin,andparacetamol.
12.2.3 spRaying of gas satuRatEd liquids
TheRESSprocessandtheantisolventprocessesaretypicallycarriedoutindiscon-tinuousorsemicontinuousmode.ThePGSSprocessmayrathereasilybeoperatedinacontinuousmodeand,therefore,isalsosuitableforproductsmanufacturedinlargerquantities[54–57].
Figure12.4illustratesaprincipleflowschemeofthePGSSprocess.Tosprayagas-saturatedliquid,theSCFhastobeadmixedwiththeproducttobepowderizedunder elevated pressures. Typically, the product has to be melted or liquefied byaddinga solvent inadvanceat low-pressureconditions.Subsequently thisfluid ispumpedviahigh-pressurepumpstoamixingdevice(mostlystaticmixers),wheretheSCFisadmixed.Underhigh-pressureconditionstheSCFispartlysolubleinthemelt,dispersionor solution.The solubility causesa reductionofviscosityandof
Gas
Powder
Gas
FIGure 12.4 PGSSprocessscheme.
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374 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
interfacialtension.Botheffectsresultinimprovedsprayability,evenforsubstancesthatnormallycannotbeatomizedbysprayprocesses.Afterward,thegas-enrichedfluidsaredepressurizedviaanozzleintoaspraytower.Normally,thespraytowerisoperatedatambientpressure.Duetothevolumeincreaseoftheexpandinggas,theproductisdisintegratedintofinedroplets.Simultaneously,thegascoolsdownimmediatelyduringexpansion.Althoughthetemperatureinspraytowercaninprin-ciplebeadjustedbyadditionalheatingor cooling,most applicationsdonotneedthis temperaturecontrol.The temperature in thespray tower ismostly setby thepreexpansionconditionsinstaticmixer.Typicaltemperaturesinthespraytowerliein the range of –20°C to 100°C. If the resultant temperature is low enough, theliquid/melt reaches the solidification point and the droplets freeze. Particle sizeandparticlesizedistributionoftheobtainedpowderscanbeadjustedbychangingtheSCF,thepressureinthemixingdevice,thetemperaturebeforeexpansion,andthegeometryofthenozzle.
Asanexample,differentmorphologiesandparticlesizesofpowdersareillus-trated in Figure12.5. The technique can be used for the powderization of manydifferent systems. In addition to organic substances such as citric acid and poly-ethylene glycol (PEG), certain pharmaceuticals (e.g., nifedipine and tobramycin)weresuccessfullymicronized.Moreover,compositesandevenreactivesystemslikepowdercoatingscanbehandledwiththistechnique[58].
Oneadvantageofthisprocess,comparedwithotherhigh-pressuretechniques,isthelowtomoderateconsumptionofSCF.Typically,0.5to5kgofSCFarenecessary
FIGure 12.5 ParticlemorphologiesofPGSSparticles.
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Preparation and Processing of Micro- and Nano-Scale Materials 375
toproduce1kgofpowder.ThelowgasdemandtogetherwiththerelativelysimpleconstructionofthePGSSplantsallowstheproductionofhugequantitiesofproducts.Thedesignoftheprocessandthecontroloftheproductpropertiesdependsonthethermodynamicandfluiddynamicbehavior,forexample,thesolubilityofthecom-pressedgasesintheproducttobepowderized,theviscosity,andflowbehaviorofthegas-containingmelts.
12.2.4 Economics
Figure12.6presentsestimatedcostsforindustrialPGSS(non-GoodManufacturingPractice [GMP])production facilities.Thediagramgives the total costs for 1kgofproduct,includingcostsforinvestment,personnel,energy,andgasconsumption[57,59].Dependingonthehourlycapacityandtheannualproductionhours,costsrangefromthirtycentsto1€(about$0.75)perkilogramofpowder.Theestimatedcosts aremainlydue topersonnel (40%)andcarbondioxideconsumption (40%).Comparedwiththecostsofclassicalmicronizationtechniques,suchasmillingorspraydrying,thecostsareonthesameorderofmagnitude.
Coststudieshavebeenpublishedforotherhigh-pressureprocesses,likeRESSandGAS.RantakyläanalyzedtheantisolventprocessSAS[60].Estimatedmanufac-turingcostsforanewGMPplantarearound50to300€/kg(38to230$/kg)productwithoutafeedstockprice.Thisisfora4000to8000kg/yearproductionrateand5to10wt%feedconcentrationofthestartingmaterialinanorganicsolvent.Aneffectivewaytodecreasethemanufacturingcostsistoincreasetherawmaterialconcentrationinsolvent.Weberetal.[61]providedataforanon-GMPPCAprocess.Foraninitialsolventconcentrationof10wt%andaproductionof11.25kg/hr(correspondingto87MT/year),thecostsperkilogramofpowderarearound8€($6).Ifthecapacityisdoubled,thespecificcostsarereducedtoapproximately5€/kg($3.80/kg).
0.54
0.390.33
0.0
0.2
0.4
0.6
0.8
1.0
2000 3000 4000 5000 6000
Annual Production (hours/year)
Cos
t (E
UR
/kg
Pow
der)
200 kg/h
350 kg/h
500 kg/h
FIGure 12.6 Production cost for PGSS process (gas consumption 2 kg/kg product).DiagramcourtesyofNatex,Austria.
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376 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
Türk[62]hasgivenvaluesforRESSplantswithafixedCO2flowof2.35MT/hr.Inthecaseofasubstancewithlowsolubility,theannualproductionis1.78MTandthespecificcostsarebetween100and140€/kg(dependingonthetimeofdepre-ciation).Forhighlysolublesubstances,theproductioncapacityinthesameplantisconsiderablyhigher(uptosomehundredtons),leadingtoreducedspecificcoststhatmightreachtherangeof1€/kgorevenlower.NeverthelessithastobenotedthatonlyalimitednumberofsubstanceshaveahighsolubilityinCO2.
The costs for RESS and antisolvent processes are dominated by relativelyhigh investmentcosts for largepressurevesselsandconsiderablegasconsumption.Therefore,thesetechniquesarepreferablyappliedforhigh-pricedproducts,suchaspharmaceuticals.PGSSisalreadyappliedindustriallyinplantsizesofsomehundredkg/hr.Fats,fatderivatives,polymers,andchocolatearealreadyprocessedinindustry.
Amainfocusofthepastyearsofresearchanddevelopmentinthefieldofsuper-criticalmicronizationhasbeenonthegenerationoftailor-madeparticlesfromsinglecomponents.Insomecases,thesetechnologieshavealreadybeentransferredsuc-cessfullyintoindustrialscale.Recently,thefocushaswidenedtowardtheformationofcomposites.TechnologieswithSCFsofferanincreasednumberofpossibilitiestogeneratecompositeswithnewfunctionalities.ThefollowingsectionhighlightstwoexampleshowSCFcanbeusedtoproducesuchproducts.
12.3 ComPosItes wItH HIGH-Pressure sPray ProCesses
Forcommercialsuccess, ithasbecomemoreandmoreattractivetodesigntailor-madeparticlesystemsthatallow,forexample,thecontrolledreleaseofactiveagentsorofferdurableprotectionof sensitive ingredients.Classicalprocesses like spraydrying,crystallization,andin-situpolymerizationprocessesareinprincipleabletoproducesuchcomposites.Inspraydrying,thehightemperaturelevellimitsthetech-niquetoinsensiblesubstances.Incrystallizationprocesses,completeencapsulationishardtoachieveandtheparticleshapeisdifficulttocontrol.Forpolymerizationprocesses,onlyafewmaterialcombinationsaresuitable.Inthisfield,afewtech-niquesusingSCFsareestablishedandtheresultsareverypromising.TheseSCFtechnologies allow thegenerationofpowderswithproperties that aredifficult orevenimpossibletoachievebyclassicalmethods.
