Including ASE Accelerated Solvent Extraction€¦ · Comparison of ASE to the Current Official Method ... AOAC Method 997.08: Determination of Fructans in Food Products ANALYTICA-EBC

BETTER SOLUTIONSFOR FOOD AND

BEVERAGE ANALYSISTHIRD EDITION

Including ASE® Accelerated Solvent Extraction

LPN 0666-05

CarboPac and MicroBead are trademarks, and ASE, AutoSuppression, IonPac,and OmniPac are registered trademarks of Dionex Corporation.

Hydromatrix is a trademark of Varian Corporation.

All rights reserved. No part of this publication may be reproduced, stored in a retrievalsystem, or transmitted, in any form or by any means, electronic, mechanical, photocopying,recording, or otherwise, without the prior written permission of the publisher.

This book is published by:

Dionex CorporationP.O. Box 3603Sunnyvale, CA 94088-3603

Copyright © 2003 by Dionex Corporation.Printed in the United States of America.

LPN 0666-05 PDF 8/03

Second Edition printed January 1997.Revised and reprinted May 2000.Third Edition, August 2003.

INTRODUCTION ........................................................................................................... 1

OFFICIALLY APPROVED IC METHODS ....................................................................... 2

CHAPTER ONE: INORGANIC ANIONS AND CATIONS .............................................. 5Municipal Drinking Water ....................................................................................................................... 6Bromate in Drinking Water and Baked Goods ...................................................................................... 7Cations in Mineral Water and Drinking Water ...................................................................................... 7Cations in Soft Drinks and Wine ............................................................................................................. 7Transition Metals in Food ......................................................................................................................... 8Sulfite in Dried Apricot ............................................................................................................................ 8Iodide in Whole Milk ................................................................................................................................ 9Nitrate/Nitrite in Ham ............................................................................................................................. 9Polyphosphates ....................................................................................................................................... 10

CHAPTER TWO: ORGANIC ACIDS ............................................................................11Organic Acids in Fruit Juice ................................................................................................................... 12

Organic Acids in Cranberry Juice and Tomato Juice ..................................................................... 12Simultaneous High-Resolution Profiling of Organic Acids and Inorganic Ions in Fruit Juice .... 13

Anions and Organic Acids in an Irish Stout ......................................................................................... 14Food Dyes ................................................................................................................................................ 14

CHAPTER THREE: AMINES AND OTHER ORGANIC BASES .................................... 15Cations and Methylamines .................................................................................................................... 16Inorganic Cations, Choline, and Acetylcholine ................................................................................... 16Flavor Constituents and Additives ....................................................................................................... 17Amines as Indicators of Seafood Spoilage ........................................................................................... 17Triazine Herbicides in Raw Fruits and Vegetables .............................................................................. 18Water-Soluble Vitamins .......................................................................................................................... 18

CHAPTER FOUR: CARBOHYDRATES ........................................................................ 19Oligo- and Polysaccharides Derived from Hydrolyzed Glucose Syrup ........................................... 21Sugar Alcohols ......................................................................................................................................... 21

Sugar Alcohols in Dietetic Hard Candy and Chewing Gum ....................................................... 22Simultaneous Determination of Sugars and Sugar Alcohols ....................................................... 22

Nutritive Sweeteners .............................................................................................................................. 23Impurities in Sweeteners .................................................................................................................. 23Sugars in Molasses ............................................................................................................................ 23Sugars in Foods ................................................................................................................................. 24Sugars in High-Fat Foods ................................................................................................................. 24

Determining Authenticity or Adulteration with Sugar and Oligosaccharide Profiles .................... 25Coffee Adulteration ........................................................................................................................... 25Oligosaccharide Profiling of Beverages and Sweeteners .............................................................. 26Establishing Geographic Origin ...................................................................................................... 26

TABLE OF CONTENTS

Fermentation Monitoring ....................................................................................................................... 27Sugar and Oligosaccharide Profiles During Beer Production ...................................................... 27

Alternative Sweeteners, Bulking Agents and Fat Substitutes ............................................................ 28Sucralose ............................................................................................................................................. 28Inulin Products .................................................................................................................................. 28Artificial Sweetener from Japan ....................................................................................................... 29Maltodextrins ..................................................................................................................................... 29Amylopectins ..................................................................................................................................... 30

Fruit and Fruit Juice ................................................................................................................................ 31Sugars in Orange Juice ...................................................................................................................... 31Oligogalacturonic Acids from Citrus Pectin ................................................................................... 31

CHAPTER FIVE: DIONEX LC TECHNOLOGIES ......................................................... 33Column Technologies ............................................................................................................................. 35Detector Technologies ............................................................................................................................. 36

Suppressed Conductivity Detection ............................................................................................... 36Pulsed Amperometric Detection ..................................................................................................... 36

Pump Technology ................................................................................................................................... 37

CHAPTER 6: ACCELERATED SOLVENT EXTRACTION (ASE®) ................................. 39Overview of ASE Technology ................................................................................................................ 41

ASE System Features ........................................................................................................................ 42ASE 100 Accelerated Solvent Extractor ........................................................................................... 42ASE 200 Accelerated Solvent Extractor ........................................................................................... 42ASE 300 Accelerated Solvent Extractor ........................................................................................... 42

Extraction of Pesticides from Grains ..................................................................................................... 43Study No. 1: Pesticides ..................................................................................................................... 43Study No. 2: Pesticides, Herbicides, and Fungicides .................................................................... 44

Extraction of Organochlorine Pesticides From Fruits and Vegetables .............................................. 45Extraction of Organophosphorus Pesticides from Baby Food ........................................................... 45Selective Extraction of PCBs from Fish Tissue ..................................................................................... 44

A Comparison of “Nonselective” and “Selective” ASE Extractions ............................................ 47PCBs in Large-Volume Fish Tissue Samples ........................................................................................ 48Extraction of PCBs From Oyster Tissue ................................................................................................ 49

Sample Preparation and Analysis ................................................................................................... 49Analysis and Quantification ............................................................................................................ 49

TABLE OF CONTENTS

TABLE OF CONTENTS

Determination of Fat in Various Food Matrices ................................................................................... 50Comparison of ASE to Soxhlet Method .......................................................................................... 50Comparison of ASE to Mojonnier Method ..................................................................................... 51Extraction of Fat from Chocolate ..................................................................................................... 52Determination of Fat in Dried Milk Products ................................................................................ 52Extraction of Fat from Liquid Dairy Products ............................................................................... 53

Extraction of Oils From Oilseeds ........................................................................................................... 54Comparison of ASE to the Current Official Method ..................................................................... 54

APPENDIX ONE: AOAC INTERNATIONAL, OFFICIALLY APPROVEDHPLC METHODS .................................................................................................. 55

APPENDIX TWO: RECOMMENDED READING ......................................................... 59Journal Articles ........................................................................................................................................ 60Dionex Presentations, Application Notes, and Technical Notes ........................................................ 64

INDEX ...................................................................................................................... 67

1

The development of rapid, automat-

able methods for analyses of foods and

beverages is one of the most challenging

areas of analytical chemistry. Food and

beverage matrices can be very complex,

and analytes of interest may only be

present at trace levels. Methods are often

prone to interferences due to lack of

detector specificity and sensitivity, and

complex sample cleanup procedures may

be required.

The examples presented in Chapters

1–4 show analytical solutions for the

determination of carbohydrates, organic

acids, amines, and inorganic ions in

foods and beverages. The methods

employed are simple, direct, and inter-

ference-free, and require only minimal

sample cleanup. These solutons

achieved by combining specific, high-

sensitivity detection with column

selectivities tailored to the analytes.

Chapter 5 provides a brief overview of

these technologies.

INTRODUCTION

SIMPLER, INTERFERENCE-FREEANALYSIS

is the result of

High-Sensitivity DetectionSamples can be diluted 100–10,000-fold

for analysis, which greatly reduces theconcentration of matrix interferencesand the potential for column fouling.

plus

Specific DetectionAnalytes of interest can be detected at lowlevels even in the presence of much higher

levels of matrix components. Extensivesample cleanup is reduced or eliminated.

plus

Analyte-Specific SeparationsHigh-resolution ion-exchange and mixed-mode columns with selectivities tailoredto specific classes of analytes providein-situ cleanup by eliminating other

compound classes.

Chapter 6 focuses on Accelerated

Solvent Extraction (ASE®), a technique

introduced by Dionex in 1995 that

provides fast, automated extraction of

food matrices and requires only small

amounts of solvent.

2

OFFICIALLY APPROVED IC METHODS

The methods listed below employ either suppressed conductivity or pulsed

amperometric detection. See Appendix I for "AOAC International: Approved

HPLC Methods". All methods listed are suitable for Dionex systems, columns,

and reagents.

AOAC INTERNATIONAL OFFICIAL METHODS BOARD, 1ST ACTION APPROVALAOAC Method 993.30: Determination of Inorganic Anions in Water by Ion ChromatographyAOAC Method 996.04: Determination of Sugar in MolassesAOAC Method 995.13: Determination of Carbohydrates in Soluble (Instant) Coffee:

Anion-Exchange Chromatographic Method with Pulsed Amperometric DetectionAOAC Method 997.08: Determination of Fructans in Food Products

ANALYTICA-EBC INTERNATIONAL METHODDetermination of Anions in Beer by Ion Chromatography

Collaboratively Tested and Approved by the American Society for Brewing Chemists,the European Brewing Convention, and the Brewery Convention of Japan

INTERNATIONAL ORGANIZATION FOR STANDARDIZATIONISO 11292: Instant Coffee: Determination of Free and Total Carbohydrates—Method by

High Performance Anion-Exchange ChromatographyISO 10304-1: Anions in Natural and Contaminated Waters

INTERNATIONAL COMMISSION FOR UNIFORM METHODS OF SUGAR ANALYSIS (ICUMSA)Determination of Sugar in Molasses

AMERICAN SOCIETY FOR TESTING MATERIALS (ASTM)ASTM D4327-91: Anions in Water by Chemically Suppressed Ion Chromatography

U. S. NATIONAL INSTITUTE FOR OCCUPATIONAL SAFETY AND HEALTH (NIOSH)NIOSH 4110: Determination of Anions by Ion Chromatography

3

UNITED STATES ENVIRONMENTAL PROTECTION AGENCY (U.S. EPA)Method 218.6: Determination of Dissolved Hexavalent Chromium in Drinking Water,

Groundwater, and Industrial Wastewater Effluents by Ion ChromatographyMethod 300.0: Determination of Inorganic Anions in Water by Ion ChromatographyU.S. EPA Method 300.1: The Determination of Inorganic Anions in Drinking Water

by Ion Chromatography ........................................................................................ 0983U.S. EPA Method 314.0: Determination of Perchlorate in Drinking Water Using

Ion Chromatography ............................................................................................. 1195U.S. EPA Method 317.0: Determination of Inorganic Oxyhalide Disinfection By-Products

in Drinking Water Using Ion Chromatography with the Addition of Postcolumnreagent for Trace Bromate Analysis ...................................................................... 1259

5

CHAPTER ONE:INORGANIC ANIONS

AND CATIONS

6

The determination of inorganic and organic anions and cations in food and

beverages is important for meeting nutritional labeling requirements and for

processing and quality control. In many cases, the concentration levels of certain

ions have a direct bearing on food quality and flavor. Health concerns associated

with ions such as nitrite, bromide, bromate, iodide, cyanide, and chromium (VI)

necessitate their determination at trace levels in many foods and beverages. Ion

chromatography (IC) offers the food chemist multiple capabilities for automated

analysis of ionic and ionizable compounds, while eliminating time-consuming

sample preparation steps.

MUNICIPAL DRINKING WATERWater is an important raw material for

many beverages and food products. Theanalysis of incoming water is importantboth for quality control and flavor consid-erations, and for nutritional and healthconcerns. Nitrate and nitrite—present inmany water sources—are particularlyproblematic since nitrate can be reduced tonitrite in the human body and ultimatelyform carcinogenic nitrosamines by reactionwith amines. Nitrate and nitrite can bedetermined at low-µg/L (ppb) levels indrinking water either by ion chromatogra-phy with UV detection or by IC with sup-pressed conductivity detection accordingto United States Environmental ProtectionAgency (U.S. EPA) Method 300.0. IC analy-sis of municipal drinking water from twodifferent locations illustrates the significantvariations in ion concentrations thatcan occur.

7

75

0

µS3

5

0 5 10

0.0

2.5

µS 1

2 5

Minutes0 5 10

12 4

5

6

2.5

0.0

µS

Minutes

75

0

µS

Column: IonPac® AS12A and guardEluent: 2.7 mM sodium carbonate/

0.3 mM sodium bicarbonateFlow Rate: 1.5 mL/minInj. Volume: 10 µLDetection: Suppressed conductivity

10046

Peaks: 1. Fluoride2. Carbonate3. Chloride4. Bromide5. Nitrate6. Phosphate7. Sulfate

1

3

75

Union City Water Sunnyvale Water

30:1 Expanded Scale 30:1 Expanded Scale

Municipal Drinking Water

0 5 10Minutes

0 5 10Minutes

INORGANIC ANIONS & CATIONS

7

BROMATE IN DRINKING WATERAND BAKED GOODS

Bromate, a by-product of the ozonationdisinfection process for drinking water, hasbeen cited by the U.S. EPA and the WorldHealth Organization as a potential carcino-gen, even at low-µg/L concentrations.Bromate is also commonly used as a doughstabilizer for bread and other baked goods,and trace amounts may remain in the finalproduct. In both of these cases, trace levelsof bromate must be determined in the pres-ence of much higher concentrations ofchloride and other common inorganic ions.The IonPac AS9-HC column was developedspecifically to provide the high capacity andselectivity needed to quantitatively deter-mine bromate at low-µg/L concentrationlevels using a simple isocratic procedure.

CATIONS IN MINERAL WATERAND DRINKING WATER

Group I and Group II cations are easilyand rapidly determined by IC as shown forvarious water samples, even when thesodium concentration is very high relativeto ammonium and other cations present.

CATIONS IN SOFT DRINKS AND WINEGroup I and II cations are easily deter-

mined in soft drinks and other beveragessuch as wine. The high specificity of sup-pressed conductivity detection providesa very simple chromatogram, free frominterferences. Sample preparation is straight-forward—simply vacuum degas if thebeverage is carbonated, and then dilute.


0 5 10 15

3

2

4

5 6

Minutes

1

3

2 4

5 6

Mineral Water (310:1)* Drinking Water (680:1)*

8108-02

Column: IonPac CS12Eluent: Methanesulfonic acid,

step change at 5.1 minFlow Rate: 2 mL/minInj. Volume: 25 µLTrap Column: CTC-1Detection: Suppressed conductivity

Peaks: 1. Lithium2. Sodium3. Ammonium4. Potassium5. Magnesium6. Calcium

* Sodium:Ammonium Ratio

Cations in Mineral Water and Drinking Water

µS µS

0 5 10 15Minutes

10751/10747

Column: IonPac CS12 and guard,Cation Trap

Eluent: Water/methanesulfonic acid gradient

Flow Rate: 1 mL/minInj. Volume: 25 µL

Cations in Soft Drinks and Wine

1

2

3

4

5

10

µS

0

A Diet Cola (1:10)

0 2 4 6 8 10 12

4 5

1

23

0

µS

10

Minutes

B White Bordeaux Wine (1:160

0 2 4 6 8 10 12Minutes

Detection: Suppressed conductivity

Peaks: 1. Sodium2. Ammonium3. Potassium4. Magnesium5. Calcium

Peaks:1. Fluoride 1.0 mg/L2. Chlorite 0.013. Bromate 0.0054. Chloride 50.05. Nitrite 0.16. Bromide 0.017. Chlorate 0.018. Nitrate 10.09. o-Phosphate 0.1

10. Sulfate 50.0

Sample also contained 150-mg/L bicarbonate

0.4

µS

0.0

0

1

23

4

5

67

8

9

10

10Minutes 12952

20 30

Common Anions and Oxyhalides

Columns: IonPac AG9-HC and AS9-HCEluent: 9 mM sodium carbonateInj. Volume: 200 µLDetection: Suppressed conductivity, ASRS®,

AutoSuppression® ext. water mode

8

TRANSITION METALS IN FOODTransition metals in food products are

often determined by AA or ICP spectroscopy.However, IC with postcolumn derivatiza-tion and visible absorbance detection is anattractive alternative that also provides in-formation on speciation. An extension ofthis technique incorporates Chelation IC,a matrix elimination, and preconcentrationprocedure, which eliminates interferencefrom calcium and magnesium, elementsthat are often problematic with AA and ICPspectroscopic techniques. Chelation IC al-lows determination of transition metalsat the part-per-billion level in matricescontaining high levels of other ions.

