Reverse phsase chromatography 1

ByAmol D Sagulale

By Amol D Sagulale Sr. Reseach Associae-II Macleod Pharmaceuticals, Mumbai Email: amol.sagulale@gmail.com

Normal phase chromatography

•It was one of the first kind of HPLC that chemist developed .

•Also known as adsorption chromatography.

•This method uses a polar stationary phase and a non-polar, non-aqueous mobile phase, and works effectively for separating analytes readily soluble in non-polar solvents.

SiSi

SiOHOH

OHOHOH

OH

surface of silica gel

Packing material

•The most popular packing material is silica gel.

•It is believed that silanol radicals ( -Si-OH ) on the surface of silica gel act as the active site and the sample is separated.

Normal Phase Chromatography : Separation mode

Stationary Phase in NPC•Bare silica or alumina that have polar hydroxyl group on the surface

--•silica is preferred over alumina due to its low cost, known performance and ready availability -•For very basic compounds (e.g., amines) alumina is a better choice because amines are retained longer on silica

Si

OH

Al

OH

Silica Alumina

Si

OH

Al

OH

Silica Alumina

Si

OH

Al

OH

Silica Alumina

Si

OH

Al

OH

Silica Alumina

Si

OH

Al

OH

Silica Alumina

Si

OH

Al

OH

Silica Alumina

-A variety of bonded phases (BP) can be prepared for NPC. However, functional groups in BP are less polar than the bare silica column

Reversed phase HPLC Reversed phase HPLC • In the 1970s most liquid chromatography was done on non-modified

silica or alumina with a hydrophilic surface chemistry and a stronger affinity for polar compounds - hence it was considered "normal".

• The introduction of alkyl chains bonded covalently to the support surface reversed the elution order. Now polar compounds are eluted first while non-polar compounds are retained - hence "reversed phase".

Reverse Phase ChromatographyReverse Phase Chromatography

• The term “Reverse Phase Chromatography” was used because RP is a form of partition chromatography where chemically bonded phase is

hydrophobic or non-polar (e.g. octadecyl group), and the starting mobile phase (e.g. water) must be more polar than the stationary phase.

• It is the most widely used technique in HPLC.

• .

Reversed Phase ChromatographyReversed Phase Chromatography

It operates on the principle of hydrophobic interactions which result from repulsive forces between a relatively polar solvent, the relatively non-polar analyte, and the non-polar stationary phase.

The components of the analyte mixture pass over stationary-phase particles bearing pores large enough for them to enter, where interactions with the hydrophobic surface removes them from the flowing mobile-phase stream.

Reversed Phase Chromatography :Separation mode

CH3 CH2COOCH3

CH3 CH2COOCH3

Silica-C18 (ODS)

Hydrophobic InteractionHydrophobic Interaction

Reverse Phase MechanismReverse Phase Mechanism

• Reversed phase chromatography is an adsorptive process by experimental design,which relies on a partitioning mechanism to effect separation.

• The solute molecules partition (i.e. an equilibrium is established) between the mobile phase and the stationary phase.

• The distribution of the solute between the two phases depends on the binding properties of the medium, the hydrophobicity of the solute and the composition of the mobile phase.

Reverse Phase ChromatographyReverse Phase Chromatography

• One common stationary phase is a silica which has been treated with RMe2SiCl, where R is a straight chain alkyl group such as C18H37 or C8H17.

• With these stationary phases, retention time is longer for molecules which are more non-polar, while polar molecules elute more readily.

Reverse Phase ChromatographyReverse Phase Chromatography

• strong attraction between the polar solvent and polar molecules in the mixture being passed through the column.

• much attraction between the hydrocarbon chains attached to the silica (the stationary phase) and the polar molecules in the solution.

• Polar molecules in the mixture will therefore spend most of their time moving with the solvent.

Reverse Phase ChromatographyReverse Phase Chromatography

• Non-polar compounds in the mixture will tend to form attractions with the hydrocarbon groups because of van der Waals dispersion forces.

• They will also be less soluble in the solvent because of the need to break hydrogen bonds as they squeeze in between the water or methanol molecules.