12.3.1 spRay agglomERation with a high-pREssuRE spRay pRocEss
The processes described above lead to reduced particle size of the raw material.SCFprocessesarenot limited toparticle size reduction.Different shape-formingmethodshavebeenestablished in the last fewyears (e.g.,coating,agglomeration,impregnation,anddispersionprocesses)[63,64].
TheCPFtechniqueisasprayagglomerationtechniquethatallowstheproduc-tionofliquid-loadedcompositeswithloadingsofupto90wt%.Theagglomeratesobtained have a high mechanical stability and good flow behavior. Figure12.7illustratestheflowschemeoftheCPFprocess.
The liquid to be powderized is dosed with a high-pressure pump from thestoragevesseltoastaticmixer.Here,asecondstreamofSCF,mostlyCO2,isadded.
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Preparation and Processing of Micro- and Nano-Scale Materials 377
Underpressuresofupto200bar,thegasandliquidaremixedandsubsequentlydepressurizedviaanozzletoatmosphericpressure.Thevolumeincreaseofthegasleads to theformationofveryfinedroplets.Temperatures in thespray towerarecontrolledbythepreexpansionconditionsinthestaticmixerandaretypicallyintherangeof–20°Cto60°C.Therefore,thistechniqueisespeciallysuitableforprocess-ingoftemperature-sensitiveorvolatilesubstances.Byaddingasolidcarrierwithapneumaticconveyingsystemintothespraytower,theliquiddropletsarebound.Solid,free-flowingagglomeratesareformedthatcanhaveamaximumof90wt%liquidcontent.Theliquidisboundbyadsorptiontothesurfaceofthecarrierandbycapillaryforcesbetweenthesingleparticlesintheagglomeratesoreveninporousstructuresofthesingleparticles(Figure12.8).Theformationofsuchagglomerateswastestedwithmanydifferentsubstances(e.g.,naturalextractsofbasil,pepper,lemonoil,α-tocopherol,whiskey).Silicicacid,celluloses,andstarcheswereusedascarriers.Inalloftheseexperiments,thefirsttaskwastogetfree-flowingpowdersthatcanbeeasilyhandledinpostprocessing.Inadditiontotheflowabilityoftheproducts,thereleaseoftheboundliquidisofimportance.Byvaryingthecarriermaterial, products with defined release behaviors can be achieved for the food,pharmaceutical,andcosmeticindustries.
Figure12.9 illustrates the controlled release of a CPF product [65]. For thisproduct, vitamin B2 was sprayed on a potato starch using the CPF technique.
Powder
Carrier
Gas
Powder
FIGure 12.7 CPFprocessscheme.
Adsorption Agglomeration Impregnation
FIGure 12.8 BindingmechanismofCPFproducts.
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378 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
Afterward,theproductwaspouredwithwaterandwasmixedwithamagneticstirrerbarwithandwithoutheating.Thereleaseofthecoloredvitaminwasmeasuredbytheextinctionoftheaqueoussolution.Thelowerflatgraphindicatesthereleaseofthevitaminbymixingwithcoldwater.Onlysmallamountsofthecoloredvitaminare released. If thewater isheated, the releaseoccurs (as indicatedby theuppercurve).At thebeginning, it is comparable to theexperimentwithcoldwater,butwhenthetemperaturefinallyreaches80°C,anearlycompletereleaseisobvious.
12.3.2 liquid-fillEd compositEs with a high-pREssuRE spRay tEchnology
BasedonthePGSSprocess,ahigh-pressurespraytechniquewasinvestigatedthatallowstomanufacturecompositesconsistingofacorematerial,whichcouldbealiquidorasoliddispersedinaliquid,andashellmaterialthatmustbesolidunderstorage conditions [66–68].Aprincipleflowschemeof thisprocess is illustratedinFigure12.10.Bothcomponentshavetobeprovidedinapumpableform.High-pressurepumpsareusedtofeedtheshellmaterialsandtheliquidcorematerialstothestaticmixer,wherethecomponentsaredispersed.Inaddition,aSCFispumpedintothemixer.Dependingonthesystemandthemixersize,amoreorlessstabledispersion isobtained.Subsequently, thisdispersion isdepressurized intoaspraytower.Theshellmaterialsolidifiesduetotemperaturereductionoftheexpandinggas.Thecorematerialisencapsulatedintheshell.Figure12.11showsinprinciplethemorphologiesthatcanbeobtainedontheonesidewithaliquiddispersedinameltandontheothersidewithadispersionoftwoimmisciblemelts.Oneadvantageofthistechniqueisthatthedispersionoremulsionsformedinthestaticmixercanbeeitherstableorunstable.Theresidencetimeaftermixingisextremelyshort(somemillisecondstoseconds)sothataphasesplitdoesnotoccurbeforeexpansion.Aftertheexpansion,solidificationoftheshellmaterialhappensinstantly;thedispersionisstabilizedbysolidification.
As an example for the manufactured composites, three scanning electronmicroscope (SEM) pictures of a solid wax/liquid PEG composites are shown inFigure12.12.Thelightergrayregionsconsistofwax;thedarkregionsinthepicturesshowtheboundliquidPEG.Intherangeof50to60wt.-%,achangefromclosedtoopencompositesisobserved.
0
20
40
60
80
100
0 20 40 60 80 100 120t(min)
Cont
rol R
elea
se (%
)
Complete Release at 80°C By Mixingand Heating
Release at 25°C byMixing
FIGure 12.9 Temperature-triggeredreleaseofaCPFproduct.
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Preparation and Processing of Micro- and Nano-Scale Materials 379
Components
Gas
PowderousComposites
A B
FIGure 12.10 Processschemeofcompositeprocess.
Solid-liquid DispersionEncapsulated Microdroplets
Solid-solid Dispersion
FIGure 12.11 Morphologiesofcomposites.
54 wt.–% 20 µm 20 µm 20 µm
57 wt.–% 64 wt.–%
FIGure 12.12 SEMpicturesofcompositeparticles(wax/PEGsystem).
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380 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
Thesurfaceofthesphericalcompositeswith54wt.-%ofPEGontheleft-handside of Figure12.12 is completely closed and uniform. No darker regions, whichwould indicate the presence of liquid PEG on the surface, can be detected. Byincreasingtheamountof liquidPEG,whichmightnotsufficientlybeadmixedinthestaticmixer,thesurfaceofthesphericalcompositesisstillclosedanduniform.Nevertheless, in thatcase, free liquidPEGmaycoexistwithdroplets thatconsistofwaxanddispersedPEG.Thefreeliquidisboundbycapillaryforcesinbetweenthesolidifiedwaxparticles.AgglomeratesareformedwithdispersedPEGencapsu-latedinthewaxandPEGascapillaryliquidbetweenthewaxparticles.Byfurtherincreaseoftheliquidcontent,thevolumefractionoftheshellmaterialistoolowtoallowcompleteencapsulation.Thephotographontheright-handsideshowsapar-ticlewithanopenstructure,wheretheliquidPEGisboundinporesofthewax.
The morphologies of the composites show that agglomerates, single particlesas well as closed and open-structured composites, can be produced. The highestconcentrationofPEGthatstillallowstheformationofcompletelyclosedcompositeswasapproximately60wt.-%.Arisingconcentrationoftheliquidfavorsthegenera-tionofopencomposites.Thiscanbeunderstoodbyfocusingonthebasicsofparticleformation.Toformacomposite,theliquidhastobeadmixedtotheshellmaterialasthedispersedphaseofanemulsion.Subsequently,themixtureissprayedandsolidi-fiedusinganexpandinggas.Themainfactorforthegenerationofclosedoropencompositesisthedifferencebetweenthespeedofsolidificationandphasesepara-tionoftheemulsion.Anincreasingamountofliquidleadstoarisingdropdiameterofthedispersedphaseorarisingnumberofdisperseddroplets.Arisingnumberofdisperseddropletsleadstoanacceleratedbreakageoftheemulsion.Withconstantprocessparameters(i.e.,temperatureandgastoproductratio),thesolidificationtimewillbecomparablebuttheseparationoftheemulsionismuchfaster.Thisresultsinthegenerationofopencomposites.