SULFITE IN DRIED APRICOTSulfite is commonly used as a preserva-

tive in many foods and beverages. Levelsare closely regulated in many countriesbecause of reported allergic reactions exper-ienced by some individuals. U.S. Food andDrug Administration regulations requirethat any product containing 10 mg/kg ormore sulfite must be labeled as such. Thedetermination shown here is based on theprocedure of Kim and Kim1; however, pulsedrather than dc amperometric detection wasused because it provides greatly improvedreproducibility of detector response. Sampleswere diluted and homogenized with theeluent and then filtered prior to injection.


1Kim, H. J.; Kim, Y. K. J. Food Sci. 1986, 51, 1380.

11853

Sulfite in Dried Apricot

Flow Rate: 1 mL/minInj. Volume: 50 µLDetection: Pulsed amperometry,

Pt electrodePeaks:1. Mannitol —2. Sulfite 10.4 mg/L

Sample Preparation:20 g dried apricot blended in 100 mL mannitol buffer.Sulfite conc. 0.8 mg/g of dried apricot sample.

Column: IonPac ICE-AS1Eluent: 20 mN Sulfuric acid

200

nC

Minutes0 1263 9

300

1

2

Column: IonPac CS5A, CG5AEluent: MetPac™ PDCAFlow Rate: 1.2 mL/minInj. Vol.: 50 µLDetection: Absorbance, 530 nm,

with PAR in MetPacPostcolumn ReagentDiluent0.2

AU

0

0 2 4 6 8 10 12 14

1

2

3

45

6

7

Minutes

8

Peaks:1. Iron (III) 1.3 mg/L 2. Copper 1.3 3. Nickel 2.64. Zinc 1.35. Cobalt 1.36. Cadmium 6.07. Manganese 2.68. Iron (II) 1.3

Transition Metals in Food

11873

9


IODIDE IN WHOLE MILKThe determination of inorganic ions

such as nitrite, nitrate, and iodide in dairyproducts is particularly important becauseof potential health implications. Simpleprotein precipitation procedures followedby ion chromatography usually provide agood analytical solution. The determinationof iodide in whole milk in the low-µg/Lrange is shown here.

NITRATES AND NITRITES IN HAMCommonly used methods for deter-

mining nitrate and nirite in food are timeconsuming and involve a series of samplepretreatment steps using protein precipitat-ing reagents or solid-phase extraction(SPE) cartridges.

The method used here greatly simpli-fies sample preparation. Homogenizedmeat samples are extracted with water at70–80 °C for 15 min, and then centrifugedand filtered. An aliquot of the filtrate isinjected directly without further cleanup.At the end of each run, a 5-min wash with100 mM sodium hydroxide prevents col-umn fouling.

The IonPac AS11 column is ideal forthis application because it provides selec-tivity, not only for the separation of nitrateand nitrite, but also for the separation ofthe analytes from potentially interferingUV-absorbing matrix components, thatelute close to the column void.

10926

0 2 4 6 8 10

12

0

30

nA

Minutes

Iodide in Whole Milk

Column: IonPac AS7 and AG7 guard

Eluent: 200 mM Nitric acidFlow Rate: 1.5 mL/minInj. Volume: 100 µLDetection: DC Amperometry,

Pt electrode, 0.8VPeaks: 1. Iodide 38.5 µg/L*

2. Unidentified*Milk diluted 1:4. Actual conc. in milk sample is 154 µg/L.

0 5 10

0.02

0

AU1

2

Minutes 12622

Peaks:1. Nitrite 1.16 mg/L2. Nitrate 0.54

Nitrate and Nitrite in Ham

Column: IonPac AS11Eluent: 5 mM Sodium

hydroxideFlow Rate: 1 mL/minInj. Volume: 25 µLDetection: UV, 225 nm

Sample Preparation:Homogenize 10 g of sample with 100 mL of water. Heat to 75 °C for 15 min. Centrifuge. Filter through1.2 µm filter, then 0.2 µm filter.

10

POLYPHOSPHATESPolyphosphates are widely used addi-

tives in products such as fruit juices andcanned goods to prevent discoloration andoff-flavors. They are also used for curingham, tenderizing vegetables, as emulsionstabilizers for cheese, and to retain mois-ture in frozen entreés. The functionality ofpolyphosphates in these applications isstrongly dependent on their sequesteringpower and buffering capacity, which isrelated to the polyphosphate chain length.

Commercial polyphosphates are mix-tures of polyphosphates with differentchain lengths. The most widely acceptedmethod for characterizing these productshas been to determine average chain lengthby end-group titration.

Microbore ion chromatography is in-creasingly being adopted for lot-to-lotquality control and for identification ofpolyphosphate products in unknownsamples because it provides a “fingerprint”of the actual chain-length distribution.The chromatographic profiles shown are oftwo 50% sodium hexametaphosphatesolutions that were prepared from the samelot of dry powder but with producedcheese products having significantly differ-ent characteristics.2 These chromatogramsindicated a different degree of hydrolysis ata critical point in the processing and pin-pointed a problem with solution handling.


B “GOOD” BATCH

2 Reproduced from Baluyot, E.; Hartford, C.G.J. Chromatogr., A. 1996, 739, 217–222.

A “BAD” BATCH

Phosphates in Cheese Products

Column: IonPac AS11 (2 mm), AG11 guard (2 mm), and ATC trap (2 mm)

Eluent: Sodium hydroxidegradient

Flow Rate: 0.3 mL/minInj. Volume: 10 µLDetection: Suppressed con-

ductivity, ASRS,AutoSuppression

recycle modePeaks: 1. PO4

2. P2O73. P3O94. P3O105. P4O126. P4O13

11

CHAPTER TWO:ORGANIC

ACIDS

12

ORGANIC ACIDS

Organic acids are important flavor components in foods and also can be

indicators of product quality or deterioration due to storage. Trace levels of organic

acids in foods are best determined by ion chromatography because suppressed

conductivity detection is approximately 10 times more sensitive than low UV

detection. Inorganic ions and organic acids in food and beverage products can

usually be determined in the same analysis.

ORGANIC ACIDS IN FRUIT JUICEProfiling of the organic acids in fruit

juices is important both for establishingfreshness and detecting adulteration. Ra-tios of certain organic acids are often deter-mined because they are characteristic of aparticular juice.

Organic Acids in Cranberry Juice andTomato Juice

Quinic acid is a specific marker forcranberry juice and is used as a measure ofpurity and authenticity. A simple isocraticseparation by ion exclusion chromatographyand detection by suppressed conductivityprovides a rapid method to determinequinic acid.

Other fruit juices also show character-istic organic acid profiles that can be usedto monitor product purity.

Analysis of food products such as tomatojuice are greatly simplified since the highconcentration of salt does not interfere.Chloride elutes in the void along with otherinorganic ions. Only dilution and filtrationof the sample is required.

9990/11397

A StandardsPeaks: 1. Oxalate 2. Tartrate 3. Citrate 4. Malate 5. Glycolate 6. Formate 7. Lactate 8. HIBA 9. Acetate 10. Succinate 11. Fumarate 12. Propionate 13. Glutarate

Organic Acids in Cranberry Juice and Tomato Juice

Column: IonPac ICE-AS6Eluent: 0.4 mM hepta-

fluorobutyric acidFlow Rate: 1.0 mL/minInj. Volume: 50 µLDetection: Suppressed conductivity

0 5 10 15 20 25 30 35

30

µS

0

3

12

4

Minutes0 5 10

0 5 10 15 20 25 30 35 40

15 20

5

µS

0

12

µS

0

2 56

4

3

75

4

8

6

11 1213

109

12

3

B Cranberry JuicePeaks: 1. Oxalate 2. Tartrate 3. Citrate 4. Quinate

C Tomato JuicePeaks: 1. Inorganics

2. Malonate3. Citrate4. Malate5. Formate6. Unknown

Minutes

Minutes

13

ORGANIC ACIDS

Simultaneous High-Resolution Profilingof Organic Acids and Inorganic Anionsin Fruit Juice

As shown here for orange juice, grapejuice, and apple juice, high-resolutionprofiles of major and minor organic acids,as well as inorganic anionic components,can be determined simultaneously usinggradient high-performance anion-ex-change chromatography. The IonPac AS11column functionality is tailored for gradi-ent elution with a sodium hydroxide elu-ent and can be reequilibrated to initialconditions in approximately 5 min. Metha-nol is incorporated in the eluent to opti-mize selectivity for separation of certainorganic acid pairs.

8508A/8589/11398

0 2 4 6 8 10 12 14 16

4

7

15

17

13

2 56 8

1916910

14

11

18

20

115

µS

02

34

5

6

7 8

9

12

13

10

15

14

1617

2018

5

µS

0

5

µS

0

1214

1615

17 20

11

10

987

645

21 3

Minutes

C Apple Juice

B Grape Juice

A Orange Juice

Peaks: 9. Glutarate10. Succinate11. Malate12 Malonate

13 Tartrate14. Sulfate15. Oxalate16. Phosphate17. Citrate18. Isocitrate19. cis-Aconitate20. trans-Aconitate

Organic Acids in Orange Juice, Grape Juice,and Apple Juice

Column: IonPac AS11Eluents: Sodium hydroxide/

Methanol gradientFlow Rate: 2.0 mL/minDetection: Suppressed conductivityPeaks: 1. Quinate

2. Lactate3. Acetate4. Glycolate5. Formate6. d-Galacturonate7. Chloride8. Nitrate

0 2 4 6 8 10 12 14 16Minutes

0 2 4 6 8 10 12 14 16Minutes

14

ANIONS AND ORGANIC ACIDS IN ANIRISH STOUT

Organic acids and inorganic anions areimportant flavor constituents in brewingliquors. Inorganic anions also affect physi-cal appearance. A complete high-resolutionprofile of both organic acids and inorganicanions in beer can be obtained in less than18 min.

ORGANIC ACIDS

FOOD DYESSynthetic food dyes are widely used in

foods and beverages. They usually fall intoone of four classes: azo (mono-, di-, and tri-),indol, triphenylmethane, and methin dyes.For the most part, these dyes are acidic oranionic, and contain sulfonate, carboxyl,or phenolic groups. These strongly ioniccompounds are not easily separated byconventional reversed-phase HPLC andrequire the use of ion-pairing reagents. Byusing a multiphase column (as shown),excellent separations are achieved withoutinvoking the use of ion-pairing reagents.

Column: OmniPac® PCX-500Eluent: Perchloric acid/

Sodium perchlorate/Acetonitrile gradient

Detection: UV, 254 nmPeaks: 1. Indigo carmine

2. Orange G3. Tropaeolin O4. Orange I5. Alizarian red S6. Orange II

5399-020 5 10 15 20

Minutes

1

2

3

4

5

6

7 8

9

10

11

12

1314

15

16

17

Peaks: 7. Chrome azurol S8. Acid blue 409. Thymol blue

10. Acid blue 11311. Fluorescein 12. Methyl green13. Acid red 11414. Acridine orange15. Nile blue16. Rhodamine B17. Malachite green

Food Dyes

107450 2 4 6 8 10 12 14 16 18

16

171 236

1454 8

910

1112

13 15

714

µS

0

Minutes

Peaks: 1. Fluoride2. Lactate3. Acetate4. Formate5. Unknown6. Pyruvate7. Chloride8. Nitrate9. Unknown

10. Unknown11. Succinate12. Malate13. Maleate14. Sulfate15. Oxalate16. Phosphate17. Citrate

Column: IonPac AS11 & AG11 guardEluent: Sodium hydroxide/ methanol gradientFlow Rate: 2 mL/minInj. Volume: 25 µLDetection: Suppressed conductivity

141312111098

4

µS

08

9

1112

13

14

15

10

Anions and Organic Acids in an Irish Stout

7

15

CHAPTER THREE:AMINES AND OTHER

ORGANIC BASES

16

AMINES & OTHER ORGANIC BASES

The determination of amines in foods and beverages is important because

evidence shows that amines can act as precursors in the formation of carcinogenic

nitrosamines, and may be indicators of food spoilage.

CATIONS AND METHYLAMINESLow-molecular-weight amines such as

trimethylamine and dimethylamine areindicators of quality in fish and other foodproducts. By tailoring the column selectivity,low-molecular-weight amines and inor-ganic cations can be determined simulta-neously. Methyl-, dimethyl-, and trimethyl-amines are all resolved from the commoncations with a run time of approximately12 min.

INORGANIC CATIONS, CHOLINE,AND ACETYLCHOLINE

Choline is essential to proper metabo-lism and is often added to infant formulaand vitamin formulations. Separation ofcholine using silica reversed-phase HPLCwith ion-pairing and detection by low UVis shown in Panel A. Ion chromatographyprovides an alternative column selectivityand has the advantage of highly sensitiveand specific detection. Nonionic UV absorb-ing matrix components that are oftenpresent in food samples do not interfere.Panel B shows a separation of choline andacetylcholine by cation exchange usingsuppressed conductivity detection. Theelution order of the choline and acetyl-choline has been reversed, and sodium andpotassium are determined simultaneously.

Peaks:1. Lithium 0.5 mg/L2. Sodium 23. Ammonium 2.54. Methylamine 105. Dimethylamine 106. Potassium 57. Trimethylamine 308. Magnesium 2.59. Calcium 5

Column : IonPac CS14Eluent: 10 mM methanesulfonic

acid/0.3% acetonitrileFlow Rate: 1 mL/minInj. Volume: 18 µLDetection: Suppressed conductivity

0 5 10 15Minutes

98

7

65

4

3

2

1

5

µS

0

8919-01

Cations and Methylamines

0 5 10 15 20

1

2

3

4

430.2

AU

0.0

2

µS

0

Minutes 7130

A Reversed Phase HPLCColumn: C-18Eluent: 5 mM heptanesulfonic

acid, pH 4.0/1% acetonitrile

Detection: UV, 190 nmInjection: Choline, acetylcholine

(10 µg each)

B Cation Exchange ICColumn: OmniPac PCX-100Eluent: 75 mM hydrochloric

acid/1% methanolDetection: Suppressed

conductivityInjection: Choline,

Acetylcholine (100 ng each)Sodium, potassium (10 ng each)

Peaks(A & B): 1. Sodium

2. Potassium3. Choline4. Acetylcholine

A Reversed- Phase HPLC

B Cation- ExchangeIC

Inorganic Cations, Choline, and Acetylcholine

0 5 10Minutes

17

FLAVOR CONSTITUENTS AND ADDITIVESNaturally occuring alkaloids such as

caffeine, theophylline, and theobromine areimportant bitter flavor constituents in coffee,tea, cocoa, and cola-type beverages. Thisseparation of 10 alkaloids was performed ona multiphase polymeric pellicular columnthat gives different retention characteristicsto a typical C-18 reversed-phase separation.The difference in selectivity may provide abetter separation from potentially interferingmatrix components.

AMINES AS INDICATORS OF SEAFOOD SPOILAGE

Biogenic amines in fish are used as in-dicators of quality and spoilage. VariousHPLC methods have been developed, butderivatization techniques are requiredbecause of the lack of a suitable chromophore.An improved method developed by agroup at the Laboratorio Alimenti, InstitutoSuperiore di Sanità in Rome allows biogenicamines to be determined directly at µg/Llevels without derivatization by usingintegrated pulsed amperometry. Panel Ashows the chromatogram of aminesextracted from spoiled canned herrings;Panel B shows the same extract spikedwith 300 µg/g of each amine.


7023-020 5 10

98

21

4

35

6

7

Minutes

Peaks: 1. Theobromine2. Theophylline3. Caffeine4. Morphine5. Colchicine6. Strychnine7. Papaverine8. Nicotine9. Cinchonine

10. Quinine10

Column: OmniPac PCX-500Eluent: Hydrochloric acid/

Potassium chloride/Acetonitrile gradient

Flow Rate: 1.0 mL/minDetection: UV, 254 nm

Flavor Constituents and Additives

10758Minutes0 5 10 15 20

1

23

45

4321

3

µC

0

3

µC

0

Column: IonPac CS10 and guard

Eluent: Acetonitrile/Perchloric acid/Sodium perchlorategradient

Flow Rate: 1.0 mL/min

Reproduced with permission from Draisci, R., et al., Chromatographia 1993, 35, No. 9–12, 584–590.

Amines as Indicators of Seafood Spoilage

A Canned Herring Sample

B Spiked Sample (300 µg/g of each amine)

Detection: Integrated amperometryPeaks: 1. Putrescine 16 µg/g 2. Histidine 103 3. Cadaverine 187 4. Histamine 172 5. Spermidine 294

5

Minutes0 5 10 15 20

18


Column: OmniPac PCX-500Eluent: Hydrochloric acid/

Ammonium acetate/Acetonitrile gradient

Flow Rate: 1.0 mL/minUV, 254 nm

0 5 10 15

0.2

0.0

AU1

2

3 45

8

6

7

Minutes 7214

Peaks: 1. Picloram2. Aminen3. Simazine4. Atrazine5. Propanine6. Prometryn7. Trifluralin,

Planavin Component8. Planavin

Triazine HerbicidesTRIAZINE HERBICIDES IN RAW FRUITSAND VEGETABLES

Due to their widespread use, triazineherbicides are typically monitored in foodssuch as raw fruits and vegetables by usingmultiresidue methods. Both gas chroma-tography (GC) and C-18 silica reversed-phase HPLC methods have been devel-oped. Potential interferences vary for eachfood product, and a different column selec-tivity may be advantageous for a particularmatrix. The common triazine herbicidesshown here were separated on a multi-phase cation-exchange reversed-phasepolymeric column that provides alternativeselectivity to silica reversed-phase. Theeluent used in this separation is compatiblewith mass spectrometry if positive peakidentification is required.

WATER-SOLUBLE VITAMINSVitamin assays in foods are needed for

nutritional labeling, quality assurance, andmonitoring changes due to processing,storage, and so on. HPLC using silicareversed-phase with ion pairing is mostcommonly used for the determination ofwater-soluble vitamins. Separations can beachieved on a multiphase column withoutrequiring ion-pairing reagents—this alsopermits two analytical determinations tobe combined into a single run, as shown inPanels A and B, by connecting UV andconductivity detectors in series.

11.5

µS

0.0

0.65

AU

0.00

12

3

7

56

8

9114

10

Na+

K+ Mg2+Ca2+

7027-02

Column: OmniPac PCX-500Eluent: Hydrochloric acid, DAP/

Acetonitrile gradientDetection: A: UV, 215 nm

B: Conductivity

Peaks: 1. Ascorbic acid2. Pantothenic acid3. Riboflavin4. Biotin5. Niacin6. Niacinamide7. PABA8. Adenine9. Folic acid

10. Pyridoxamine11. Thiamine

Inorganic Cations and Water-Soluble Vitamins

A UV Detection of Water-Soluble Vitamins

B Conductivity Detection of Inorganic Cations

0 5 10 15Minutes

0 5 10 15Minutes

19

CHAPTER FOUR:CARBOHYDRATES

20

Carbohydrates are important constituents in many food and beverage products

and are determined for a variety of reasons, including quality control; monitoring

of food-labeling claims; establishing authenticity; analysis of sweeteners, bulking

agents, and fat substitutes; and fermentation monitoring in the production of

alcoholic beverages.

HPLC on aminopropyl-bonded silica and polymeric phases, or metal-loaded

cation-exchange resins in conjunction with refractive index (RI) or low-wave-

length UV detection, often provide simple isocratic methods for determining

common sugars. The need for improved methods has been recognized3 because

these approaches are unsatisfactory for some applications due to inadequate

resolution of sugars from sugar alcohols and organic acids, lack of detector

specificity, and insufficient sensitivity. Improved methods are particularly

important for nutritional labeling since total sugar content must be stated.

Sodium chloride interference and the use of acetonitrile have also been cited as

additional problems.3

High-performance anion-exchange chromatography at high pH coupled with

pulsed amperometric detection (HPAE-PAD) solves these problems. Sugars,

sugar alcohols, oligo-, and polysaccharides can be separated with very high

resolution in a single run without derivatization, and quantified down to picomole

levels. The technique is being applied to a wide variety of routine monitoring and

research applications and official methods have been approved by the International

Standards Organization and other official regulatory agencies. Alcohols, glycols,

and aldehydes can also be determined with this technique.

CARBOHYDRATES

3 De Vries, J. W.; Nelson, A. L. Food Technology 1994, July, pp 76–77.

21

CARBOHYDRATES

107440 10 20 30 40 50

12

10

11

678 9

5

4

30.14

µC

0.00

Minutes

Column: CarboPac MA1Eluent: 500 mM sodium hydroxideFlow Rate: 0.4 mL/minInj. Volume: 10 µL

Detection: Pulsed amperometry

Peaks: 1. myo-Inositol2. Glycerol3. i-Erythritol4. Xylitol5. Arabitol6. Sorbitol7. Dulcitol8. Mannitol9. Glucose

10. Fructose11. Sucrose

Sugar Alcohols

Column: Amino-PropylEluent: 75% acetonitrile/

WaterFlow Rate: 2.0 mL/minDetector: Refractive indexTemp.: 25 °CPeaks: 1. DP-1

2. DP-23. DP-34. DP-45. DP-5

0 5

1

2

34 5

0 5 10 15Minutes

123

4

5

8576-8579

0 10

1

2

34 5

Column: Reversed-phaseEluent: WaterFlow Rate: 0.5 mL/minDetection: Refractive indexTemp.: Ambient

Column: CarboPac PA1Eluent: Sodium hydroxide/

Sodium acetate gradient

Flow Rate: 1.0 mL/minDetector: Pulsed amperometryTemp.: Ambient

0 8 12

54

32

1

Column: Cation-exchange, Calcium form

Eluent: WaterFlow Rate: 0.6 mL/minDetector: Refractive indexTemp.: 80 °C

Oligo- and Polysaccharides Derived fromHydrolyzed Glucose Syrup

OLIGO- AND POLYSACCHARIDES DERIVEDFROM HYDROLYZED GLUCOSE SYRUP

This comparison of chromatographictechniques used for the analysis of ahydrolyzed glucose syrup illustrates theremarkable resolving power of theHPAE-PAD technique. Elution order isreversed with the CarboPac® PA1 columnas compared with conventional metal-loaded cation-exchange columns. Thatis the higher homologues elute later asresolved peaks rather than in a poorlyresolved group at the beginning of thechromatogram.

SUGAR ALCOHOLSNutritional labeling requirements for

sugar alcohols are presently optional in theU.S., but as with sugars, the total sugaralcohol content is listed. Minor sugaralcohols must therefore be determined.Gas chromatogarphy (GC) methods forsugar alcohols have been developed, butare complicated by the requirement forderivatization. The only official method inexistence is AOAC 973.28, a GC method forsorbitol. A simpler, more direct approachthat does not require derivatization isshown here. The selectivity of the columnwas designed to allow sugar alcohols as agroup to elute ahead of sugars with highresolution.

22

CARBOHYDRATES

12236

Column: CarboPac PA10, PA10 Guard,Borate Trap™

Eluent: 52 mM sodiumhydroxide

Flow Rate: 1.5 mL/min

Inj. Volume: 5 µLDetection: Pulsed amperometry,

gold electrodePeaks: 1. Glycerol 4.0 µg/mL

2. Xylitol 2.03. Sorbitol 1.54. Mannitol 3.05. Glucose 5.06. Fructose 8.07. Sucrose 2.08. Lactose 8.0

Isocratic Separation of Food Sugarsand Sugar Alcohols

0

123

4

5

6 78

42 6 12108 14

40

nC

Minutes

0

8587-02/8850-01

A Dietetic Hard Candy

0 10 20 30

1

3

2

4

Minutes

Column: CarboPac MA1Eluent: 500 mM sodium

hydroxideFlow Rate: 0.4 mL/minDetection: Pulsed amperometry,

Au electrode

Peaks A:1. Sorbitol 106 µg2. Mannitol 17

Peaks B:1. Glycerol2. Sorbitol3. Mannitol4. Glucose

B Chewing Gum

Sugar Alcohols in Dietetic Hard Candyand Chewing Gum

µC

2

15

00 10 20 30

Minutes

µC

Sugar Alcohols in Dietetic Hard Candyand Chewing Gum

Use of sugar alcohols as alternativesweeteners is increasing rapidly particularlyfor dietetic foods and in products such aschewing gum because of their noncariogenicproperties. In these cases, sugar alcoholsmust be routinely determined in foods tomeet regulatory requirements. Simple pro-cedures for sugar alcohols in hard candy(boiled sweets) and chewing gum areshown here. Sorbitol and mannitol can beeasily determined, interference-free, in hardcandy; sample preparation consists of dis-solution and dilution in water (Panel A).Panel B shows the determination of glycerol,sorbitol, mannitol, and glucose in a chewinggum sample; sample preparation consistsof sonication with deionized water,OnGuard® A cartridge pretreatment, follow-ed by filtration through a 0.45-µm filter.

Simultaneous Determination of Sugarsand Sugar Alcohols

Sugars and sugar alcohols commonlyfound in food and beverage samples can beroutinely determined in the same run on theCarboPac PA10 column. Under isocraticconditions, glycerol, xylitol, sorbitol, andmannitol elute rapidly followed by themono- and disaccharides.

23

CARBOHYDRATES

0 2 4 6 8 10 12

12

3

Minutes 7660

Column: CarboPac PA1 and guard

Eluent: 160 mM sodium hydroxide

Flow Rate: 1.0 mL/minDetection: Pulsed

amperometry, Au electrode

Peaks: 1. Glucose2. Fructose3. Sucrose

µA

Impurities in Sweeteners

0 2 4 6 8

1

23

4

8

µA

0

Minutes 10930



Flow Rate: 1.0 mL/minInj. Volume.: 50 µLDetection: Pulsed amperometry,

Au electrodePeaks: 1. Glucose 4.39%

2. Fructose 6.673. Lactose (int. std.)4. Sucrose 30.8

Sugars in Molasses

10

NUTRITIVE SWEETENERSHPLC is in daily use in the sugar

industry to determine organic acids andcarbohydrates. Strong cation-exchangecolumns in various metal forms have beenmost commonly used; however, HPAE-PAD now offers a powerful alternativewith significant advantages.

Impurities in SweetenersThe common crystalline sugar products

—sucrose, maltose, lactose, dextrose andfructose—have highly predictable func-tionality when used in food products be-cause of their high purity. Trace impuritiesare easily and rapidly determined, asshown in this typical sucrose analysis.

Sugars in MolassesThe determination of sugars in molas-

ses using the official method of the Interna-tional Commission for Uniform Methodsof Sugar Analysis (ICUMSA) approved in1994 is shown here. Approval of themethod was based on an internationalcollaborative study involving 11 laborato-ries. Excellent reproducibility was obtainedand the results were in close agreementwith a parallel GC collaborative study.

Among the advantages cited for thenew method were: (1) lack of coelutionwith nonsugar impurities; (2) greatlyreduced possibility of overestimation ofsugars due to coeluting impurities; and(3) no column heater is required.

24

Sugars in FoodsSugars can easily be determined in foods

such as tomato ketchup by a simple extrac-tion followed by dilution and filtration, asillustrated in Panel A. In Panel B, glucose,fructose, maltose, and maltotriose contentwere determined in a butterscotch candysample. Samples were diluted 1:2000 andpassed through a 0.2-µm filter prior to injec-tion. In Panel C, sugars present in a flavoredpotato chip extract were determined directlyfollowing a simple extraction. The unidenti-fied peaks may be other sugars or easilyoxidizable ingredients such as aldehydes.

0 5 10 15 20 25

3

1

2

200

0

nC

Minutes 10753

0 10 20Minutes

1

2

3

5

1

4µC

0

10755

0 10 20Minutes

1

2

3

4

5

4133

B Butterscotch CandyColumn: CarboPac PA1Eluent : Sodium hydroxide/

sodium acetate gradient

C Flavored Potato ChipColumn: CarboPac PA1Eluent: Sodium hydroxide

Flow Rate: 1.0 mL/minDetection: Pulsed amperometry,

Au electrodePeaks: 1. Arabinose

2. Glucose3. Fructose4. Lactose5. Sucrose

Flow Rate: 1.0 mL/minInj. Volume: 25 µLDetection: Pulsed amperometry,

Au electrodePeaks: 1. Glucose 6.8%

2. Fructose 3.0% 3. Unidentified 4. Maltose 4.5%

5. Maltotriose 3.9%

Column: CarboPac PA1Eluent: 150 mM sodium hydroxideFlow Rate: 1 mL/minInj. Volume: 50 µLDetection: Pulsed amperometry, Au electrodePeaks: 1. Glucose 950 µg/mL

2. Fructose 5903. Sucrose 4.1Sample diluted 1:100 with DI water.

Sugars in Food

A Ketchup

µC

0 2 4 6 8 10

12

1.5 3

0

µC

Minutes 10172-01

Flow Rate: 1.0 mL/minInj. Volume: 50 µL (~1 g in 16 L)Detection: Pulsed amperometry,

Au electrodePeaks:1. Glycerol not quantified2. Lactose 1.3 µg3. Sucrose 2.9

Sugars in Milk Chocolate



Sugars in High-Fat Foods A challenge in determining sugars in

high-fat food samples is that the fat mayinterfere with the chromatography. Thisproblem can be overcome by extracting thefat prior to analysis. Supercritical fluidextraction was used in this example.

CARBOHYDRATES

25

0 10 20 30 40Minutes

0

12

34

5 6 7

6007-02

Carbohydrates in Instant Coffee

Inj. Volume: 25 µL of 10 g/L solutionDetector: Pulsed amperometry,

Au electrode; postcolumnaddition of 0.3 M NaOH

Peaks: 1. Mannitol 21 mg/L2. Arabinose1403. Galactose 764. Glucose 445. Xylose 266. Mannose 517. Fructose 93Sample Preparation: Phenolics removed with On-Guard P cartridge.

Column: CarboPac PA1Eluent: 150 mM sodium

hydroxide/deionizedwater gradient

1000

0

nA

CARBOHYDRATES

DETERMINING AUTHENTICITY ORADULTERATION WITH SUGAR ANDOLIGOSACCHARIDE PROFILES

Coffee AdulterationThe fraudulent addition of cheaper

coffee substitutes to commercial productscan be detected by determination of freeand total carbohydrate profiles. For ex-ample, high levels of free mannitol andtotal xylose are indicative of adulterationwith coffee husks and parchments,whereas so-called “pure” soluble coffeescontaining cereals or caramelized sugarcontain high levels of glucose.

Previous methods for determination ofcarbohydrates in coffee have been limitedeither by complex sample preparation,enzyme availability, or lack of specificity.Complete carbohydrate profiles could onlybe obtained by combining results fromdifferent techniques. Using HPAE-PAD, allthe major carbohydrates present in solublecoffee can be determined in a single run.

The separation shown here employs a“reverse” gradient. A similar method hasbeen validated by a collaborative studyinvolving 11 laboratories from differentcountries and has been approved as anofficial ISO method (ISO 11292). Severalpapers have been published relating to ISO11292:

• Prodolliet, J; Bruelhart, M; Lador, F; Mar-tinez, C; Obert, L; Blanc, M. B; Parchet, J-M. “Determination of Free and Total Carbohydrate Profile in Soluble Coffee”J. Assoc. Off. Anal. Chem. Int. 1995, 78,749–761.

• Prodolliet, J; Blanc, M. B; Leloup, V;Cherix, G; Donnelly, C. M; Viani, R.“Adulteration of Soluble Coffee withCoffee Husks and Parchments”J. Assoc. Off. Anal. Chem. Int. 1995, 78,761–767.

• Prodolliet, J; Bugner, E;Feinberg, M.“Determination of Carbohy-drates in Soluble Coffee by AnionExchange Chromatography with PulsedAmperometric Detection:Interlaboratory Study” J. Assoc. Off. Anal. Chem. Int. 1995, 78,768–782.

For additional literature of interest,see Appendix Two: Recommended Reading.

26

CARBOHYDRATES

0 10 20 30

0

µA

5

11399

B Orange Juice Adulterated with Beet Medium Invert Sugar

Oligosaccharide Profiles of Pure and Adulterated Orange Juice

Minutes

Column: CarboPac PA-100Eluent: Sodium hydroxide/


Flow Rate: 1 mL/minInj. Volume: 25 µLDetection: Pulsed

amperometry,Au electrode

0

µA

5A Pure Orange Juice

0 10 20 30Minutes

1

4938-01/4937-01

Sucrose

0

µA

Minutes

0

µA

1

Oligosaccharide Profile of Beet Sugar Molasses

C British Column: CarboPac PA1Eluent: 100 mM sodium

hydroxide/20 mM Sodium acetate

Flow Rate: 1 mL/minDetection: Pulsed

amperometry, Au electrode

0 5 15

D U.S.