Theprocessdescribedabovehasalreadybeensuccessfullyappliedtodifferentproductsinthechemical,food,andcosmeticindustries.Waterhasbeenencapsulatedinfat;liquidaromasandantioxidantshavebeenboundinafatmatrixtoreducethelosses during storage, different vegetable oils have been encapsulated in PEGS, aparaffinwaxhasbeenboundinpolyester,andkirschwasencapsulatedinachocolatematrix[69,70].Themicronizedchocolatewiththeencapsulatedkirscharomacouldbeused,forexample,toenhancetheflavorofhotcocoaortobringadditionalaromaintoanychocolateproduct.Thereleaseofthearomaforthesediffersfromproductsonthemarketwhereliquorsareencapsulatedinmacroscopicstructures,likepralines.Theverysmallsizeofthechocolateparticles(some10tosome100microns)leadstoimmediatemeltinginthemouth.Thereby,thearomasofchocolateandkirscharereleasedtogethertoformaflavorthatcombinesthebestofboth(Figure12.13).
12.4 ProCessInG oF nutraCeutICals wItH suPerCrItICal FluId teCHnoloGy
Manyoftheprocessesdescribedabovearedesignedandoperatedtosubstituteoneclassicalprocesstask(e.g.,milling).Togainlargerbenefits,differentprocesstasks
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Preparation and Processing of Micro- and Nano-Scale Materials 381
canbecombinedandsolvedinoneSCF-assistedprocess[71,72].Table12.1givesanoverviewofsomenutraceuticalsprocessedwithSCFs.
SCFtechnologyistypicallyalow-temperaturetechniqueandisnormallycarriedout in completely inert atmospheres. Therefore, these techniques are especiallysuitableforthermo-andoxygen-sensitivesubstances.Greenteaandespeciallypoly-phenolextractsfromgreentealeavesarewidelyusedinnutraceuticalapplications.Polyphenols,substancesknowntostabilizeoilandfatproducts,areantioxidantsthathavebeendiscussedforcancerpreventionandfordentalcariesprevention,tonamejusttwopositiveeffects.Toisolatethesepolyphenolsfromgreentealeaves,awaterextractionismade.Afterfiltration,thisaqueousextractisdriedwithclassicalspraydrying techniques. Inspraydrying,high temperaturesarenecessary toevaporatewater.Inaddition,mostspraydryersworkwithheatedairandthereforethepoly-phenolsmaysufferfromthermalandoxidativestressduringprocessing.Resultantgreen teaproductsmaycontain lower concentrationsof antioxidants thancanbeachievedwithgentlerprocessing.
OnepossibilityforobtainingsolidgreenteaproductswithoutdegradationoftheantioxidantsistouseSCFtechnology.Therefore,aprocessthatcombinesthedryingstepofaqueousgreenteaextractswithparticleformationwasdesigned.TheflowschemeofthisprocessispresentedinFigure12.14.Thegreenteaextractusedforthedryingandpulverizationexperimentswasobtainedbyanextractionperformedat 60°C, by mixing 1 kg of extract in 10 kg of deionized water for 15 minutes.Thisextract isdosed toavesselbyahigh-pressurepumpthroughastaticmixer.Here,preheatedCO2isaddedunderelevatedpressures.Theresidencetimeinthemixerisextremelyshort(<1sec);therefore,itispossibletoraisethetemperatureabove100°C,evensometimesashighas180°C,withoutdegradationoftheproduct.Subsequently, themixture isdepressurized intoa spray towerand thusquenched
FIGure 12.13 Chocolate–kirschcomposite.
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382 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
immediatelytolowtemperatures.Finedropletswithlargesurfacesareformed.ThewaterisextractedalreadyinthestaticmixerathightemperaturesoristakenupbythedryCO2afterexpansionand,finally,solidgreenteaextractisprecipitatedinthespraytower.TheobtainedgreenteapowdersareshowninFigure12.15.Theevapo-ratedwatercanbewithdrawnwiththeexpandedCO2.
Table12.2showsthepolyphenolconcentrationsandwatercontentofthediffer-entproducts.Therawmaterial(tealeaves)hashadawatercontentof2.97weight%.Thegroundleaveswereextractedwithwater(leaves:water/1:10[g/g]),andthewaterextractwasdriedwithalow-temperaturevacuumevaporation(40°C)andwiththeSCFprocess.ThemainprocessparametersofthesupercriticaldryingprocessareshowninTable12.3.Theresidualwatercontentinthevacuumprocessafter6hourswasdeterminedto8.82weight%.InSCFprocessing,5.09weight%wasobtained.Thepolyphenolconcentrationafterwaterextractioncouldbeincreasedforallthreetypesofpolyphenolscomparedwiththerawmaterial.ThePGSSdryingstepshowsthesameorslightlyhigherconcentrationsofpolyphenolsthanthewaterextractdried
table 12.1nutraceuticals Processed with supercritical FluidsProcess substance reference
RESS Benzoicacid [76]
Ibuprofen [77,78]
Aspirin [79]
Caffeine [76]
Griseofulvin [38,80]
Lidocaine [81]
GAS Mefenamicacid [82]
Copper-Inomethacin [78,83]
Insulin [84–86]
Paracetamolandascorbicacid [47]
β-carotene [87]
SAS Amoxycilin [25,88]
Dextran,cholesterol [89–91]
Inulin [92]
Lecithin [93]
Organicpigments [94]
PGSS Felodipine [55]
Glucose [79]
Albuterolsulphate [79,95]
Cromolynsodium [79,95]
Glucoseoxidase [96]
CPF Flavourextracts [24,97]
Emulsions [98]
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Preparation and Processing of Micro- and Nano-Scale Materials 383
FIGure 12.15 PictureandSEMofpowderousgreenteaextract.
table 12.2Polyphenol Content at different Process steps
Process step
water Content (%) Polyphenols (g/100 g dry raw material)
residue (%)epicatechin
(eC) epigallocatechin-Gallate (eGCG)
epicatechin-Gallate (eCG)
Rawmaterial 2.97 0.97 3.92 1.41
Waterextract(1:10)vacuumdriedforanalyses
8.82 2.31 4.07 1.50
Supercriticalfluiddried
5.09 2.16 4.90 1.70
Gas
Green Tea
Gas + Solvent
Green Tea Solution
FIGure 12.14 Greenteaprocessingwithsupercriticalfluids.
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384 Supercritical Fluid Extraction of Nutraceuticals and Bioactive Compounds
invacuum.ThisdemonstratesthatduringprocessingwiththeSCF,nopolyphenols(exceptasmalldecreaseoftheepicatechinconcentration)weredegradedcomparedwithavacuumdryingprocess.Inaddition,SCF-assistedtechnologyallowsthepro-ductionofparticulatesystemswithlargesurfacesthatcanbeeasilyredissolvedinwater.Particlemorphologyandparticlesizecanbeadjustedbyvaryingtheprocessparameters[73–75].Invacuumdrying,abulkyproductisobtainedthathastobegrindedtogetfineparticles.