Sucrose

Minutes0 5 15

Oligosaccharide Profiling of Beveragesand Sweeteners

The determination of oligosaccharidecomposition profiles is a powerful tech-nique for detection of adulteration of natu-ral fruit juices by inexpensive sweetenerssuch as beet sugar (Panels A and B).

Profiles can be rapidly determined, asshown in this comparison of pure orangejuice (A) and orange juice adulterated with20% beet medium invert sugar (B).

Establishing Geographic OriginOligosaccharide profiling is also a useful

tool for establishing the geographical origin ofcarbohydrate food ingredients such as molas-ses. Panels C and D show the difference inoligosaccharide profiles for beet sugar fromBritain (C) and the United States (D).

27

CARBOHYDRATES

0 5 10 15 2520Minutes

0

nC

70 2

34

5 6

78

910

11

2000

nC

0

2

34

56 7 8 9 1011

10927/11358

800

nC

0

1

23

45

6 7 8 9 10 11

Column: CarboPac PA-100 and guardEluent: Sodium hydroxide/

Sodium acetate gradientFlow Rate: 1.0 mL/minInject Vol.: 10 µLDetection: Pulsed amperometry,

Au electrode

Sugars and Oligosaccharide ProfilesDuring the Beer Brewing Process

Peaks: 1. Ethanol2. Glucose

3. Maltose 4. Maltotriose 5. Maltotetraose

6. Maltopentaose7. Maltohexaose8. Maltoheptaose9. Maltooctaose

10. Maltononaose11. Maltodecaose

C Wort

A Maltose Oligomers

D Finish

0 5 10 15 2520Minutes

1600

nC

0

2

3

4

5 6

B First Mash

0 5 10 15 2520Minutes

0 5 10 15 2520Minutes

FERMENTATION MONITORING

Sugar and Oligosaccharide ProfilesDuring Beer Production

Determining the levels of fermentableand nonfermentable sugars at every stage ofbeer production is important because fer-mentable sugars determine the final alcoholcontent, and nonfermentable sugars contrib-ute to the flavor and “body” of the finalproduct. Sugars, sugar alcohols, alcohols,and glycols can be rapidly determined withhigh resolution at all phases of beer, wine,or cider production, as shown here.

A separation of maltose oligomers upto DP10 (“DP” refers to their degree ofpolymerization) with baseline resolution isshown in Panel A, and sugar and oligosac-charide profiles at various stages of thebrewing process in Panels B, C, and D.All samples were diluted 1:10.

Similarly, oligosaccharide profiles canbe determined for comparing regular andlow calorie beers.

28

CARBOHYDRATES

0 10 20 30 40 50

1

Minutes

2

3780

nA

0

10898

Column: CarboPac MA1Eluent: 150 mM sodium

acetate/0.2% (v/v) Acetic acid, pH 5.5

Flow Rate: 0.4 mL/minInj. Volume: 25 µLDetection: Pulsed amperometryPeaks:1. 4-Cl-Galactose 7.8 mg/L2. 1,6-di-Cl-Fructose 2.43. Sucralose

Sucralose

0 10 20 30 40Minutes

10

20

50

3550/3073

3

0

µA


Sodium acetate gradientFlow Rate: 1 mL/minDetection: Pulsed amperometry,

Au electrodeSample*: 0.3% Water washed

Inulin (polyfructose) in 0.1 M sodium hydroxide

*Sample courtesy of Dr. C. Mitchell. California Natural Products, Manteca, CA

Purified Inulin

β

CH2

O

H HO

OH HCH2OH

O

OO

O OO

HHOCH2

HOH2Cαβ

CH2

β

H HH

OH

HOCH2

H

HOCH2

HH HO

OH H

H HO

OH H H HO

OH H

n

ALTERNATIVE SWEETENERS, BULKINGAGENTS, AND FAT SUBSTITUTES

Increasing public concern over healthand nutrition has led to the developmentof new low-calorie sweeteners and fat sub-stitutes. Products have been developed notonly to emulate the sweetness of sucrose,or act as a fat substitute, but also to provideother important properties such as textureand bulk.

SucraloseSucralose is a new high-intensity sweet-

ener with 400–800 times the sweetness ofsucrose. Sucralose is manufactured by selec-tive chlorination of sucrose and is currentlythe only nonnutritive sweetener based onsucrose. The product was approved inCanada in September 1991 for use in avariety of food and beverage categories. Itis presently under review in other countries,including the United States and the UnitedKingdom. A sucralose sample shows traceimpurities when analyzed by HPAE-PAD.

Inulin ProductsInulin-based products derived from

chicory root and Jerusalem artichokes aremarketed as fat replacers and dietary fiberadditives to food formulations. These inulinproducts are mixtures of linear polyfructosechains incorporating a few glucose units.Their degree of polymerization (DP) istailored for a particular end use, so it iscritical to determine the chain-length distri-bution to maintain quality control of theend product. Determination of the chain-length distribution is shown here for DPvalues up to 50 and more.

29

CARBOHYDRATES

0 5 10 15

123

4

5

Minutes 4940-01/6055

0

µA

1

Column: CarboPac PA1Eluent: Sodium hydroxide/sodium acetateFlow Rate: 1 mL/minDetector: Pulsed amperometry,

Au electrodePeaks: 1. Glucose

2. Fructose3. Sucrose4. 1-Kestose5. Nystose

Kestose — Artificial Sweetener from Japan

OH

HO

HO

HOCH2

HOCH2

HOCH2 O

OHOH

OH

OH

O

O

CH2

O

CH2

O

Sucr

ose

HigherHomologs

1α

2β

2β

1-Kestose

OH

HO

HO

HOCH2

HOCH2

HOCH2 O

OHOH

OH

OH

O

O

CH2

O

CH2

O

Nystose

HO

HOCH2

OH

O

CH2OH

O

0 5 10 15 20 25 30 35 0

175

nC

Minutes

0 5 10 15 20 25 30 35 0

175

nC

Minutes

Maltodextrins — Maltrin® MO40 & M700

Inj. Volume: 25 µLFlow Rate: 1 mL/minDetection: Pulsed amperometry,

Au electrode

Maltrin is a trademark of Grain Processing Corporation.



A Maltrin MO40

B Maltrin M700

Artificial Sweetener from JapanKestoses improve the flavor of products

such as yogurt, and are also used as alterna-tive sweeteners with a sweetness 0.4–0.6times that of sucrose. Commercial productsare obtained from sucrose by an enzymaticprocess that results in a mixture of 1-kestose,nystose, glucose, sucrose, and fructose.HPAE-PAD provides an excellent method formonitoring the enzymatic process and forquality control of the final product.

MaltodextrinsMany commercial low-calorie bulk

sweeteners, bulking agents, and fat substi-tutes are polysaccharide or polyol materi-als derived from various types of starch.Determination of the distribution of poly-saccharide chain lengths in these productshas a direct bearing on the product func-tionality. HPAE-PAD is the method ofchoice because other approaches cannotseparate the higher DP polysaccharidechains. Chain-length distribution profilesfor two maltodextrins derived from corn-starch are shown here. Maltrin® MO40 isused in film-forming applications and toprovide a smooth texture. Maltrin M700 isprocessed to produce very low density par-ticles with excellent dissolution character-istics. The differences in physical proper-ties of the two maltodextrins are clearly re-flected in the different chain-length distri-bution profiles.

30

CARBOHYDRATES

D Edible Canna

Column: CarboPac PA1Eluent: Sodium hydroxide/sodium acetate gradientFlow Rate: 1.0 mL/minDetection: Pulsed amperometry, Au electrodeFrom Koizumi, K.; Fukuda, M.; Hizukuri, S. J. Chromatogr. 1991, 585, 233–238.

Amylopectin Chain-Length Distribution

504030

20

12

A Rice

B Corn

C Sweet Potato

6

6

12

20

3040 50 60

6

12

20

3040 50 60

0 10 20 30 40Minutes 9661

6

12

20

3040 50 60

0 10 20 30 40Minutes

0 10 20 30 40Minutes

0 10 20 30 40Minutes

AmylopectinsStarch from various sources varies in its

functional properties as a consequence ofdifferences in chemical structure. Anunderstanding of the relationship betweenmolecular structure and functional proper-ties is important for fundamental researchand in selecting and developing starch-derived additives for food formulations.Chain-length distribution is an importantparameter in characterizing both the amy-lose and amylopectin portion of starch.Shown here are complete chain-lengthdistributions up to DP60 for debranchedamylopectin from different sources.

31

FRUIT AND FRUIT JUICEHPLC is a powerful technique for the

analysis of carbohydrates, organic acids,and preservatives in fruit juice to ensureproduct quality, support nutritional label-ing claims, and detect adulteration.

Sugars in Orange JuiceAs shown, sugars can be determined

directly in juices, free of interference fromorganic acids. The only sample preparationrequired in this case is dilution and filtra-tion.

Oligogalacturonic Acidsfrom Citrus Pectin

Pectin has been used for years to thickenor gel products such as jams and jellies, butnew applications are being discovered.One specialty pectin product used as a fatsubstitute consists of partially methylatedpolygalacturonic acid extracted from citruspeel. Pectin from different sources has aunique oligogalacturonic acid “fingerprint”that can be used for identification andquality control purposes. A typical profileof oligogalacturonic acids from citrus pec-tin can be obtained in less than 40 min.Sample preparation consists of direct injec-tion of the diluted pectin hydrolysate.

CARBOHYDRATES

0 5 10 15 20 25 30 35 40 45

6

5

78 9

1210

1311 14

1516

1720

18

19

4939-01Minutes

10

µA

0

Oligogalacturonic Acids from Citrus Pectin

Column: CarboPac PA1Eluent: Sodium acetate/

Sodium hydroxide gradient

DP

Flow Rate: 1 mL/minDetection: Pulsed amperometry,

Au electrode

Sugars in Orange Juice

0 2 4 6 8 10 12

3

2

1

Minutes

0

40

nC

11400

Column: CarboPac PA1Eluent: Sodium hydroxideFlow Rate: 1 mL/minDetection: Pulsed amperometry,

Au electrodePeaks: 1. Glucose

2. Fructose3. Sucrose

33

CHAPTER FIVE:DIONEX LIQUID

CHROMATOGRAPHY

TECHNOLOGIES

34

Dionex LC instrumentation accommodates all of the conventional HPLC

techniques. New detector, column, and pump technologies

have been developed—often as a result of working with our customers to

solve specific analytical problems. Some of our most unique and effective

innovations include:

• Ion-exchange stationary phases with unique selectivities tailored to provide

fast, high-resolution separations of ionic and polar compounds.

• Metal-free HPLC system flow paths that guard against corrosion and

protein denaturation.

• Advanced suppressed conductivity detection technology for quantification

of organic and inorganic anions and cations with high specificity and

sensitivity.

• Pulsed amperometric detection (PAD) technology that greatly simplifies

the determination of carbohydrates.

• A revolutionary HPLC pumping system that integrates unique feedback

control with digital signal processing and artificial intelligence to eliminate

pulsation and provide exceptional flow accuracy—even under the changing

backpressure of gradient elution.

DIONEX LC TECHNOLOGIES

35


COLUMN TECHNOLOGIESResolution in HPLC is highly depen-

dent on column selectivity, which, in turn,depends on the stationary phase.

Dionex MicroBead™ high-efficiency,high-polymeric stationary phases weredeveloped specifically to optimize theseparation of specific classes of analytes.These stationary phases offer:• High-speed equilibration for gradient

elution.• Excellent mass transport characteristics

that result in higher efficiency.• Compatibility with high flow rates for

rapid separations.• Complete pH compatibility (pH 0 to 14).• High mechanical (4000 psi) and chemical

stability for exceptionally long column life.• Noncompressible resins that simplify

linear scale-up for preparative-scaleapplications.

The MicroBead stationary phase is madeby agglomerating a nonporous, noncom-pressible, polymeric substrate with beads ofquaternary-functionalized latex. The resultis a highly stable particle with a thin surfacelayer rich in ion-exchange sites that can betailored to separate specific analytes.

A variation on the MicroBead station-ary phases, also developed at Dionex, arepackings in which ion-exchange resins aregrafted in a thin layer on the surface of thecore polymer particle. Packings of this typehave been developed for applications suchas ion analysis of drinking water and wastewater.

Dionex has also developed multiphasepackings that incorporate both reversed-phase and ion-exchange functionality,which often eliminate the need for the ion-pairing reagents used in conventionalHPLC reversed-phase separations.

Figure 1Construction of IonPac’spellicular anion-exchangeresin bead:• Highly cross-linked inert,

nonporous core providesHPLC solvent compatibilityfor cleanup and formodifying eluent selectivity.

• Surface-sulfonated regioncompletely covers the core.

• Submicron pellicularMicroBead layerconcentrates a vast numberof ion-exchange sites into avery thin layer.

0.1-µmDiameter

3183-02

Core Ion-ExchangeSurface

SO3–

SO3–

SO3–

SO3–

SO3–

SO3–

SO3–

R3N+

R3N+

N R3+

N R3+

N R3+

R3N+

N R3+

N R3+

N R3+

N R3+

N R3+

N R3+

R3N+

N R3+

N R3+

R3N+

IonPac's Pellicular Anion-Exchange Resin Bead

5 µm

36


DETECTOR TECHNOLOGIESThe simplicity of many of the applica-

tion solutions shown in this book can, inmany cases, be attributed to the combina-tion of unique column selectivities witheither suppressed conductivity or pulsedamperometric detection.

Figure 3 Electrolysis of water within the SRS produces thehydronium and hydroxide ions required for eluent neutralization.Effluent from the detector cell is recycled to provide a continuouswater source.

8416-03

Eluent

Waste

Detector CellSRS

Recycled to SRS

ASRS Eluents: Hydroxide, carbonate/bicarbonate, boric acid/tetraborateCSRS Eluents: Methanesulfonic acid (MSA). (Eluents containingCl– or N0

3– cannot be used in this mode).

AutoSuppression Recycle Mode

Time

E1

Delay Integration

Poten

tial

E2

E3

t2

t3

5031-01

Pulsed Amperometry Triple-Pulse Sequence

t1

Figure 4 The triple-pulse potential sequence employed inpulsed amperometric detection ensures a clean electrode foraccurate, consistent detection results.

Figure 2 The principle of AutoSuppression® suppressedconductivity with the SRS® Self-Regenerating Suppressor.

–2

11309

AnalyticalColumn

Time

µS

Cl–F– SO4–2

Anion Suppressor

Waste

µS

SO4Cl–F–

Eluent (NaOH)Sample F–, Cl–, SO4

–2

H2O

NaF, NaClNa2SO4 in NaOH

Without Suppression

With AutoSuppression

The Role of AutoSuppression

Time

Suppressed Conductivity DetectionThe principle of suppressed conductivity

detection is illustrated in Figure 2. Thesuppressor reduces the eluent conductivityto a very low level while enhancing theanalyte conductivity. The result is excep-tional sensitivity and high specificity forionic analytes.

The SRS Self-Regenerating Suppressoris a self-contained unit that operates unat-tended and requires no maintenance(Figure. 3).

Pulsed Amperometric DetectionAnalytes such as carbohydrates cannot

be determined by DC amperometry due toelectrode fouling. Pulsed amperometryovercomes this problem by using a repeat-ing triple-pulse voltage sequence thatmaintains a clean electrode surface, thusensuring a consistent detector response(Figure. 4). This technique is now wellestablished for the determination ofcarbohydrates, and provides high sensitiv-ity and specificity without the need forderivatization.

37

PUMP TECHNOLOGYAccuracy and precision of retention

times and peak areas can be adverselyaffected with conventional HPLC pumpdesigns due to flow rate variations. Thesechanges result from the effect of changingsystem backpressure on the compressibilityand viscosity of the mobile phase, systemcompliance, and other factors such as mi-nor seal leakage. Flow rate variations areparticularly problematic during gradient

Primary Information Pathways of the PumpDigital Control System


elution where large changes in systembackpressure often occur. The Dionexquaternary gradient pumping system wasdesigned to directly address these prob-lems by using a flow control system engi-neered around state-of-the-art digital sig-nal processing (DSP) and fuzzy-logic (arti-ficial intelligence) algorithms. This engi-neering results in accurate, precise andpulse-free flow over a broad range of mo-bile phase compositions and systembackpressures.