12.5 ConClusIons
In the last15 to20years,numerousprocessesforparticlegenerationusingSCFshavebeenproposed and applied for substances from the food, polymer, pharma-ceutical,lifescience,andnutraceuticalindustries.Themainfocusoftheseapplica-tionshasbeenon themicronizationofpure substances.The thermodynamicandfluid-dynamic properties of certain single-component model systems (e.g., PEGs,triglycerides,naphthalene)inthepresenceofcompressedgases,mostlyCO2,havebeenstudiedintensively.Thisfundamentalresearchhasledtoanimprovedunder-standingoftheprocessesforparticlegeneration.Asaresult,high-pressuretechnologybecomesmoreandmoreestablished in industry.Meanwhile,high-pressureplantswithcapacitiesofsomegramsperhour tosomehundredkilogramsperhourcanbedesignedandbuiltbyseveralspecializedplantconstructors.Costanalysisshowsthatthesetechniquescanbecompetitivetoclassicalmicronizationtechniques.SCFsnotonlyopennewpossibilitiesforprocessingsubstancesthataredifficulttohandle(e.g., substanceswith lowmeltingpoints, highviscosities, or sticky surfaces) butalsoallowgenerationofcompositeswithcustomizedproperties.Thesenewchancesmotivateresearchforimprovedunderstandingoftheprocessesandthedevelopmentofnewproducts.
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391
Indexα-Carotene, sources of, 281α-Linolenic acid, 57, 78α-Tocopherol, 113, 115β-Carotene Chrastil parameters of from fish oil, 160 fruit and vegetable oil extraction and, 86 overview of, 56, 196–197 physical properties of, 53 separation of isomers of, 196–198 sources of, 281β-Cryptoxanthin, 281
AAbsorption, 26Acetone, 34Acorns, 63, 70Activity coefficients, 13Adlay seed oil, 223–224Adsorption chromatographic separations and, 156 concentrated powder form process and, 377 extraction process and, 26, 327–328 liquid feed extraction and, 321 procyanidin extraction and, 233 solute separation and, 218Aerosol solvent extraction systems (ASES),
372–373Agglomeration, 371, 376–378, 380Aging, free radical theory of, 276–277Aglycons, 280Aguaribay, 245Ajwain, 355Algae, 192. See also MicroalgaeAlkaloids, 342Almonds, 52, 57, 59, 62, 63, 66, 69–71Amaranthus grain, 80, 81, 85Ammonia, 3Andreadoxa, 249Anethole, 261, 314Angelica sinensis, 228–230Anise, 261Annatto, 245Anthocyanadins, 280Anthocyanins, 342Anticaking agents, 343Antimicrobial activity, 285Antioxidants A. sinensis, L. chuanxiong hort and, 228 carotenoids as, 281, 284 conventional solvent extraction of, 292–293
determination of activity of, 285–286 effect of pressure and temperature on
extraction of, 289–292 lycopene as, 56 overview of, 275–276 overview of natural, 277–282 phenolics as, 280, 282–283 SC-CO2 extraction of, 286–292 spices and, 342 terpenoids as, 280–281, 283 tocols and, 59–60 types and regulation of, 276–277 vitamin E as, 281–282, 284Antisolvent extraction. See Supercritical
antisolvent extractionAntisolvent processes, 369, 372–375.
See also Specific processesApricots, 73, 79Aqueous alkaline extraction, 346Arachidonic acid, 57Arnica, 249Aroeira, 249Aromatic compounds, 11, 245–247Arruda de serra, 249Artemisia, 249Artemisinin, 312Arteriosclerosis, 142Arthritis, 142–143Arthrospira (Spirulina) spp., 205–209ASES. See Aerosol solvent extraction systemsAstaxanthin, 193, 195, 198–201ATBC study, 56Atherosclerosis, 56, 60, 228Atomization, 33–34Avocado, 249Ayurveda, 338
BBaccharia, 249Bacuri, 244, 248Bamboo piper, 245Basil, 245, 249Batch reactors, 20Bergamot peel oil, 328BHA. See Butylated hydroxyanisoleBHC. See HexachlorocyclohexanesBHT. See Butylated hydroxytolueneBinaries behavior, 15–16Binding, 377Binodal curves, 19
7089_Index.indd 391 10/8/07 11:48:22 AM
392 SupercriticalFluidExtractionofNutraceuticalsandBioactiveCompounds
Bioethanol, 15Bioflavonoids, 342Biofuels, 126, 192Black mustard, 345Black oreo, 147, 148Black pepper bioactive compounds from, 244, 245 biologically active constituents of, 345 extraction of bioactive compounds from, 359 solubility of oils in SC-CO2 and, 363, 364Black wattle, 249Blends, 346Blowout discs, 41Boldo, 249Borage, 73, 79Botrycoccus braunii, 191–193Brazil, 244Brazilian ginseng, 249, 261Breakage, 324Broken-intact cells, 324, 325–326Buriti fruit, 87, 248Bushy lippia, 245Butylated hydroxyanisole (BHA), 276, 277Butylated hydroxytoluene (BHT), 276, 277
CCacao, 249Caking, 343Calcium stearate, 343Campesterol, 58Camphor, 313Cancers adlay seed oil and, 223–224 carotenoids and, 284 lycopene and, 56–57 omega-3 fatty acids and, 142–144 phenolics and, 282, 283 sitosterol and, 59 squalene and, 58 terpenoids and, 283 tocols and, 60 Vitamin A and, 146 vitamin E and, 284Candida antarctica lipase, 158Canthaxanthin, 193, 195, 200, 281Cap automation mechanisms, 29–30Caprylic acid methyl ester, 78Capsaicin, 252, 342, 356–357Capsules, 52Caraway, 360–361Cardamon, 344, 359–360, 361Cardiovascular disease carotenoids and, 284 phenolics and, 282 polyunsaturated fatty acids and, 57 terpenoids and, 283
tocols and, 60 vitamin E and, 284CARET study, 56Carnahan-Starling equation, 7Carotenes, 56, 193. See also β-CaroteneCarotenoids algae and, 193–195, 198–201, 206–209 as antioxidants, 280, 281 biological properties of, 284 cosolvents and, 89 fruit and vegetable oil extraction and, 86 rice germ extraction and, 84 solubility of in SC-CO2, 290 in specialty oils, 56–57 spices and, 342Carrier materials, extraction process and, 26Carrots, 87, 90Caryophyllene, 313, 316Cascading extraction vessels, 29Cashews, 250Cassia, 344Catalysts, 105–106Catechins, 280Celery seed extraction, 354–356Cell cycle, 284Cellular location, 324Cellulosic structure, 252–254Cereal oils, 80–84Chamazulene, 314Chamomile bioactive compounds from, 245 composition of essential oil from, 315–316 essential oil extraction from, 314 overall extraction curve for, 259Charge time, 29–30Cherry, 58, 72, 73, 79, 80Chile, 244Chilean hops, 250Chili extracts, 356–357Chili peppers, 346Chlorella vulgaris, 192, 193–196, 202Chocolate-kirsch composites, 380, 381Cholesterol, 58, 59, 145, 338Chrastil correlation, 159, 160, 363–364Chromatography gas chromatograph with electrical conductivity
detector analysis, 233–234 high-speed countercurrent, 224 overview of, 156 supercritical fluid, 36–37, 173–175Chromobacterium viscosum lipase, 157Cinnamon, 341, 344, 355, 362Citronella, 245Cleaning-in-place, 35Cloudberries, 87Clove bud oils, 225–228, 244, 245, 314–316