Artificial Intelligence

Memory

MotorControlCircuit

TachCircuit

Motor

Sensor

Mechanism

EncoderDisk

Piston A&

Piston B

ProportioningValves

PressureTransducer

DSP

Eluents

To Column

39

CHAPTER SIX:ASE® ACCELERATED

SOLVENT

EXTRACTION

40

ACCELERATED SOLVENT EXTRACTION

Extraction is often a necessary first step in the analysis of food samples.

Traditional extraction methods such as Soxhlet use large volumes of solvent and

are slow, often requiring hours to obtain a satisfactory recovery. Sonication is a

faster technique, but it too requires large amounts of solvents and is not easily

automated. The high initial cost of solvents and their subsequent disposal makes

these methods less than desirable for routine use.

Accelerated Solvent Extraction (ASE) is an automated extraction method

developed by Dionex that takes advantage of the temperature dependence of

extraction kinetics and requires only small amounts of solvent. Extraction times

are reduced from hours to just minutes by operating at higher temperatures and

pressures than are typically used for traditional solvent-based extraction tech-

niques. ASE significantly streamlines sample preparation and is being rapidly

adopted for extraction of environmental, food, polymer, and other types of solid

and semisolid samples. For example, ASE meets all of the requirements of U.S.

EPA SW-846 Method 3545A for Pressurized Fluid Extraction of bases, neutrals,

and acids (BNAs), chlorinated pesticides and herbicides, polychlorinated

biphenyls (PCBs), and organophosphorus pesticides.

41


OVERVIEW OF ASE TECHNOLOGYAccelerated Solvent Extraction is

achieved by using organic or aqueous sol-vents at elevated temperature and pressure.The solvent is pumped into an extractioncell containing the sample, which is thenbrought to a specified temperature (fromambient to 200 °C) and pressure. Followingextraction, the extract is transferred from theheated cell, through a filter, to a standardcollection vial for cleanup and/or analysis.

Oven

Collection Vial

Extraction Cell

Solvent

Pump

Vent

Load sample into cell.

Fill cell with solvent.

Heat and pressurize cell.

Hold sample at pressure and temperature.

Pump clean solvent into sample cell.

Purge solvent from cell with N2 gas.

Extract ready for analysis.

Schematic of the Accelerated Solvent Extraction Process

Increased temperature accelerates theextraction kinetics, while elevated pressureprevents boiling at temperatures above thenormal boiling point of the solvent. Onlysmall amounts of solvent are used andextraction times can be as short as 15 min.Time and solvent consumption are thussignificantly reduced compared to othersolvent extraction techniques. Methoddevelopment is generally straightforwardbecause the same solvent used for a Soxhletor other current methods is generally usedwith ASE.

42


ASE System FeaturesAll ASE Systems Feature:• Automated sample extraction• Automatic extract filtration• Easy-to-fill sample cells with hand-tight

fittings• Easy-to-use collection vials and bottles• Convenient front-panel operation with

multiple method storage• Sensors for temperature, pressure, and

solvent vapors ensure safe operation atall times

• Easy method transfer between systems• Patented technology (patent numbers

5,843,311; 5,647,976; 5,660,727; and5,785,856)

• Established methodologies andEPA approval

• Temperature range from ambientto 200 °C

ASE 100 Accelerated Solvent ExtractorThe ASE 100 is the entry-level ASE

system designed for use in lower-through-put labs. This system is priced economi-cally and offers fast and efficient extractionfor a large range of sample sizes.• Automated extraction of a single sample• Sample cell sizes: 10, 34, 66, and 100 mL• Collection bottle: 250 mL• Operating pressure: 1500 psi (100 bar)• Small footprint requires less than 36 cm

(14 in.) of bench space ASE 200 Accelerated Solvent Extractor

The ASE 200 is designed for high-throughput labs with sample sizes of 1 to30 grams. With automation capabilities forup to 24 samples, the ASE 200 maximizes

sample throughput and extraction effi-ciency. The ASE 200 is ideal for standardenvironmental analysis, pharmaceuticalprocesses, and routine food and polymerapplications.• Unattended extraction for up to

24 samples• Samples cell sizes: 1, 5, 11, 22, and 33 mL• Scheduling programming for automated

method optimization• Collection vial sizes: 40 and 60 mL• Automatic rinsing of system between

sample extractions• Compatible with AutoASE® computer

control software and ASE SolventController

• Operating pressure: 500–3000 psi(30–200 bar)

ASE 300 Accelerated Solvent ExtractorThe ASE 300 is designed for high-

throughput labs with large sample volumerequirements. With automation capabilitiesfor up to 12 cells and sample cell volumesup to 100 mL, the ASE 300 is ideal for thebusy environmental and food analysis lab.• Unattended extraction for up to

12 samples• Samples cell sizes: 34, 66, and 100 mL• Collection bottles: 250 mL• Automatic rinsing of system between

sample extractions• Scheduling programming for automated

method optimization• Compatible with AutoASE computer

control software and ASE SolventController

• Operating pressure: 1500 psi (100 bar)

43

EXTRACTION OF PESTICIDES FROM GRAINSThe determination of pesticides, herbi-

cides, and other related compounds infoods and agricultural commodity productsis important to ensure the safety of the foodsupply. Increasingly, multiresidue analysistechniques are being used that require aninitial extraction of groups of pesticideswith high efficiency.

The data shown here are from collabora-tive studies conducted with two independentresearchers on the extraction of various classesof pesticides from wheat. Ground wheatsamples were sent to Dionex for extraction byASE. The extracts were then returned to theresearchers for cleanup and/or analysis.

Study No. 1: PesticidesTables 1 and 2 show the data from the

first study. Table 1 compares the currentand ASE extraction conditions used toobtain the recovery data shown in Table 2.ASE provides a more efficient extraction ina much shorter time with greatly reducedsolvent usage, and does not require a post-extraction SPE cleanup step.


TABLE 1. PESTICIDES IN WHEATStudy No. 1: Comparison of Current

and ASE Extraction Conditions

Current Method ASESample Size 3–20 g 3–20 gSolvent Volume 130 mL 15 mLPost Extraction SPE cleanup NoneTotal Time 60 min 15 minSample Analysis GC-FPD GC-FPD

TABLE 2. PESTICIDES IN WHEATStudy No. 1: Comparison of Recoveriesfor Current and ASE Extraction Methods

Malathion Methylchlorpyrifos(µg/L) (µg/L)

Sample Current ASE Current ASEMethod Method Method Method

1 40 50 70 90

2 40 50 80 100

3 60 70 50 60

5 40 100 30 70

10 60 80 60 80

11 60 70 70 90

44


TABLE 4. ASE EXTRACTION OF WHEATStudy No. 2: Recoveries of Spiked

Pesticides, Herbicides, and Fungicides

Spike Recovery(µg/L) (%)

OrganophosphorusPesticidesAzinphos-methyl 56 94.2Chlorpyrifos 20 60.1Chlorpyrifos-methyl 8 115.7Demeton-S 38 96.7Diazinon 26 96.9Dichlovos 18 60.5Dimethoate 58 87.8Disulfoton 22 87.9Disulfoton-sulfone 98 77.7Omethoate 74 85.4Parathion 84 101.2Parathion-methyl 40 115.7Phorate 18 92.8Phorate-sulfone 32 105.7

Chlorinated PesticidesEndosulfan-alpha 56 94.1Endosulfan-beta 68 93.3Endosulfan-sulfate 20 77.0Methoxychlor-o,p 48 89.9Methoxychlor-p,p’ 50 114.9

Carbamate PesticidesCarbaryl 92 54.1Carbofuran 22 96.6

HerbicidesAtrazine 14 92.8Diclofop-methyl 36 81.8Linuron 102 83.6Triallate 68 87.8Trifluralin 44 99.6

FungicidesImazalil 40 108.8Thiabendazole 44 158.8

TABLE 3. PESTICIDES IN WHEATStudy No. 2: ASE Extraction Conditions

Sample Size 3.0 gStatic Time 5 minSolvent AcetonitrileTotal Time 12 minPressure 2000 psiSolvent Volume 14 mLTemperature 100 °CSample Analysis GC-MSHeatup Time 5 min

Study No. 2: Pesticides, Herbicides,and Fungicides

Table 3 summarizes the ASE conditionsused in the second study and Table 4shows the recovery data. In this study,samples were spiked at 2 times the limit ofquantification, as determined by theresearcher using GC-mass spectrometry inthe single-ion monitoring mode. In thiscase, extracts were cleaned up prior toanalysis. The data indicates that ASE givesexcellent recoveries for a wide variety ofpesticide classes.

45

EXTRACTION OF ORGANOCHLORINEPESTICIDES FROM FRUITS & VEGETABLES

Diatomaceous earth is typically addedto fruit and vegetable samples because oftheir high water content. In the examplesshown, all the pesticides were spiked at100 µg/kg. The same extraction conditionswere used for both banana and potato, andall extracts were analyzed by GC. Goodrecoveries and RSDs were obtained for allthe organochlorine pesticides, as shown inTables 5 and 6.

TABLE 5 . ASE EXTRACTION OF ORGANOCHLORINEPESTICIDES FROM BANANASa,b

Compound Avg. Rec. Std. RSD(n = 3) Dev. (%)

alpha-BHC 100.3 2.3 2.3beta-BHC 102.2 2.3 2.3gamma-BHC 98.9 3.2 3.2Heptachlor 89.2 7.6 8.5Aldrin 89.4 2.2 2.5Heptachlor Epoxide 93.5 2.1 2.2Dieldrin 93.7 1.6 1.74,4'-DDE 92.1 1.8 1.92,4'-DDD 95.4 2.5 2.6Endrin 94.4 2.7 3.04,4'-DDD 88.0 2.7 3.04,4'-DDT 89.6 5.8 6.4a100 µg/kg per compoundbConditions: 10-g samples mixed with 6-g diatomaceous earth, 100 °C,10 MPa (1500 psi); 5-min heatup, 5-min static, 60% flush; 60-s purge,hexane/10% acetone.


Compound Avg. Rec. Std. RSD(n = 3) Dev. (%)

alpha-BHC 96.3 6.3 6.6beta-BHC 108.6 2.3 2.1gamma-BHC 97.4 6.6 6.8Heptachlor 93.9 3.5 3.7Aldrin 95.9 3.3 3.4Heptachlor Epoxide 95.2 2.4 2.6Dieldrin 97.1 0.55 0.574,4'-DDE 95.4 0.67 0.702,4'-DDD 95.7 0.85 0.89Endrin 97.8 1.8 1.94,4'-DDD 93.7 1.8 1.94,4'-DDT 93.0 4.5 4.8a100 µg/kg per compoundbConditions: 10-g samples mixed with 6-g diatomaceous earth, 100 °C,10 MPa (1500 psi); 5-min heatup, 5-min static, 60% flush; 60-s purge,hexane/10% acetone.

TABLE 6. ASE EXTRACTION OF ORGANOCHLORINEPESTICIDES FROM POTATOESa,b

EXTRACTION OF ORGANOPHOSPHORUSPESTICIDES FROM BABY FOOD

Sample of 30 g of baby food carrots andapples were weighed out. For this study,7.5 mL of a pesticide mixture at 0.2 mg/mLwas added to the baby food for a finalconcentration of 50 mg/kg on a samplemass basis. The samples were mixed withenough Hydromatrix™ to make them easyto work with and load into the extractioncells, usually around 1:1 (w/w). Table 7shows the results.

These results confirm that pesticideresidues can be easily extracted from large-volume food samples using the ASE 300.

46


Traditional extraction methods would takefrom one to several hours for each sampleand several hundred milliliters of solventwould be used for each sample. With theASE 300, these samples can be extracted in

about 15 min each, with about 160 mL ofsolvent for each sample. In addition, theASE 300 can extract up to 12 samples se-quentially without user intervention.

50 µg/kg per compound. Conditions: 30-g samples mixed with 30-g Hydromatrix, 100 °C, 10 MPa (1500 psi); 5-min

heatup, 5-min static, 2 cycles, 60% flush; 180-s purge, methylene chloride/acetone (1:1, v/v).

TABLE 7. PERCENT RECOVERY OF ORGANOPHOSPHORUS PESTICIDES FROMSPIKED APPLE OR CARROT PUREE

Carrot Puree Apple PureeAvg. Rec. % RSD Avg. Rec. % RSD

(n = 12) (n = 12)

Dichlorvos/Naled 82 8 87 12Mevinphos 94 8 100 12TEPP 90 14 121 16Demeton-O 97 11 65 19Ethoprophos (Ethoprop) 89 8 95 12Sulfotep 85 6 95 10Phorate 88 7 86 9Demeton-S 93 18 59 18Dimethoate 125 9 128 15Diazinon 86 7 93 10Disulfoton 97 11 63 18Parathion-methyl 87 8 95 10Fenchlorphos 84 7 91 10Malathion 86 8 94 15Fenthion 88 7 86 8Chlorpyrifos 83 8 91 10Parathion-ethyl 84 12 99 11Trichloronat 86 7 89 10Tetrachlorvinphos 85 7 91 9Prothiofos 84 7 85 11Merphos 87 9 82 10Fensulfothion 91 8 98 11Sulprofos 86 8 80 10EPN 89 7 97 11Azinphos-methyl 95 9 98 11Coumaphos 90 7 98 8

47


SELECTIVE EXTRACTION OF PCBSFROM FISH TISSUE

The analysis of extracts containing PCBcontaminants from fish tissue and fishhomogenates can be hindered by the pres-ence of coextracted fatty materials thatinterfere with the chromatographic analy-sis. Cleanup procedures, including size-exclusion chromatography (SEC), columnchromatography, and acid treatment, areusually required to remove the coextractedlipids from such samples prior to analysis.These procedures are time consuming andincrease the potential for analyte losses. Analternative selective extraction procedureusing accelerated solvent extraction hastherefore been developed.

Selective ASE extractions can be per-formed with the proper choice of solventand sorbent in the extraction cell. An im-portant benefit of using a sorbent, such asalumina in this case, is that the extracts, ascollected, may not require further cleanupprior to analysis by gas chromatography.

The fish tissue sample was obtainedfrom the National Research Council ofCanada (NRC-CNRC). It is characterizedas a ground whole Carp reference materialfor organochlorine compounds (CARP-1).

A Comparison of “Nonselective” and“Selective” ASE Extractions

A chromatogram of a "nonselective" hex-ane ASE extract of the fish tissue is showncompared to that of a "selective" ASE extractof the same sample. The use of alumina in

the outlet end of the extraction cell adsorbslipids and other coextractable materials, thusyielding a much cleaner extract and greatlysimplifying the quantification of PCBs.

In general, the selective extraction usingASE gives acceptable results (see Table 7),and eliminates the need for additionalcleanup, such as sulfuric acid treatment orsize-exclusion chromatography.

Using this method, both sample prepa-ration time and the potential for analytelosses are decreased significantly.

0

1000

mV

0 10 20 30Minutes

40 50

0

1000

mV

12018

ASE Extraction of Fish Tissue

A Nonselective Hexane Extract

B Selective Extract with Alumina in Cell

Columns: DB-608 & DB-1701 (J&W)30-m × 0.53-mm i.d.

Carrier: Helium, 30 cm/sInjector: Splitless at 220 ˚C, 5 µLDetector: Electron capture at 320 ˚COven: 60–200 ˚C at 28 ˚C/min after

1-min hold, then to 265 ˚C at 10 ˚C/min and 20.5-min holdAnalyses performed byMountain States Analytical, Inc.

0 10 20 30Minutes

40 50

48


Congener Cert.a Value Extract 1 Extract 2 Extract 3 Average Std. Dev. RSD (%)

52 124 ± 32 100 107 99 102 4.4 4.3101/90 124 ± 37 101 103 100 101 1.5 1.5

105 54 ± 24 124 128 125 126b 2.1 1.7118 132 ± 60 107 109 107 108 1.2 1.1

138/163/164 102 ± 23 48 48 48 48b 0.0 N/A153 83 ± 39 48 48 48 48 0.0 N/A

170/190 22 ± 8 30 31 31 31 0.58 1.9180 46 ± 14 65 62 64 64b 1.5 2.4

187/182 36 ± 16 30 30 30 30 0.0 N/A

a 95% confidence limits are given. b Values fall outside the 95% confidence limitsConditions: 3-g samples mixed with 15-g sodium sulfate, dried, then combined with 5-g alumina; hexane; 100 °C,10 MPa (1500 psi), 5-min heatup, 5-min static, 60% flush, 90-s purge, 2 static cycles, 17-min per sample total time.