7089_Index.indd 392 10/8/07 11:48:22 AM
Index 393
Cloves biologically active constituents of, 344 cost of manufacturing and, 261 extraction of by various methods, 353 therapeutic benefits of, 341 yields and concentrations of active
ingredients from, 355CMC-Na, 222–223CO2 (SC) advantages of processing with, 52, 158, 276,
338 cereal oil extraction in, 80–84 cosolvents, TCM processing and, 220–221,
222 fruit and vegetable oil extraction in, 84–90 liquid-liquid immiscibility and, 11 nut oil extraction in, 62–72 processing of TCM and, 220 seed oil extraction in, 72–80 solvent properties of, 3 specialty oil extraction in, 61–62 TCM processing and, 217–219Coca, 250Cod, 143, 153Codex Alimentarius, 277Coffee, 26, 246Cold processing, specialty oils and, 52Collection, high-pressure spray processes and, 370Color algae and, 193 fruit and vegetable oil extraction and, 90 nut oil extraction and, 71 paprika extraction and, 357 seed oil extraction and, 78, 80 spices and, 343Composites, 376–380Compounds, 28–30, 31–34Concentrated powder form (CPF) process, 370,
376–378, 382Concretes, 310, 312, 316–318Conjugated double bonds, 56Constant extraction rate periods, 257–260Control systems, 41–42Conventional solvent extraction of antioxidants, 292–293 cereal oil extraction and, 84 fruit and vegetable oil extraction and, 90 nut oil extraction and, 72 seed oil extraction and, 80 specialty oils and, 52Copaiba, 250Copper Reduction Assay (CUPRAC), 286Coriander, 246, 344, 355, 360–361Coronaridine, 261Cosolvents A. sinensis, L. chuanxiong hort and, 229–230 algal extraction and, 200–201
BHC extraction from radix ginseng and, 235–236
cereal oil extraction and, 83 essential oil extraction and, 308 fruit and vegetable oil extraction and, 86, 89 heat treatment for removal of, 91 nut oil extraction and, 69, 71 polarity and, 6, 349 processing of TCM and, 220–221, 222 procyanidin extraction and, 231–232 SC-CO2 extraction and, 166–168 seed oil extraction and, 77–78 solubility and, 123 vegetable oils as, 89 vitamin E and, 352Cost estimates cellulosic structure and, 252–254 industrial process implementation and, 44–48 for PGSS, 375–376 for selected Latin American plants, 260–261 selection of parameters for, 254–260 SFE for Latin American plants and, 243,
254–262 spice oil extraction and, 350–351Cost of manufacturing (COM). See Cost estimatesCountercurrent extraction columns, 18, 31–32,
36, 328Couplings. See Drive couplingsCPF (concentrated powder form) process, 370,
376–378, 382Critical curves, 115Critical point, 2Critical pressure, 115Critical properties, 3, 5, 30Crossover effects, 30, 348Croton, 246Cryoprotection, 58Crystallization, 151–152, 152–156, 372–373Cumin, 341, 344, 353, 355CUPRAC. See Copper Reduction AssayCupuassu, 248Curcuma longa, 365Curcumin, 343Cuticular waxes, 310, 315–316
DDecaffeination, 26Degradation fish oil extraction and, 148–149, 151 green tea leaves and, 381 hydrodistillation and, 309 lipid oxidation and, 276–277 molecular distillation of tocopherols and, 105Degree of extraction, 71Degree of saturation, 152–156Dehydration, 11–15
7089_Index.indd 393 10/8/07 11:48:23 AM
394 SupercriticalFluidExtractionofNutraceuticalsandBioactiveCompounds
Dense-gas fractionation, 17–18Density fog phenomenon and, 126 nut oil extraction and, 71 overview of, 4 phase equilibrium and, 2–3 solvent power and, 1Deodorizer distillate (DOD), 104, 108.
See also TocopherolsDepressurization rate, 27–29, 67–68Deterpenation, 320DHA. See Docosahexaenoic acidDiacyl glycerol ethers, 144–146, 176–178Diallyl sulfides, 361–362Diffusion coefficients, 1, 4Diffusion-controlled rate periods, 257–260Dilophus ligulatus, 192Diphtheria, 338Discharge time, 29–30Dispersal, 369Dissolution, 369Distillation celery seed essential oil and, 356 essential oils and, 309 of fish oils, 149–151 of menhaden oil, 150 problems with tocopherol concentration using,
104–105 SFE and for TCM processing, 223–224 spice constituent extraction and, 353 spice extraction and, 346–347Distribution coefficients, 113, 115Diterpenes, 341Docosahexaenoic acid (DHA), 57, 142–144,
168–169Docosapentanoic acid (DPA), 57DOD. See deodorizer distillate.
See also TocopherolsDPA. See Docosapentanoic acidDPPH radicals, 286Drive couplings, 32, 33DSS, 222–223Dunaliella spp., 191, 192, 196–198Dynamic axial columns, 36–37
EEbers Papyrus, 338Ecdysterone, 261Echium, 73, 79Economics. See Cost estimatesEDTA. See Ethylenediamenetetraacetic acidEicosanoids, 57Eicosapentanoic acid (EPA) algae and, 201–202, 202–205 extraction of from fish oils, 142–144 overview of, 57
SC-CO2 extraction of, 168–169 structure of, 144Emulsions, 346Encapsulation, 380Entrainment, 105, 201, 209Enzymatic transformation, 156–158, 175–176Ephedrine, 222Equilibration time, 66, 76Equilibria, 151–152Equilibrium calculations, 8–9Equipment, 34–35, 219–220Erva baleeira, 246Essential oils antisolvent extraction and, 322–323 celery seed extraction and, 356 examples of, 320–322 extraction of from flowers, 314, 316–318 extraction of from leaves, 312–313 extraction of from seeds, 314 flower concretes fractionation and, 316–318 ginger extraction and, 358 liquid feed extraction and, 318–319 mathematical modeling of extraction of,
324–328 operating parameter selection for, 319–340 overview of, 305–307, 343, 346 solids processing and, 307–312 sources of, 311 spices and, 341, 342Esterification, 105–106, 156–157Estragole, 314Ethane, 3, 11Ethanol. See also Cosolvents cereal oil extraction and, 83 as cosolvent, 91 fruit and vegetable oil extraction and, 89 nut oil extraction and, 69 processing of TCM and, 220–221 seed oil extraction and, 77–78 squalene extraction and, 168 urea inclusion complexation and, 155–156Ethylenediamenetetraacetic acid (EDTA), 277Eucalyptus, 246Eugenia carophyllata, 225–228Eugenol, 225–227, 316Evening primrose, 73, 79Expansion. See Rapid expansion of supercritical
solution (RESS) processExplosives, 373Extract materials, 26Extraction. See also Conventional solvent
extraction of compounds from liquid feed, 31–34 of compounds from solid matrix, 28–30 control systems for, 41–42 equipment design and, 34–35 heat exchangers for, 38–39
7089_Index.indd 394 10/8/07 11:48:23 AM
Index 395
industrial process implementation for, 42–48 overview of, 48–49 of oxychemicals, 11–15 piping, valves and, 39–41 process development and, 218 process overview, 26–28 processing parameters for solids extraction,
30–31, 308–309 pumps and compressors for, 37–38 from vegetable matrices, 18–19 vessels for, 34, 35–37Extraction time, 68–69, 77, 83Extraction vessels, 29, 34, 35–37
FFalling extraction rate periods, 257–260FAME. See Fatty acid methyl estersFanshensu, 225Fatty acid methyl esters (FAME), 105–106,
106–107, 126–136Fatty acids cereal oil extraction and, 83–84, 85 Chrastil parameters of from fish oil, 160 of fish oils, 143 fruit and vegetable oil extraction and, 89–90 nut oil extraction and, 70 seed oil extraction and, 78, 79Feed materials, 26Fenchone, 314Fennel, 246, 261, 314, 360–361Fermenters, algae and, 191Ferric Reducing Antioxidant Power (FRAP), 286Ferulic acid, 228–230Fish oils chromatographic separations of, 156 distillation of, 149–151 enzymatic transformation of, 156–158 low-temperature crystallization of, 151–152 omega-3 fatty acids of, 142–144 overview of, 141–142 overview of separation and fractionation
technologies for, 147–149 phase equilibria of in SC-CO2, 158–168 polyunsaturated fatty acids and, 57, 168–176 squalene and diacyl glycerol ethers of,
144–146, 176–178 urea crystallization of, 152–156 vitamin A and, 146, 178–181 wax esters of, 146–147, 181Fixed costs, 260–261Flammability, 18–19Flavanones, 280Flavones, 280, 313Flavonoids, 280Flavonols, 280Flax, 74, 79
Flow control valving, 39–40Flow rates and directions cereal oil extraction and, 83 fruit and vegetable oil extraction and, 86, 89 nut oil extraction and, 68 seed oil extraction and, 77 solids extraction processing parameters and,
31Flowers, 251, 313, 314, 316–318Fog phenomenon, 126Fractional extraction processes, 26–27, 150–151Fractionation of essential oil extracts, 306, 310 FAME removal during tocopherol
concentration and, 126–136 of oils, 17–18 of rose concrete, 317–318 spice extraction and, 354Fragrances, 316–318, 358, 359, 361FRAP. See Ferric Reducing Antioxidant PowerFree fatty acid esters, 163Free fatty acids, 105–106Free radicals, 276–277, 285–286French paradox, 282Fruit oils, 84–90Fugacity, 4–6Fugacity coefficients, 6
GGallates, 276, 277–278Gamma-linoleic acid (GLA), 57 (all-cis-6,9,12-octadecatrienoic acid), 205–206Garlic, 338–340, 344, 361–362GAS (gas antisolvent) process, 15, 372–374, 382Gas Chromatograph with Electrical Conductivity
Detector (GC-ECD) analysis, 233–234Gas recycling, 370Gas salting out effect, 4Gas saturated liquids (PGSS), 369, 373–375, 382Gases, physical properties of, 4Gas-liquid alternating circulation system, 107–109General expenses, 260–261Genetic engineering, 191Ginger bioactive compounds from, 244, 246, 344 cost of manufacturing and, 261 extraction of bioactive compounds from, 358 extraction of by various methods, 353 overall extraction curve for, 259, 260 return on investment and, 47, 48 therapeutic benefits of, 341 yields and concentrations of active ingredients
from, 355Ginseng, 233–236, 249, 261GLA. See Gamma-linoleic acidGlycerides, 105–106
7089_Index.indd 395 10/8/07 11:48:23 AM
396 SupercriticalFluidExtractionofNutraceuticalsandBioactiveCompounds
Glycosides, 342Good Manufacturing Practice (GMP)
compliance, 47–48, 375Grapefruit, 250Grapes, 74, 79, 230–233, 250Green pepper basil, 246Green tea leaves, 381–384Green-lipped mussel oil, 181Grinding, 76, 86, 368Group contribution equation of state (GC-EOS)
model, 7Guaco, 250Guarana, 250
HHaematococus pluvialis, 192, 198–201Halibut, 143HAT. See Hydrogen atom transferHazard and Operatability (HAZOP) studies, 38Hazelnuts, 63, 66, 69, 70HD. See HydrodistillationHeat exchangers, 38–39Heat treatment, 91Helmholtz residual energy, 7Hemicellulose, 69Herring, 143Hexachlorocyclohexanes (BHC), 233–236Hexane, 80, 347, 353, 355Hibiscus, 74, 79High-critical temperature (high Tc) fluids, 3High-pressure spray processes, 368–376, 376–380,
384High-speed countercurrent chromatography
(HSCCC), 224Hiprose, 74, 87Hoki liver oil, 172Horsetail (giant), 246HSCCC. See High-speed countercurrent
chromatographyHybrid hibiscus, 74, 79Hydrocarbons, 55, 160, 192–193Hydrodistillation (HD), 309, 346–347, 356Hydrogen atom transfer (HAT), 286Hydrolysis, 151, 156–157, 309Hyperforin, 312Hypertension, 228, 338Hypnea charoides, 192, 201–202
IImpregnation, 377Industrial process implementation, 42–48Inflammation, 57, 282Interactions, 324Interfacial tension, 4
Isochrisis galbana, 192Isoflavones, 280Isofugacity criterion, 4–5Isolation valving, 39–40Isomerization, 151Isoprenoids. See TerpenoidsIsopropanol, 15
JJackfruit, 250Jalapeno peppers, 250Jojoba, 248, 341
KKanglaite Injection, 223–224Khoa, 246Kinetics, 257–258Kirsch, 380, 381Koenen and Gaube diagrams, 12
LLabor, 47Latin American plants cost estimates and, 243, 254–262 examples of SFE from, 244–252 overview of SFE of bioactive compounds
from, 243–244 SFE process for, 252–254Lavender, 314Leaves, 312–313Lecithin, 33–34Lemon verbena, 247Lemongrass, 247Licorice, 346Limonene, 314, 316, 320, 327Linalool, 314, 321, 327, 361Lingusticum chuanxiong hort, 228–230Linoleic acid, 55, 57Linolenic acid, 69, 78, 90, 201–202, 230–233Lipases, 156–158Lipids, 18–19, 190, 248, 276–277, 281–282Lippia sidoides, 247Liquid feeds, 31–34, 306Liquid-filled composites, 378–380Liquid-liquid equilibrium, 7Liquid-liquid immiscibility, 9–11Liquids, 4Lobenzarit preparation, 16Low-critical temperature (low Tc) fluids, 3Low-temperature crystallization, 151–152Lunaria, 79Lutein, 53, 84, 86, 200
7089_Index.indd 396 10/8/07 11:48:24 AM
Index 397
Lycopene, 53, 56–57, 86, 89, 281Lyprinol, 181
MMace, 346Macela, 247Mackerel, 143Macroalgae, 190Macroporous resin adsorption technology, 233Magnesium stearate, 343Mangos, 250Manufacturing costs. See Cost estimatesMarigolds, 247, 250Marine macroalgae, 190Mass transfer models, 255, 325–327Mastranto, 247Matricine, 314MD extraction, 224Mechanical mixing, 32–33ME-DOD. See Methyl esterified DODMelting, 369Menhaden, 143, 150Mesityl oxide, 34Methanol, 3, 155–156Methanolysis, 105–106Methyl esterified DOD (ME-DOD).