TABLE 8. RECOVERY OF PCBS FROM FISH TISSUE USING SELECTIVE ASE(concentration expressed as µg/kg)

PCBS IN LARGE-VOLUME FISH TISSUESAMPLES

The fish tissue used in this study wascod fillet obtained from a local source. Thesample had a fat content of 0.25% and amoisture level of 81%. The samples werepremixed with 20 g of pelleted diatoma-

ceous earth (Hydromatrix) prior to cellloading. Extraction results are shown inTable 9. Average recovery for the nine PCBcongeners was 96.9% with an averagepercent RSD of 6.1 (n = 5).

TABLE 9. RECOVERY OF SPIKED PCB CONGENERS FROM30-g FISH TISSUE SAMPLES

Congener BZ # Spike (µg) % Recovery % RSD2-Chlorobiphenyl 1 2.5 99.8 3.02,3-Dichlorobiphenyl 5 2.5 103.8 8.82,4,5-Trichlorobiphenyl 29 2.5 107.1 3.12,2’,4,6-Tetrachlorobiphenyl 50 5.0 98.4 2.42,2’,3,4,5’-Pentachlorobiphenyl 87 5.0 92.3 7.92,2’,4,4’,5,6’-Hexachlorobiphenyl 154 5.0 89.0 5.92,2’,3,4’,5,6,6’-Heptachlorobiphenyl 186 7.5 91.1 8.52,2’,3,3’,4,5’,6,6’-Octachlorobiphenyl 200 7.5 96.0 6.5Decachlorobiphenyl 209 12.5 94.2 8.7

Conditions: 125 °C, 1500 psi, 5-min heatup, 3-min static, 60% flush, 120-s purge, 3 static cycles, methylene chloride solvent.

100-mL cells containing 10 g alumina.

49


TABLE 10. PCB RECOVERYFROM OYSTER TISSUEa

PCB Congener Avg. Rec., n = 6 RSD(as % of Soxhlet) (%)

PCB 28 90.0 7.8PCB 52 86.9 4.0PCB 101 83.3 1.5PCB 153 84.5 3.5PCB 138 76.9 3.0PCB 180 87.0 4.3

a Analyte concentration range: 50–150 µg/kg per component.Conditions: 5–10 g, 100 °C, 14 MPa (2000 psi); 5-min heatup,5-min static, 60% flush; 60-s purge, hexane/acetone (1:1), (v/v)

EXTRACTION OF PCBS FROMOYSTER TISSUE

Table 10 shows the ASE extraction ofPCBs from oyster tissue. The table showsthe average recoveries and percent RSDsfor PCB congener content.

Sample Preparation and AnalysisOyster tissue samples were obtained

from the National Oceanographic andAtmospheric Administration (NOAA)Laboratory (Seattle, Washington, USA).Samples were mixed in equal proportionswith Hydromatrix to bind water.

Analysis and QuantificationASE extracts were passed first through

a silica gel loaded with silver nitrate/sulfuric acid; then through alumina col-umns; and concentrated to 1 mL for GCanalysis with ECD using a 30-m × 0.25-mmi.d. Rtx-5 (or equivalent) column.

50


TABLE 14. EXTRACTION OF FATFROM SWEET CEREAL

Method Solvent % Fat Std. RSD(wt. %) Dev. (%)

Soxhlet Methanol/ 10.0-12.0 N/A N/ACHCl3 (2:1)

ASEa Hexane/ 11.6 0.09 0.73IPA (3:2)

a Conditions: same as Table 5

DETERMINATION OF FAT IN VARIOUSFOOD MATRICES

The fat content of foods has been ofgrowing concern worldwide. In the U.S.,the Nutrition Labeling and Education Actrequires the labeling of total saturated andunsaturated fats contained in foods. Foodmanufacturers also require a method forroutine determination of fat content forquality-control purposes.

In currently used methods, such asSoxhlet and automated Soxhlet, the fatcontent is determined gravimetrically afterextraction with organic solvents such aschloroform or petroleum ether. Thesemethods require large volumes of solventsand time periods ranging from 2 to 16 h.Faster techniques requiring less solvent aretherefore needed.

Comparison of ASE to Soxhlet MethodsASE was applied to the determination of

fat content in various solid or semisolidfoods. The fat content was determined bycollecting the extracts in preweighed vials,evaporating the solvent with a nitrogenstream, and reweighing the vials.

Samples were provided by a number ofdifferent food companies. In all cases, theSoxhlet results shown were determined bythe food company supplying the sample.

Table 11 shows the results of extractingvarious snack foods by ASE and determin-ing their fat content. Recoveries were equi-valent to Soxhlet. Precision, expressed asrelative standard deviation (RSD), is good.

Tables 12–16 compare results for otherfoods. In most cases, there is close agreementbetween Soxhlet and ASE using the sameextraction solvent. Good agreement may

TABLE 11. EXTRACTION OF FATFROM SNACK FOODS

Sample Avg. % Fat Std. RSD(n = 5) (wt. %) Dev. (%)

Potato Chips 34.0 0.11 0.33Corn Chips 32.8 0.08 0.25Cheese Snacks 33.3 0.17 0.51Tortilla Chips 21.5 0.07 0.34Snack Chip 19.2 0.10 0.53

*Conditions: 3-g samples, 125 °C, 6.7 MPa (1000 psi), 6-min heatup,25-min static, 60% flush, 60-s purge, chloroform, 3 static cycles.

Method Solvent Avg. % Fat Std. RSD(wt. %) Dev. (%)

Soxhlet Methanol/ 20.0–22.0 N/A N/ACHCl3 (2:1)

ASE, n=3a Hexane/ 20.8 0.18 0.85IPA (3:2)

a Conditions: 3-g samples, 125 °C, 6.7 MPa (1000 psi), 6-min heatup,25-min static, 60% flush, 60-s purge, 1 static cycle.

TABLE 12. EXTRACTION OF FATFROM COOKIES

Sample Method Avg. % Fat Std. RSD(wt. %) Dev. (%)

Cracker 1 Soxhleta 15.4 N/A N/ACracker 1 ASEb, n=3 14.6 0.09 0.65Cracker 2 Soxhleta 28-30 N/A N/ACracker 2 ASEb, n=3 28.1 0.20 0.70

TABLE 13. EXTRACTION OF FATFROM SNACK CRACKERS

a After acid hydrolysisb Conditions: 5-g samples, 125 °C, 6.7 MPa (1000 psi), 6-min heatup,25-min static, 60% flush, 60-s purge, 1 static cycle, hexane/isopropanol (3:2).

51


also be obtained with a different solvent.For fat extraction of sweet cereal (Table 14),methanol/chloroform mixture was usedfor the Soxhlet extraction. However, thecompany providing the sample wanted toremove the methanol from the productionline and eliminate the use of chlorinatedsolvents (e.g., chloroform). Equivalent datawas generated for the ASE extraction bychanging the solvent to hexane/isopro-panol (3:2).

Comparison of ASE to the MojonnierMethod

The traditional Mojonnier method for thedetermination of fat in dairy products in-volves a base pretreatment (usually ammo-nium hydroxide) followed by extraction withdiethyl ether/ethanol, and petroleum ether/ethanol. The purpose of the base petreatmentis to dissolve casein and release interstitialfat. The method is both labor and time inten-sive, and requires multiple extraction steps.Comparison of ASE with Mojonnier forextraction of a variety of high-fat foods andcheeses indicates that a base pretreatment isnot required for ASE. The results obtainedwith a 30-min ASE extraction show goodagreement with the Mojonnier method.

Sample Method Solvent Avg. % Fat Std. RSD(wt. %) Dev. (%)

SRMa Soxhlet Petroleum 8.80 0.50 5.7ether

SRM ASEb Petroleum 9.12 0.15 1.6ether

Brand X Soxhlet Petroleum 10.3 N/A N/Aether

Brand X ASEb Hexane/ 10.4 N/A N/AIPA (3:2)

TABLE 15. EXTRACTION OF FATFROM DOG BISCUITS

a SRM = Standard Reference Materialb Conditions: 7-g samples, 125 ˚C, 6.7 MPa (1000 psi), 6-min heatup,25-min static, 60% flush, 60-s purge, 1 static cycle.

ASE vs Mojonnier Fat Extraction (Gravimetric)High-Fat Content Foods Common Cheeses

0

20

40

60

80

Salad Mayonnaise Cream Processed PeanutDressing Cheese Cheese Butter

Wei

ght %

Fat

ASE

Mojonnier

0

10

20

30

Wei

ght %

Fat

Mild Cheddar Monterey MozzarellaCheese Jack Cheese String Cheese

ASE/Acetone

Mojonnier

ASE/Hex:IPA

TABLE 16. EXTRACTION OF FATFROM LOW-FAT SNACK CRACKERS

Method % Fat Std. RSD(wt. %) Dev. (%)

Soxhlet 1.40 N/A N/AASEa 1.43 0.03 2.1

a Conditions: 5-g samples, 125 °C, 6.7 MPa (1000 psi), 6-min heatup,25-min static, 60% flush, 60-s purge, 1 static cycle, hexane/isopropanol(3:2).

52


Extraction of Fat from ChocolateThe extraction of fat from chocolate by

ASE is compared with an acid hydrolysis/ether extraction Mojonnier method (AOAC922.06). The ASE extraction is completed inonly 18 min and uses 20 mL of solvent ascompared with the Mojonnier method thattakes over 2 h and uses more than 110 mLof solvent. See Table 17.

TABLE 17. EXTRACTION OF FAT FROMCHOCOLATE

Sample (n = 3) Avg. % Fat SD % RSDBaking Chocolate ASE 52.80 0.35 0.67Mojonnier 51.69 0.26 0.50Milk Chocolate ASE 31.80 0.32 1.02Mojonnier 32.34 0.33 1.02Cocoa Powder ASE 11.82 0.12 1.01Mojonnier 11.52 0.15 1.33

ASE Conditions: 1-g samples mixed with Hydromatrix, 125 °C, 10 MPa

(1500 psi), 6-min heatup, 3-min static, 60% flush, 60-s purge, 3 static

cycles, Petroleum ether 100%.

Mojonnier conditions: Followed AOAC Method 922.06.

Determination of Fat in Dried MilkProducts

The samples range from very low-fatproducts such as skim milk powder to veryhigh-fat products such as cream powder.The extraction of fat from these matrices israpid and the results are equivalent to thereferenced traditional methods. Samples of2 grams are placed directly into 11-mLextraction cells. See Tables 18 and 19. Datasupplied courtesy of New Zealand DairyResearch Institute.

Samples Extraction Solvent Ratio(hexane:dichloro-

methane:methanol)Whole Milk Powder 5:2:1Cream Powder 5:2:1Skim Milk Powder 3:2:1Whey Protein Concentrate 2:3:3Whey Protein Isolate 2:3:3Sodium Caseinate 2:3:3

TABLE 18. GRAVIMETRIC COMPARISON OF ASEAND ROESE-GOTTLIEB METHODS

Sample % Fat ASE % Fat RGCream Powder 54.88 54.96Whole Milk Powder 29.41 29.45Skim Milk Powder 0.96 0.95

Conditions: 80 °C, 1500 psi, 5-min heatup, 1-min static, 100% flush, 40-s

purge, 3 static cycles, solvent as noted above.

TABLE 19. GRAVIMETRIC COMPARISON OF ASEWITH SBR AND SOXHLET METHODS

Sample % Fat ASE % Fat SBR % Fat SoxhletLactic Acid WPC Powder 5.47 4.95 5.50Acid WPC Powder 5.71 5.66 6.38Cheese WPC Powder 6.93 6.75 7.32Whey Protein Isolate 0.45 0.58 0.50Sodium Caseinate 0.66 0.65 0.55

Conditions: 80 °C, 1500 psi, 5-min heatup, 1-min static, 100% flush, 40-s

purge, 3 static cycles, solvent as noted above. SBR stands for Schmidt-

Bondzynski-Ratzlaff Method.

53


Extraction of Fat from Liquid DairyProducts

The current methods for determiningfat in dairy products, though acceptable,have several drawbacks. Many dairy-based products require a pretreatmentprior to extraction. The standard fat extrac-tion method, including the pretreatment, ismanual and thus very time consuming.Large amounts of solvent are required toremove the fat from each sample matrix.

ASE is an automated extraction tech-nique that uses the same solvents as cur-rent extraction techniques but in signifi-cantly smaller amounts and in a fraction ofthe time. ASE extraction of the followingliquid milk samples takes only 10 min anduses less than 30 mL of solvent. See Tables20 and 21. Data supplied courtesy of NewZealand Dairy Research Institute.

TABLE 20. SAMPLE PREPARATION FOR THE EXTRACTION OF FAT FROM LIQUIDMILK PRODUCTS

SAMPLE CELL SIZE, HYDROMATRIX EXTRACTION SOLVENT COMBINATION(HM) AMOUNT, SAMPLE AMOUNT

Cream 11-mL cell, 0.9 g HM, 1 g sample. Petroleum ether:acetone:isopropanol (3:2:1)Whole Milk 11-mL cell, 2 g HM, 1 g sample. Petroleum ether:isopropanol (2:1)Homogenized/UHT Milk 11-mL cell, 2 g HM, 1 g sample. Petroleum ether:isopropanol (3:2)Skim Milk 33-mL cell, 5.5 g HM, 3-5 g sample. Petroleum ether:isopropanol (3:2)

TABLE 21. MILK AND CREAM % FATRECOVERY: ASE VS ROESE-GOTTLIEB METHOD

Sample ASE Mean ± SD (n) RG MethodCream 40.62 ± 0.06 (3) 40.58Whole Milk 4.42 ± 0.02 (4) 4.50Homogenized Milk 3.39 ± 0.03 (6) 3.39Skim Milk 0.053 ± 0.010 (7) 0.053

ASE Conditions: 120 °C, 1500 psi, 6-min heatup, 1-min static, 100%

flush, 60-s purge, 3 static cycles.

54


EXTRACTION OF OILS FROM OILSEEDSOils for foods and cooking are derived

from oilseeds such as canola, soybeans,corn, flax, cotton, etc. The accurate determi-nation of seed oil content is therefore essen-tial for optimizing oil output.

Existing methods for extraction of oilfrom oilseeds use large volumes of solvent(typically several hundred milliliters) andlong extraction times (8 to 16 h).

Comparison of ASE to the CurrentOfficial Method

Extraction of canola seeds, which con-tain approximately 45-weight percent oil,serves as an excellent example for compar-ing ASE to the AOCS (American Oil Chem-ist Society) Official Method AM 2-93,which is based on the FOSFA (Federationof Oil Seeds and Fat Association) OfficialMethod. Table 22 gives the specifics of thismethod. Conditions used for the ASEextraction are listed in Table 23.

Results showed close agreement be-tween ASE and the official method. ForASE, the weight percent of oil in the seedswas 44.9% with 0.31% RSD (n = 3) as com-pared to 45.2% with 0.24% RSD (n = 12) forthe AOCS method. The histograms to theright show the percent of the total oil ex-tracted as a function of time. ASE givescomparable results faster and with lesssolvent usage than the FOSFA procedure.