See also Tocopherols composition of, 130 FAME removal from, 126–129 phase behavior of, 106–124, 124–126 pressure and, 131–134 pretreatment and, 105–106, 129–131Methyl oleate distribution coefficients of, 113, 115 FAME removal and, 106–107, 110–111 gas-liquid interface and, 117 separation factor of in SC-CO2 fractionation,
122–123Microalgae Botrycoccus braunii, 191–193 Chlorella vulgaris, 193–196 Dunaliella spp., 196–198 Haematococus pluvialis, 198–201 Hypnea charoides, 201–202 Nannochloropsis spp., 202–205 overview of, 189–191, 209, 244 Spirulina spp., 205–209Microemulsion. See SurfactantsMicronization, 13, 15–17. See also Supercritical
antisolvent micronizationMicro-particles, 367–368. See also High-pressure
spray processesMigration rates, 156Milk thistle, 74Minerals, 69Miscibility, 9–11, 115
Mixtures, 43Modeling, 7, 255, 324–327Modified Huron-Vidal 2 (HHV2) model, 7Modifiers, 290–291. See also CosolventsMoisture content cereal oil extraction yield and, 80 fruit and vegetable oil extraction and, 86 nut oil extraction and, 66 seed oil extraction and, 76 solids extraction processing parameters and, 31 spice extraction and, 350Molecular distillation, 104–105, 150Mongolia mushrooms, 225Monoterpenes, 341, 355Morphology, 31Mortierella sp., 192mRNA, 284Mullet, 143Multicomponent fluids, 8Multiplunger pumps, 37–38Multistage extraction, 308Munch, 72, 74, 79Myristicin, 359
NNannochloropsis spp., 192, 202–205Nano-particles, 367–368, 371. See also
High-pressure spray processesNanosuspensions, 371Natural products. See Traditional Chinese
medicines and natural productsNear-critical region, 2, 7, 8–9Nebuilizing, 32Neem, 79n-Hexane, 3, 109, 119, 168, 291Nonclassical supercritical effects, 13–14Non-Random Two Liquids (NRTL) model, 7Nut oils, 62–72Nutmeg, 346, 358–359Nutraceuticals, 342, 380–384
OOats, 81, 85Ochronomas danica, 192Odor, 208. See also FragrancesOEC. See Overall extraction curvesOils, 17–19Oleic acids, 69, 78Oleoresins cost of manufacturing of, 261 defined, 244, 342 ginger extraction and, 358 from Latin American plants, 245–247, 252 liquid solvent extraction and, 310
7089_Index.indd 397 10/8/07 11:48:24 AM
398 SupercriticalFluidExtractionofNutraceuticalsandBioactiveCompounds
overview of, 343, 346 sources of, 311 vanilla extraction and, 359Olives, 87, 248Omega-3 fatty acids, 142–144Onions, 341Operating costs. See Cost estimatesOptimization, 9ORAC. See Oxygen Radical Absorbance CapacityOrange (sweet), 247Orange roughy, 147, 148Oregano, 247, 327Organic acids, 277Organochlorine pesticide, 233–236Osteoarthritis, 60Overall extraction curves (OEC), 256, 257–260Oxidation. See also Antioxidants CO2 processing and, 52 distillation of fish oils and, 151 fish oil extraction and, 149 nut oils and, 62, 72 seed oil extraction and, 80 squalene and, 145Oxychemicals, 11–15Oxygen Radical Absorbance Capacity (ORAC),
286Oxygenated compounds, 341
PPalmarosa, 247Palmitic acid, 69, 90Palms, 248Paprika powder, 248, 357–358Paraffins, 11, 310Paragual, 244Particle shape, 37, 324–325, 368, 379Particle size A. sinensis, L. chuanxiong hort and, 228–229 cereal oil extraction yield and, 80 clove bud oils and, 226 fruit and vegetable oil extraction and, 86 nut oil extraction and, 62, 66 seed oil extraction and, 76 solids extraction processing parameters and, 31Particulates, 367–368, 368–376Passion flower, 251Passion fruit, 248PCA. See Precipitation with compressed
antisolventPeanuts, 64, 66Pecans, 64, 66, 69, 70Pectins, 69Peel oils, 320PEG, 380Pejibaye, 248Pepper, 355
Percolation method, 230Peroxidation, 281–282Pesticides, 233–236PGSS (gas saturated liquids), 369, 373–375, 382Phaffia rodozyma, 192Phase equilibria chromatographic separations and, 156 of fish oils in SC-CO2, 158–168 for Latin American bioactive compounds, 253 of methyl oleate-DOD, 124–126 multiple, 6–8 overview of, 9–11 phase equilibrium analyzers and, 43 solid solubilities and, 4–6 tocopherol concentration and, 110–113,
117–119Phase equilibrium analyzers, 43Phase equilibrium diagrams, 2Phase equilibrium engineering, 8–11, 20Phenolics as antioxidants, 280 biological properties of, 282–283 effect of pressure and temperature on yield of,
291 as primary antioxidants, 277 solubility of in SC-CO2, 290Phenylpropanoids, 342Photobioreactors, 191Photosynthetic capacity, 190–191Phthalides, 355Physical properties, overview of, 4Phytochemicals, defined, 342Phytoplankton, 189Phytyl chains, 59Pigments, 56, 193, 202–205, 281, 360Pilayella littorallis, 192Pink trumpet tree, 251Piperine, 359Piping, 39–41Piprioca, 247Pistachios, 64, 69Pitanga, 251Plant polyphenols, 230–233Polarity, 6, 217, 229, 349Polyethylene Terephthalate (PET) films, 252Polymerization, 151Polyphenols, 381–384Polysaccharides, 225Polyunsaturated fatty acids algae and, 201–202 extraction of from fish oils, 168–176 of fish oils, 142 in specialty oils, 57–58 vitamin E and, 60Potassium stearate, 343Pravastatin, 58
7089_Index.indd 398 10/8/07 11:48:24 AM
Index 399
Precipitation with compressed antisolvent (PCA), 372–373
Preservation, antioxidants and, 276Pressure. See also Depressurization rate CO2 extraction and, 218, 289–290 FAME removal during tocopherol
concentration and, 131–134 fruit and vegetable oil extraction and, 86 nut oil extraction and, 66–68 phase equilibrium and, 2 seed oil extraction and, 76–77 selection of cost estimate parameters and,
254–257 solvent recycle and, 45, 46 tocopherol SC-CO2 concentration and,
117–119Pressure reduction, 218Pressure vs. temperature diagrams, overview of, 8Pressured fractional distillation, 223–224Pretreatments, 105–106, 116–118, 129Preventative antioxidants, 277Primary antioxidants, 277Primrose, 73, 79Process design extraction from vegetable matrices and, 18–19 fractionation of oils and, 17–18 overview of, 26–28 oxychemical extraction, dehydration and,
11–15 particle micronization and, 15–17 supercritical reactions and, 19–20Processing parameters, 30–31Procyanidins, 230–233Propane, 3, 13–14, 18–19, 359–360Prostaglandins, 57Protocatechualdehyde, 225Provitamin A, 342. See also β-CarotenePseudomonas sp., 157, 158Psoralen, 224Pumpkins, 75, 79Pumps and compressors, 37–38Pupunha, 248
QQuinones, 277
RRadix ginseng, 233–236Raffinate, 31–32Rapeseed, 248Rapid expansion of supercritical solution (RESS)
process cost studies of, 375–376 nutraceuticals produced by, 382
overview of, 369, 370–371 solubility and, 15 spraying of gas saturated liquids and, 373–375Recycling, 9, 44–47, 174, 370Red chili extraction, 346, 356–357Redundancy, control systems and, 41Regulation of antioxidants, 277–278Relative volatility, 18Residence time, 31Residual oils, 48Resins, 342. See also OleoresinsResistance temperature detector (RTD) sensors, 41Respiratory tract, 338RESS. See Rapid expansion of supercritical
solution processRetinol, 146Retrograde behavior, 5Revenue estimates, 47Rice bran, 81–82, 84, 85, 248Roasting, 62Rose concrete, 317Rose hips, 75, 79, 248Rosemary, 247, 261
SSAE. See Supercritical antisolvent extractionSafety, 41, 233–236Saffron, 244, 346Sage, 312–313Salmon, 143Saponification values, 107Saponins, 342Saprolegnia parasitica, 192, 195–196Sardine oil, 171SAS. See Supercritical antisolvent micronizationSaturation degree of, 152–156SC-CO2. See CO2-SCScenedesmus obliquus, 191, 192SDS, 222–223Sea buckthorn, 75Sebum, 144Secondary antioxidants, 277SEDS. See Solution-enhanced dispersion by
supercritical fluidsSeed oils celery seed extraction and, 354–356 characteristics of products extracted from,
78–80 cosolvents and, 77–78 essential oil extraction and, 313, 314 extraction time and, 77 flow rate and direction and, 77 modeling extraction of, 327 moisture, equilibration time and, 76 overview of, 72–76 particle size and, 76
7089_Index.indd 399 10/8/07 11:48:25 AM
400 SupercriticalFluidExtractionofNutraceuticalsandBioactiveCompounds
temperature, pressure and, 76–77Selectivity, 3–4, 26–27Sensors, 41Sensory properties, 280, 352–353Separability, 3Separation, 26, 218Sequential depressurization, 27–29Sesame, 75, 79Sesquiterpenes, 314, 321, 341SET. See Single electron transferSFC. See Supercritical fluid chromatographySharks, 145Shea nut oil, 72Shell-and-tube heat exchangers, 39, 40Shogaols, 358Short-path distillation, 104–105, 150SHS, 222–223Sidda, 338Silanol, 321Silybum marianum, 72, 77Single electron transfer (SET), 286Sitosterol, 58Sitosterolemia, 58–59Skeletonema costatum, 192Small spined oreo, 147, 148Solid matrices, 28–30Solids, 30–31Solubility of β-carotene isomers, 197–198 Chrastil correlation and, 159, 160 CO2 extraction and, 61, 289–290 cosolvents and, 123 crystallization separations and, 151–152 of fatty acids from fish oils, 159–162 hydrodistillation and, 309–310 for Latin American bioactive compounds,
253, 256–257 of methyl oleate and α-tocopherol in SC-CO2,
113, 114 nut oil extraction and, 67 phase equilibrium analyzers and, 43 solids extraction processing parameters and, 30 spice oils and, 362–364, 365Solution-enhanced dispersion by supercritical
fluids (SEDS), 372–373Solvent loading, 29Solvent power, 3, 15, 26Solvent recycle, 9, 44–47, 174Solvent-feed ratios, 30–31Solvents. See also Cosolvents chlorinated, 347 crystallization and, 367 density-dependent nature of, 1 high-pressure spray processes and, 369 reaction with piping surfaces and, 39Span-80, 223
Specialty oils. See also Specific oils bioactives in, 52–55 carotenoids in, 56–57 extraction of, 61–62 overview of, 52, 90–91 polyunsaturated fatty acids in, 57–58 squalene in, 58 sterols in, 58–59 tocols in, 59–61Sperm whales, 147, 148Spice extracts, 343, 346, 348Spices antioxidants from, 277 beneficial aspects of, 339–341 bioactive compounds from, 341–343 black pepper extraction and, 359 cardamom extraction and, 359–360 celery seed extraction and, 354–356 cinnamon extraction and, 362 commercial SCFE process for, 349–351 conventional extraction of, 346–347, 351–354 defined, 338 fennel, caraway, coriander extraction and,
360–361 garlic extraction and, 361–362 ginger extraction and, 358 importance of, 338–339 nutmeg extraction and, 358–359 overview of, 337–338, 364–365 paprika extraction and, 357–358 red chili extraction and, 356–357 saleable products from, 343–346 SC-CO2 extraction of, 347–349, 351–354 solubility of oils in SC-CO2 and, 362–364 specific therapeutic benefits of, 340–341 vanilla extraction and, 359Spiny Dogfish, 143Spirulina maxima, 205–207, 244Spirulina platensis, 207–209Spirulina sp., 192Splinefitting, 258–260Spray agglomeration, 376–378Squalene amaranth oil and, 83, 84 from fish oils, 144–146, 176–178 oil fractionation and, 17 physical properties of, 55 SC-CO2 extraction of, 165–168, 176–178 in specialty oils, 58Standardization, 285Steam distillation, 346, 352–353Sterols, 54–55, 58–59, 342Steroptens, 317Stevia, 244, 247, 251Stigmasterol, 58Stripping, 18Structure, 324
7089_Index.indd 400 10/8/07 11:48:25 AM
Index 401
Sucrose, 244Sugars, 280Supercritical reactions, 19–20Supercritical antisolvent extraction (SAE),
306–307, 322–323Supercritical antisolvent micronization (SAS),
306–307, 322, 372–373, 375–376, 382Supercritical fluid antisolvent. See Supercritical
antisolvent micronizationSupercritical fluid chromatography (SFC), 36–37,
173–175Supercritical fluids, defined, 217, 338Supercritical region, 2Surface tension, 1Surfactants, 221–223Symposia, 219
TTabernaemontana, 251, 261TAG form of polyunsaturated fatty acids, 157Tannins, 342Tanshinone, 225TBHQ. See Tertiary butyl hydroquinoneTEAC. See Trolox equivalent antioxidant capacityTecanalysis software, 262Temperature clove bud oils and, 227 CO2 extraction and, 218, 289–290 distillation of fish oils and, 151 enzymatic transformation of fatty acids and,
156–157 fruit and vegetable oil extraction and, 86 nut oil extraction and, 66–68 phase equilibrium and, 2 seed oil extraction and, 76–77 selection of cost estimate parameters and,
254–257 tocopherol SC-CO2 concentration and, 117–119Terpenes, 280, 320–321, 342Terpenoids (isoprenoids), 144, 280–281, 283, 290,
291Tertiary butyl hydroquinone (TBHQ), 276Texture, 71, 290Thar Technologies, 32, 33Thermal conductivities, 4Thermowell isolation, 41Thrombosis, 142Thyme, 346Tobacco, 323Tocols, 53–54, 59–61Tocopherols binary phase equilibria of, 107–113 conventional extraction and, 72 distribution coefficients of, 113–118 effect of pressure and temperature on yield of,
293
equilibrium lines for, 123–124 fundamental research on concentration of,
106–107 molecular distillation and, 104–105 molecular structure of, 104 nut oil extraction and, 69 oil fractionation and, 17 overview of, 104, 136–138 phase behavior of ME-DOD system and,
124–126 pretreatment before concentration of, 105–106 propane solvents and, 72 regulation of, 277 rice bran oil and, 84 separation factor of with methyl oleate,
122–123 separation of with SC-CO2 fractionation,
126–136 solubilities of, 113, 290 ternary phase equilibria of, 118–122 wheat germ extraction and, 83Tocotrienols, 284Tomatoes, 75, 79, 88, 90Torulaspora delbrueckii, 192Total Peroxyl Radical-Trapping Antioxidant
Parameter (TRAP), 286Traditional Chinese medicines and
natural products of edible and medicinal ingredients from
grape seeds, 230–233 equipment made in China for, 219–220 essential oil from clove buds and, 225–228 of medical ingredients from A. sinensis and
L.chuanxiong hort and, 228–230 of organochloride pesticide from ginseng,
233–236 overview of, 216–217, 236–237 overview of active compounds extracted from,
221, 222 SFE and enhanced separation methods and,
223–224 SFE and ultrasound-enhanced extraction and,
223 SFE with CO2 in presence of solvent and,
220–221 SFE with CO2 in presence of surfactant and,
221–223 SFE with pure supercritical CO2 and, 220 supercritical fluid processing of, 217–219 use of combinations of extraction methods
and, 224–225Traditional processing methods.
See Conventional solvent extractionTRAP. See Total Peroxyl Radical-Trapping
Antioxidant ParameterTree tea oil, 341Triacylglycerols, 52, 160
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402 SupercriticalFluidExtractionofNutraceuticalsandBioactiveCompounds
Triglycerides, 69Trilinolein, 89–90Triple point, 2Trolox Equivalent Antioxidant Capacity (TEAC),
286Trout, 143Tuberose volatile oil, 318Tucuman, 248Tuna oil, 172, 175Turbines, 46–47Turmeric bioactive compounds from, 244, 251, 343, 345 solubility of essential oils from, 364 therapeutic benefits of, 341Tween-80, 223Type V phase behavior, 9–10
UUcuuba, 248Ultrasound, 223Ultraviolet radiation, 145Unani, 338Urea crystallization, 152–156Urea inclusion complexation, 155–156Urea-fatty acid ratio, 152–154
VValves, 39–41Vanilla, 346, 359, 360Vanillin, 359Vapor pressure, 149–150Vapor-liquid equilibrium (VLE), 7Vegetable matrices, 18–19Vegetable oils, 17, 84–90Vessels, 29, 34, 35–37
Vetivergrass, 247Vinca, 251Viscosity, 1, 4, 32, 134–136Vitamin A, 146, 160, 178–181Vitamin E, 277, 281–282, 284.
See also TocopherolsVLE. See Vapor-liquid equilibriumVoacangine, 261Volatile oils cost of manufacturing of, 261 from Latin American plants, 245–247, 252 liquid solvent extraction and, 310 sources of, 311–312 spices and, 341Volatility, 18, 67Volume, 2, 71
WW3 fatty acids, 201–202, 223Walnuts, 65, 70Water, 3, 13–14Wax ester oils, 146–147, 181Waxes, 146–147, 181, 310, 315–316Wertheim’s statistical association fluid theory, 7Wheat germ, 82, 85Wheat plumule, 82Workflows, 42–43
XXanthophylls, 56, 193Xylopia aromatica, 247
ZZeaxanthin, 84, 281
7089_Index.indd 402 10/8/07 11:48:26 AM
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