Peroxide value (PV) and free fatty acid(FFA) determinations show that no signifi-cant triglyceride degradation occurs dur-ing the ASE extraction. 0

20

40

60

80

100

(%) E

xtrac

ted

240 480 720Minutes

FOSFA

10258

Extraction of Oil from Oilseeds: Comparison of ASE and FOSFA

Solvent Extraction Methods

30 60 90

ASE 200

Minutes

TABLE 22. EXTRACTION OF OIL FROM OILSEEDSAOCS Method AM 2-93 Conditions

Sample Size: 4 g ground seedsOven: 130 °C, 2 hExtract: 4 h, drain solvent and coolRegrind: 7 minExtract: 2 h, drain solvent and coolSolvent: Petroleum etherTotal Vol. Solvent Used: 150–250 mLTotal Time: 10.5 h

TABLE 23. EXTRACTION OF OIL FROM OILSEEDSASE Extraction Conditions

System Pressure: 6.7 MPa (1000 psi)Oven Temperature: 105 °COven Heatup Time: 5 minStatic Time: 10 minFlush Volume: 100%Purge Time: 60 sSolvent: Petroleum etherStatic Cycles: 3

55

APPENDIX ONE:AOAC INTERNATIONAL

APPROVED

HPLC METHODS

56

Aflatoxins in Cottonseed Products 980.20 Silica, 25 cm × 4.6 mm, 5 µm

Aflatoxins M1 and M2 in Fluid Milk 986.16 C18 Reversed Phase, 25 cm × 4.6 mm, 5 µm

Antioxidants in Oils and Fats 983.15 C18 Reversed Phase, 25 cm × 4.6 mm, 5 µm

Aprinocid in Feeds 981.27 Silica, 25 cm × 4.6 mm, 5 µm

Bacitracin in Premix Feeds 982.44 C8 Reversed Phase, 15 cm × 4.6 mm, 5 µm

Benzoate, Caffeine, and Saccharin in Soda Beverages 979.08 C18 Reversed Phase, 30 cm × 4.6 mm, 5 µm

Benzoic Acid in Orange Juice 994.11 SupelcoGel™ TPR, 15 cm × 4.6 mm, 5 µm

Domoic Acid in Mussels 991.26 C18 Reversed Phase, 15 cm × 4.6 mm, 5 µm

Fenbendazole in Beef Liver 991.17 C18 Reversed Phase, 25 cm × 4.6 mm, 5 µm

Furazolidone in Feeds and Premixes 985.51 C18 Reversed Phase, 25 cm × 4.6 mm, 5 µm

Glucose, Fructose, Sucrose, and Maltose in 982.14 NH2-bonded Silica, 25 cm × 4.6 mm, 5 µmPresweetened Cereals

Glycerol in Wine and Grape Juice 991.46 SupelcoGel C-610H, 30 cm × 7.8 µm

Glycyrrhizic Acid or Acid Salts in Licorice Products 982.19 C18 Reversed Phase, 30 cm × 4.6 mm, 5 µm

Glyphosate, Technical and Formulations 983.10 Silica-SAX, 25 cm × 4.6 mm, 5 µm

Intermediates and Reaction By products in 982.28 Silica-SAX, 25 cm × 4.6 mm, 5 µmFD&C Yellow No. 5

Intermediates in FD&C Red No. 40 981.13 Silica-SAX, 25 cm × 4.6 mm, 5 µm

Intermediates in FD&C Yellow No. 8 977.23 Silica-SAX, 25 cm x 4.6 mm, 5 µm

Iodine in Pasteurized Liquid Milk and Skim Milk Powder 992.22 C18 Reversed Phase, 15 cm × 4.6 mm, 5 µm

N-Methylcarbamate Residues in Grapes and Potatoes 985.23 C8 Reversed Phase, 15 cm × 4.6 mm, 5 µm

N-Methylcarbamoyloximes and N-Methylcarbamates 991.06 C18 Reversed Phase, 25 cm × 4.6 mm, 5 µmin Finished Drinking Water

Ochratoxin A in Corn and Barley 991.44 C18 Reversed Phase, 25 cm × 4.6 mm, 5 µm

Pesticides in Water 992.14 C18 Reversed Phase, 25 cm × 4.6 mm, 5 µm

AOAC METHOD NUMBER COLUMN TYPE

* Dionex equipment can be used for all methods listed

AOAC INTERNATIONAL:APPROVED HPLC METHODS

57

AOAC INTERNATIONAL:APPROVED HPLC METHODS

AOAC METHOD NUMBER COLUMN TYPE

Phenolic Antioxidants in Oils, Fats, and Butter Oil 983.15 C18 Reversed Phase, 25 cm × 4.6 mm, 5 µm

Purity of Lactose 984.22 NH2-bonded Silica, 25 cm × 4.6 mm, 5 µm

Quinic, Malic, and Citric Acids in Cranberry Juice 986.13 C18 Reversed Phase, 25 cm × 4.6 mm, 5 µmCocktail and Apple Juice

Saccharides (major) in Corn Syrups and Sugars 979.23 Cation exchange-Ca form resin, 30 × 7.8

Saccharides (minor) in Corn Syrups and Sugars 979.23 Cation exchange-Ca form resin, 30 × 7.8

Separation of Sugars in Honey 977.20 NH2-bonded Silica, 25 cm × 4.6 mm, 5 µm

Sugars in Licorice Extracts 984.17 NH2-bonded Silica, 25 cm × 4.6 mm, 5 µm

Sulfamethazine in Raw Bovine Milk 992.21 C18 Reversed Phase, 25 cm × 4.6 mm, 5 µm

Total Malic Acid in Apple Juice 993.05 C18 Reversed Phase, 25 cm × 4.6 mm, 5 µm

Trans-Vitamin K1 in Infant Formula 992.27 Silica, 25 cm × 4.6 mm, 5 µm

Triglycerides in Vegetable Oils 993.24 C18 Reversed Phase, 25 cm × 4.6 mm, 5 µm

Vanillin, Vanillic Acid, p-Hydroxybenzaldehyde, and 990.25 C8 Reversed Phase, 15 cm × 4.6 mm, 5 µmp-Hydroxybenzoic Acid in Vanilla Extract

Vitamin A in Milk and Milk-Based Infant Formula 992.04 C18 Reversed Phase, 25 cm × 4.6 mm, 5 µm

Vitamin D in Fortified Milk and Milk Powder 981.17 Silica, 25 cm × 4.6 mm, 5 µm

Vitamin D in Infant Formula 992.26 Silica, 15 cm × 4.6 mm, 5 µm

Vitamin D in Mixed Feeds, Premixes, and Pet Foods 982.29 Silica, 25 cm × 4.6 mm, 5 µm

Vitamin D in Multivitamin Preparations 980.26 Silica, 25 cm × 4.6 mm, 5 µm

Vitamin D in Vitamin A & D Concentrates 985.27 Silica, 25 cm × 4.6 mm, 5 µm

Vitamin D in Vitamin Preparations 979.24 Silica, 15 cm × 4.6 mm, 5 µm

Vitamin E Activity in Milk-Based Infant Formula 992.03 Silica, 25 cm × 4.6 mm, 5 µm

Zearalenone and α-Zearalenol in Corn 985.18 C18 Reversed Phase, 30 cm × 4 mm, 5 µm

59

APPENDIX TWO:RECOMMENDED

READING

60

RECOMMENDED READING

JOURNAL ARTICLES

Amines and Other BasesDraisci, R.; Cavalli, S.; Lucentini, L.; Stacchini,A. “Ion Exchange Separation and PulsedAmperometric Detection for Determinationof Biogenic Amines in Fish Products”Chromatographia 1993, 35 (9–12), 584–590.

Draisci, R.; Giannetti, L.; Boria, P.; Lucentini,L.; Palleschi, L.; Cavalli, S. “Improved IonChromatography—Integrated PulsedAmperometric Detection Method for theEvaluation of Biogenic Amines in Food ofVegetable or Animal Origin and inFermented Foods.” Chromatogr., A 1998, 798,109–116.

Hagar, A. F.; Madsen, L.; Wales, Jr., L.;Bradford, Jr., H. B. J. Assoc.“Reversed-PhaseLiquid Chromatographic Determination ofVitamin D in Milk” Off. Anal. Chem. 1994, 77,1047–1051.

Amino AcidsSingleton, J. A.; Grimm, D. T.; Sanders, T. H.“Interference of Amino Acids in PulsedAmperometric Detection of Peanut Sugars”Peanut Science 1996, 23, 61–65.

CarbohydratesAlonso, S.; Setser, “Functional Replacementsfor Sugars in Foods” C. Trends Food Sci.Tech. 1994, 5 (May), 139–146.

Akers, A. A.; Hoseney, R. C. “Water SolubleDextrins from α-Amylase-Treated Bread andTheir Relationship to Bread Firming” CerealChemistry 1994, 71, 224–226.

Corradini, C.; Canali, G.; Cogliandro, E.;Nicoletti, I. “Determination of Sugars andSugar Alcohols in Dietetic Sweeteners andFood by High-Performance Anion-ExchangeChromatography (HPAEC) Coupled withPulsed Amperometric Detection (PAD)”Proceedings of EURO FOOD CHEM VIII,Vienna, Austria, September 18–20, 1995, 2,307–310.

Corradini, C.; Canali, G.; Nicoletti, I. “Appli-cation of HPAEC to Carbohydrate Analysisin Food Products and Fruit Juices” Seminarsin Food Analysis 1997, 2, 99–111.

Corradini, C.; Cristalli, A.; Corradini, D.“HighPerformance Anion-Exchange Chroma-tography with Pulsed Amperometric Detectionof Nutritionally Significant Carbohydrates” J. Liq. Chromatogr. 1993, 16, 3471–3485.

Craig, S. A. S.; Holden, J. F.; Khaled, M. Y.“Determination of Polydextrose as DietaryFiber in Foods”J. AOAC Int. 2000, 83 (4),1006–1009.

Craig, S. A. S.; Holden, J. F.; Khaled, M. Y.“Determination of Polydextrose in Foods byIon Chromatography: Collaborative Study” J.AOAC Int. 2001, 84 (2), 472–478.

Day-Lewis, C. M. J.; Schäffler, K. J.“Analysis of Sugar in Final Molasses byIon Chromatography” Proc. S. A. SugarTechnol. Assoc., June 1992.

Déséveaux, S.; Daems, V.; Delvaux, F.;Derdelinckx, G. “Analysis of FermentableSugars and Dextrins in Beer by Anion-Exchange Chromatography withElectrochemical Detection” Seminars in FoodAnalysis 1997, 2, 113–117.

Eggleston, G.; Clark, M. A. “Applications ofHPAE-PAD in the Sugar Industry” Seminarsin Food Analysis 1997, 2, 119–127.

61

RECOMMENDED READING

Garleb, A. K.; Bourquin, L. D.; Fahey, Jr., G.C. “Neutral Monosaccharide Composition ofVarious Fibrous Substances: A Comparisonof Hydrolytic Procedures and Use of Anion-Exchange HPLC with PAD Detection ofMonosaccharides” J. Agric. Food Chem 1989,37, 1287–1293.

Giese, J. H. “Alternative Sweeteners andBulking Agents” Food Technol. 1993, (January),114–126.

Hotchkiss, Jr., A. T.; Hicks, K. B.“Analysis ofOligogalacturonic Acids with 50 or FewerResidues by High-Performance AnionExchange Chromatography and PulsedAmperometric Detection” Anal. Biochem.1990, 184, 200–206.

Hoebregs, H. “Fructans in Foods and FoodProducts, Ion Exchange ChromatographicMethod: Collaborative Study” PosterPresented at the 110th AOAC InternationalMeeting, Orlando, FL, Sept. 8–12, 1996.

Lamb, J. D.; Myers, G. S.; Edge, N. “IonChromatographic Analysis of Glucose,Fructose, and Sucrose Concentrations in Rawand Processed Vegetables” J. Chromatogr.Sci. 1993, 31, 353–357.

Madigan, D.; McMurrough, I.; Smyth, M. R.“Application of Gradient Ion Chromatographywith Pulsed Electrochemical Detection to theAnalysis of Carbohydrates in Brewing” J. Am. Soc. Brew. Chem. 1996, 54 (1), 45–49.

Quemencer, B.; Thibault, J. F.; Coussement,P.“Determination of Inulin and Oligofructosein Food Products, and Integration in theAOAC Method for Measurement of TotalDietary Fibre” Lebensm.-Wiss. Technol. 1994,27, 125–132

Schäffler, K.; Day-Lewis, C. M. J.; Clarke, M.;Jekot, J. “Determination of Sugars in Beet andCane Final Molasses by Ion Chromatography:Collaborative Study”J. of AOAC Int. 1997, 80(3), 603–610.

Stumm, I.; Baltes, W. Z. “Determination ofPolydextrose in Food by Means of IonChromatography and Pulsed AmperometricDetection” Unters. Forsch 1992, 195, 246.

Swallow, K. W.; Low, N. H. “Detection ofOrange Juice Adulteration with Beet MediumInvert Sugar Using Anion Exchange LiquidChromatography with Pulsed AmperometricDetection” J. Assoc. Off. Anal. Chem. 1991, 74,341–343.

Thayer, A. M. “Food Additives” Chem. Eng.News 1992, June 15, 26–43.

Thompson, J. C. “Ion ChromatographicAnalysis of Sugars in Foods and Molasses”Proceedings of Sugar Processing ResearchInstitute Workshop on Analysis of Sugars inFoods 1992, M. C. Clarke, Ed.

Tsang, W. S. C.; Cargel, G.-L. R.; Clarke, M. A.“Ion Chromatographic Analysis of Oligosac-charides in Beet Sugar” Zuckerind. 1991, 116,No. 12, 1058–1061.

White, D. R., Jr.; Widmer, W. W. “Applicationof High Performance Anion-ExchangeChromatography with Pulsed AmperometricDetection to Sugar Analysis of Citrus Juices”J. Agric. Food Chem. 1990, 38, 1918–1921.

Wong, K. S.; Jane, J. “Effect of PushingAgents on the Separation and Detection ofDebranched Amylopectin by High-Performance Anion-ExchangeChromatography with Pulsed Ampero-metric Detection” J. Liq. Chromatogr. 1995,18 (1), 63–80.

62

Inorganic Anions and CationsBaluyot, E.; Hartford, C. G. “Comparisonof Polyphosphate Analysis by IonChromatography and by Modified End-Group Titration” J. Chromatogr. 1996, 739,217–222.

Bettler, K. M.; Chin, H. B.“ImprovedDetermination of Chlorite and Chlorate inRinse Water from Carrots and Green Beansby Liquid Chromatography and Ampero-metric and Conductivity Detection” J. Assoc.Off. Anal. Chem. Int. 1995, 78 (3), 878–883.

Buckee, G. K. “Determination of Anions inBeer by Ion Chromatography” J. Inst. Brew.1995, 101 (6), 429–430

de Bruijn, J. M.; Heringa, R. “Determinationof Anions and Cations in Sugar FactorySamples by Ion Chromatography” Presentedat the 1992 Conference on Sugar ProcessingResearch, New Orleans, LA, September 1992.

Dolenc, J.; Gorenc, D. “Ion ChromatographicDetermination of Inorganic Anions in VinegarSamples” Die Nahrung 1994, 4, 434–438.

Gaucheron, F; Le Graet, Y; Piot, M; Boyaval,E. “Determination of Anions of Milk by IonChromatography” Lait 1996, 76, 433–443.

Madigan, D; McMurrough, I; Smyth, M. R.“Determination of Oxalate in Beer and BeerSediments Using Ion Chromatography”J. Am. Soc. Brew. Chem. 1994, 52 (3), 134–137.

Pereira, C. F. “Application of IonChromatography to the Determination ofInorganic Anions in Food-stuffs”J. Chromatogr. 1992, 624, 457–470.

Perez-Cerrada, M.; Casp, A.; Maquieira, A.“Chromatographic Determination of theAnion Content in Spanish Rectified Musts”Am. J. Enol. Vitic. 1993, 44 (3), 292–296.

Sekiguchi, Y.; Matsunaga, A.; Yamamoto, A.;Inoue, Y. “Analysis of Condensed Phosphatesin Food Products by Ion Chromatographywith an On-Line Hydroxide EluentGenerator” J. Chromatogr., A 2000, 881,639–644.

Wagner, H. L.; McGarrity, M. J. “The Use ofPulsed Amperometry Combined with Ion-Exclusion Chromatography for the Simultan-eous Analysis of Ascorbic Acid and Sulfite”J. Chromatogr. 1991, 546, 119–124.

Organic AcidsBarber, E. L.“The Analysis of Organic Acidsby Ion Chromatography in Beer and Wort”J. Am. Soc. Brew. Chem. 1990, 48, 44–46.

Boyles, S.“Method for the Analysis ofInorganic and Organic Acid Anions in AllPhases of Beer Production Using GradientIon Chromatography” J. Am. Soc. Brew.Chem. 1992, 50, 61–63.

Johnson, D. C.; Ngoviwatchai “PulsedAmperometric Detection of Sulfur-Containing Pesticides in Reversed-PhaseLiquid Chromatography” Anal. Chim. Acta1988, 215, 1–12.

Saccani, G.; Gheradi, S.; Trifiro, A.; SoresiBordini, C.; Calza, M.; Freddi, C. “Use of IonChromatography for the Measurement ofOrganic Acids in Fruit Juices” J. Chromatogr.1995, 706, 395–403.

Talmond, P.; Doulbeau, S.; Rochette, I.;Guyot, J. P.; Treche, S. “Anion-ExchangeHigh-Performance Liquid Chromatographywith Conductivity Detection for the Analysisof Phytic Acid in Food0” J. Chromatogr., A2000, 871, 7–12

RECOMMENDED READING

63

VitaminsAlbalá-Hurtado, S.; Novella-Rodrígues, S.;Veciana-Nogués, M. T.; Mariné-Font, Abel.“Determination of Vitamins A and E in InfantMilk Formulae by High-Performance LiquidChromatography” J. Chromatogr, A, 1997, 778,243–246.

Chen, B. H.; Peng, H. Y.; Chen, H. E. “Changesof Carotenoids, Color, and Vitamin A Contentsduring Processing of Carrot Juice” J. Agric.Food Chem. 1995, 43, 1912–1918.

Haroon, Y.; Shearer, M. J.; Rahim, S.; Gunn,W. G.; McEnery, G.; Barkhan, P. “The Contentof Phylloquinone (Vitamin K1) in HumanMilk, Cows’ Milk and Infant Formula FoodsDetermined by High-Performance LiquidChromatography” J. Nutr. 1985, 112, 1105–1117.

Other“Application of High Performance AnionExchange Chromatography with PulsedAmperometric Detection in Food andBeverage Analysis” Seminars in Food Analysis1997, 2, 3–4.

Epler, K. S.; Ziegler, R. G.; Craft, N. E.“Liquid Chromatographic Method for theDetermination of Carotenoids, Retinoids andTocopherols in Human Serum and in Food”J. Chromatogr 1993, 619, 37–48.

Henshall, A. “Liquid ChromatographicTechniques for Detecting EconomicAdulteration of Foods” Cereal Foods World1998, 43 (2), 98–103.

Henshall, A. “Use of Ion Chromatography inFood and Beverage Analysis” Cereal FoodsWorld 1997, 42 (5), 414–419.

Koswig, S.; Fuchs, G.; Hotsommer, H. J.“The Use of HPAE-PAD for the Analysis ofThickening Agents in Fruit Juice and FoodAnalysis” Seminars in Food Analysis 1997, 2,71–83.

LaCourse, W. R.; Dasenbrock, C. O.; Zook, C.M. “Fundamentals and Applications ofPulsed Electrochemical Detection in FoodAnalysis” Seminars in Food Analysis 1997, 2,5–41.

Low, N. H. “Food Authenticity Analysis byHigh Performance Anion ExchangeChromatography with Pulsed AmperometricDetection” Seminars in Food Analysis 1997, 2,55–70.

Obana, H.; Kikuchi, K.; Okihashi, M.; Hori, S.“Determination of OrganophosphorusPesticides in Foods Using an AcceleratedSolvent Extraction System” The Analyst 1997,122, 217–220.

Okihashi, M.; Obana, H.; Hori, S.“Determination of N-MethylcarbamatePesticides in Foods Using an AcceleratedSolvent Extraction with a Mini-ColumnCleanup” The Analyst 1998, 123, 711–714.

RECOMMENDED READING

64

Dionex Presentations, Application Notes,and Technical NotesA Comparison of Conductivity andPostcolumn Derivatization Methods for theDetermination of DBP Anions in DrinkingWater Using Ion Chromatography”; DionexPresentation 2338.

“Recent Advances in IC-PAD (HPAE-PAD)Detection for the Analysis of Carbohydratesin Grain Products” Dionex Presentation 2328.

“Single Injection Determination of Sugars,Organic Acids, and Alcohols in Beveragesand Food” Dionex Presentation 1826.

“Accelerated Extraction of Vitamins fromTablets and Food Samples” DionexPresentation 2349.

Accelerated Solvent Extraction (ASE) ofMycotoxin Contaminants in Food Matrices”Dionex Presentation 2354.

“Use of Accelerated Solvent Extraction (ASE)for Analysis of Trace Contaminants in Foods”Dionex Presentation 2228.

“Use of Accelerated Solvent Extraction (ASE)for Analysis of Trace Contaminants in Foods”Dionex Presentation 2337.

AN#21: Organic Acids in Wine

AN#25: Analysis of Inorganic Anions andOrganic Acids in Carbonated Beverages

AN#37: Determination of Iodide in DairyProducts

AN#46: Ion Chromatography: A VersatileTechnique for the Analysis of Beer

AN#54: Determination of Sulfite in Foodand Beverages with Pulsed AmperometricDetection

AN#67: Determination of Plant-DerivedNeutral Oligo- and Polysaccharides

AN#70: Choline and Acetylcholine

AN#71: Determination of PolyphosphatesUsing Ion Chromatography with SuppressedConductivity Detection

AN#82: Analysis of Fruit Juice Adulteratedwith Medium Invert Sugar from Beets

AN#83: Size-Exclusion Chromatography ofPolysaccharides with Pulsed AmperometricDetection

AN#87: Determination of Sugar Alcohols inConfections and Fruit Juices by High-Perfor-mance Anion Exchange Chromatographywith Pulsed Amperometric Detection

AN#92: Determination of Sugars inMolasses by High-Performance AnionExchange Chromatography with PulsedAmperometric Detection

AN#112: Determination of Nitrate andNitrite in Meats

AN#124: Determination of Choline in Milkand Infant Formula

AN#142 : Determination of TryptophanUsing AAA-Direct

AN#143: Determination of Organic Acids inFruit Juices

AN#147: Determination of Polydextrose inFoods by AOAC Method 2000.11

AN#149: Determination of Chlorite, Bromate,Bromide, and Chlorate in Drinking Water byIon Chromatography with an On-LineGenerated Postcolumn Reagent for Sub-µg/LBromate Analysis

AN#155: Determination of Trans-Galactooligosaccharides in Foods by AOACMethod 2001.02

RECOMMENDED READING

65

AN#314: Determination of Unbound Fat inVarious Food Matrices Using AcceleratedSolvent Extraction (ASE)

AN#316: Extraction of PCBs from Environ-mental Samples Using Accelerated SolventExtraction (ASE)

AN#321: Determination of Unbound Fat inVarious Food Matrices Using AcceleratedSolvent Extraction (ASE)

AN#322: Selective Extraction of PCBs fromFish Tissue Using Accelerated SolventExtraction (ASE)

AN#325: Extraction of Oils from Oilseeds byAccelerated Solvent Extraction (ASE)

AN#329: Determination of Total Fat in InfantFormula Using Accelerated SolventExtraction (ASE)

AN#340 : Determingtion of Fat in Dried MilkProducts Using Accelerated SolventExtraction (ASE)

AN#342: Determingtion of PCBs in Large-Volume Fish Tissue Samples UsingAccelerated Solvent Extraction (ASE)

AN#343: Determination of Pesticides inLarge-Volume Food Samples UsingAccelerated Solvent Extraction (ASE)

AN#344 : Extraction of Fat from ChocolateUsing Accelerated Solvent Extraction (ASE)

AN#345: Extraction of Fat from DairyProducts (Cheese, Butter, and Liquid Milks)Using Accelerated Solvent Extraction (ASE)

AN#409: Fast Determination of Acrylamidein Food Samples Using Accelerated SolventExtraction (ASE) Followed by IonChromatography with UV or MS Detection

TN#20: Analysis of Carbohydrates by High-Performance Anion Exchange Chromatogra-phy with Pulsed Amperometric Detection(HPAE-PAD)

TN#21: Optimal Settings for PulsedAmerometric Detection of CarbohydratesUsing Dionex Pulsed Electrochemical andAmperometric Detectors

TN#50: Determination of the Amino AcidContent of Peptides by AAA-Direct

TN#55 : Screening of Sample Matrices andIndividual Matrix Ingredients for Suitabilityin AAA-Direct

RECOMMENDED READING

66

RECOMMENDED READING

The following series of journal articles is published in Seminars in Food Analysis;Hurst, W. J., Ed.; Chapman & Hall, 1997; Vol. 2, No. 1/2.

Campbell, J. M.; Flickinger, E. A.;Fahey, G., Jr. “A Comparative Study ofDietary Fiber Methodologies Using PulsedElectrochemical Detection of MonosaccharideConstituents” Dept. of Animal Sciences,University of Illinois, Urbana, IL, USA.

Coraddini, C.; Canali, G.; and Nicoletti, I.“Application of HPAEC-PAD to CarbohydrateAnalysis in Food Products and Fruit Juices”C.N.R. Istituto di Cromatografia del C.N.R.,Rome, Italy.

Deseveaux, S.; Daems, V.; Delvaux, F.;Derdelincx, G. “Analysis of FermentableSugars and Dextrins in Beer by HPAEC-PAD”University of Louvain, Centre for Maltingand Brewing Science, Leuven, Belgium.

Durgnat, J. M.; Martinez, C. “Determinationof Fructooligosaccharides in Raw Materialsand Finished Products by HPAE-PAD”Nestec Ltd., Nestlé Research Centre,Lausanne, Switzerland.

Eggleston, G. “Applications of HPAE-PAD inthe Sugar Industry” U.S. Dept. of Agriculture,Agricultural Research Service, SRRC, NewOrleans, LA; and Clarke, M., Sugar ProcessingResearch Institute, New Orleans, LA.

Koswig, S.; Fuchs, G.; Hotsommer, H. J.;Graefe, U.“The Use of HPAE-PAD for theAnalysis of Thickening Agents in Fruit Juiceand Food Analysis” Gesellschaft fürLebensmittel-Forschung mbH, Berlin,Germany.

Lacourse, W. R.; Dasenbrock, C. O.; Zook,C. M. “Fundamentals and Applications ofPulsed Electrochemical Detection in FoodAnalysis” Dept. of Chemistry and Biochem-istry, University of Maryland, BaltimoreCounty, MD, USA.

Low, N. H. “Food Authenticity Analysis byHigh-Performance Anion-ExchangeChromatography with Pulsed AmperometricDetection” Dept. of Applied Microbiologyand Food Science, University ofSaskatchewan, Saskatchewan, Canada.

67

INDEX

69

INDEX

AAccelerated Solvent Extraction 39–54

vs Mojonnier Method 51vs Soxhlet 40–41, 50–51

Additives 17Adulteration of:

Coffee 25Natural Fruit Juices 26

Amines and Other Organic Bases 15–18Biogenic 17Choline 16Vitamins 18

Amylopectins 30Anions and Cations, Inorganic 5–10Anions and Organic Acids in an

Irish Stout 14AOAC International: Officially

Approved HPLC Methods 55–57AOAC Method 973.28 21AOCS (American Oil Chemist

Society) 54Apricots (Dried) and Sulfite 8Approved IC Methods 2Artificial Sweetener from Japan 29ASE (Accelerated Solvent Extraction):

ASE 100, 200, and 300 42Schematic of Operation 41Technology and Features 41–42

BBaby Food 45–46Baked Goods and Bromate 7Bananas and Pesticides 45Beer, Wine, or Cider Production 27Beet Sugar 26Beverages and Sweeteners 26

Bread and Bromate 7Bromate in:

Baked Goods 7Drinking Water 7

Bulking Agents 28

CCandy (Hard) 22Carbohydrates 19–31Cations in:

Mineral Water and DrinkingWater 7

Soft Drinks and Wine 7Cereal, Extraction of Fat 50–51Cheese:

Extraction of Fat 51Phosphates in Cheese Products 10

Chewing Gum 22Chocolate 24, 52Coffee Adulteration 25Column Technologies 35Cookies, Extraction of Fat 50Crackers, Fat Extraction 51

DDairy Products:

Iodide in 9Fat, Extraction of 52–53

Detector Technologies 36Dionex Application and Technical

Notes 64Dionex LC Technologies 33–37Dog Biscuits, Fat Extraction 51Drinking Water 6–7Dyes 14

70

EEstablishing Geographic Origin 26EPA, U.S. 3Extraction (ASE) of:

Fat from:Cheese 51Chocolate 52Cookies 50Dog Biscuits 51Dried Milk Products 52High-Fat Content Food

(Salad Dressing, Mayonnaise,Cheeses, Peanut Butter) 51

Liquid Dairy Products 53Low-Fat Snack Crackers 51Snack Foods 50Sweet Cereal 50

Fungicides from Grain 44Herbicides from Grain 44Oils From Oilseeds 54Organochlorine Pesticides from

Fruits and Vegetables 45PCBs from:

Fish Tissue 47–48Oyster Tissue 49

Pesticides from:Baby Food 45–46Grain 41–42

FFat, Extracted from:

Cheese 51Chocolate 52Cookies 50Dog Biscuits 51Dried Milk Products 52High-Fat Content Food

(Salad Dressing, Mayonnaise,Cheeses, Peanut Butter) 51

Liquid Dairy Products 53

Low-Fat Snack Crackers 51Snack Foods 50Sweet Cereal 50

Fat Substitutes 28Fermentable Sugars 27Fish:

Extraction of PCBs 47–48Seafood Spoilage 17

Flavor Constituents and Additives 17Flavored Potato Chip Extract 24FOSFA (Federation of Oilseeds and

Fat Association) 54Fruit and Fruit Juices:

Oligogalacturonic Acidsfrom Citrus Pectin 31

Organic Acids in:Cranberry Juice 12Orange Juice 13Grape Juice 13Apple Juice 13

Sugars in Orange Juice 31Sulfite in Dried Apricot 8

Fruits and Vegetables:Extraction of Organochlorine

Pesticides 45Triazine Herbicides in 18

GGlucose, Fructose, Maltose, and

Maltotriose 23–24Glucose Syrup 21Grape Juice, Organic Acids in 13

HHam, Nitrates/Nitrites in 9Herbicides in:

Fruits and Vegetables 18Grain 44

High-Fat Foods 24HPAE-PAD, description of 20

INDEX

71

IIC Methods for Food 2–4ICUMSA 23Inorganic Anions and Cations 5–10Inorganic Cations, Choline, and

Acetylcholine 16Inulins 28Iodide in Whole Milk 9Irish Stout 14ISO/DIS 11292, for coffee

adulteration 25

JJournal Articles 60–63Juices. See Fruit and Fruit Juices.

KKestoses 29

LLow-Fat Snack Crackers,

Extraction of Fat 51

MMaltodextrins 29Mayonnaise, Extraction of Fat 51Methylamines 16Milk and Iodide 9Milk Products, Fat Extraction 52–53Molasses:

Beet Sugar, Geographic Origin 26Sugars in 23

Monjonnier Method, vs ASE 51–52Municipal Drinking Water 6

NNatural Fruit Juices 26Nitrite/Nitrate in Ham 9Nonselective vs Selective ASE 47Nutritional Labeling Requirements 21Nutritive Sweeteners 23

OOfficially Approved IC Methods 2–3Oils From Oilseeds, Extraction of 54Oligo- and Polysaccharides 21Oligogalacturonic Acid:

Fingerprinting 31From Citrus Pectin 31

Oligosaccharide Profiling 26Oligosaccharides 21, 26Orange Juice. See Fruit and Fruit Juices.Organic Acids in:

Irish Stout 14Fruit Juice 12–13

Organochlorine Pesticides in Fruitsand Vegetables 45

Organophosphorus and Baby Food45–46

Oyster Tissue, Extraction of PCBs 49

PPCBs, Extraction from:

Fish Tissue 47–48Oyster Tissue 49

Peanut Butter, Extraction of Fat 51Pectin 31Pesticides, Extraction from:

Fruits and Vegetables 45Grain 43–44

Polyphosphates 10Polysaccharides 21Potato Chips:

Extraction of Fat 50Flavor Additives 22

Potatoes and Pesticides 45Pulsed Amperometric Detection 36Pump Technology 37Purity. See Adulteration.

RRecommended Reading 25, 59–65

INDEX

72

SSalad Dressing, Extraction of Fat 51Seafood Spoilage, Amines 17Selective vs Nonselective ASE 47Snack Foods, Extraction of Fat

from 50–51Soft Drinks 7Stout, Organic Acids in 14Sucralose 28Sucrose, Maltose, Lactose, Dextrose

and Fructose 23Sugar Alcohols 21–22Sugar Alcohols in Dietetic Hard

Candy and Chewing Gum 22Sugars in:

Beer 27Foods 24High-Fat Foods 24Molasses 23Orange Juice 31

Sulfite in Dried Apricot 8Suppressed Conductivity Detection 36Sweet Cereal, Extraction of Fat 50Sweeteners 23–24, 26, 28–29Syrup, Glucose 21

TTomato Ketchup 24Transition Metals 8Triazine Herbicides in Raw Fruits

and Vegetables 18

UUnited States Environmental

Protection Agency (U.S. EPA) 3

VVitamins, Water-Soluble 18

WWater:

Drinking 6–7Mineral 7

Wheat and Pesticides 43–44Wine 7

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