• They therefore spend less time in solution in the solvent and this will slow them down on their way through the column.

Reverse Phase ChromatographyReverse Phase Chromatography

• The retention time can be increased by adding more water to the mobile phase; thereby making the affinity of the hydrophobic analyte for the hydrophobic stationary phase stronger relative to the now more hydrophilic mobile phase.

• Similarly, the retention time can be decrease by adding more organic solvent to the eluent.

Si

Si

O － Si － CH2(CH2)16CH3

CH3

CH3

O － Si － CH2(CH2)16CH3

CH3

CH3

O － Si － CH2(CH2)16CH3

CH3

CH3

CH3

CH3

O － Si － CH3

Commonly used packing materials are hydrocarbons having 18 carbon atoms (called the Octadecyl radical) which are chemically bonded to silica gel (Silica-ODS).Since the surface of the Silica-ODS is covered with hydrocarbon, the polarity of the packing material itself is very low.

Reversed Phase Chromatography :Separation mode

Principle of reverse phase chromatographyPrinciple of reverse phase chromatography

Gradient elution

• To equilibrate the column packed with reverse phase medium under suitable initial mobile phase conditions of –

• pH

• Ionic strength

• Polarity ( mobile phase hydrophobicity)

• The polarity of the mobile phase is controlled by adding organic modifiers or ion –pairing agents.

• Polarity of initial mobile phase ( usually reffered to as mobile phase A ) must be low enough to dissolve the partially hydrophobic solute

• Yet high enough to ensure binding of the solute to the reverse phase chromatographic matrix.

• Sample containing the solutes to be separated is applied .

• The sample is applied to the column at a flow rate where optimum binding will occur.

• Chromatographic bed is washed further with mobile phase .

• Bound solutes are next desorbed from the reverse phase medium by adjusting the polarity of mobile phase so that the bound molecule will sequentially desorbs and elute from column.

• Removing the substances not previously desorbed.

• Re-Equilibration of the chromatographic medium from 100% mobile phase B back to the initial mobile phase conditions.

Ion-pairing agents• Ion-pairing agents are ionic compounds that

contain a hydrocarbon chain that imparts a certain hydrophobicity so that the ion pair can be retained on a reversed-phase column.

• Ion Pairing agents are added at concentrations of 0.05 to 0.2.

• All ion-pairing agents are potentially capable of ion-pairing with the positively charged basic residues of peptides or proteins, thus reducing hydrophilicity and increasing their retention time

• Hydrophobic counterions such as TFA and HFBA in addition to ion-pairing with the positively charged solute also increase the affinity of the solute (peptide or protein) for the hydrophobic stationary phase.

• While hydrophilic counterions such as following ion-pair formation with positive charged residues would be unlikely to interact with the stationary phase.

Trifluoroacetic acid (TFA).Heptafluorobutyric acid (HFBA).Hexafluoroacetone (HFA).Formic Acid (FA)Phosphoric Acid.Hydrochloric Acid.Triethylamine Phosphate (TEAP).

Organic modifiers• Additive that changes the character of the mobile

phase. In RP chromatography, water is the weak solvent, and acetonitrile, the strong solvent is added gradually to generate a gradient.

• Acetonitrile.• Isopropanol.• Methanol.• Ethanol

• Acetonitrile is the reverse phase solvent of choice because the UV cut off for acetonitrile is190 nm, allowing detection at lower wavelengths.

• It is less viscous than methanol, thus causing less fluctuations in pressure.

• Less bubble formation occurs when it is mixed with water. It has also better selectivity for peptides and proteins.

• Isopropanol is used either alone or in combination with acetonitrile to elute large or

hydrophobic proteins.

Quality of Stationary phasesQuality of Stationary phases

Determined by their physical and chemical properties Physical Properties :PorositySpecific surface areaParticle sizeParticle shapePore size

Greatly determines the efficiency of packing.

Quality of Stationary phasesQuality of Stationary phases

• Must be controlled in narrow tolerances to enable manufacturer for reproducible packing materials.

• Porosity : Determines the surface area & others parameters.

• Retention

• Selectivity

Chemical properties Chemical properties

• Result of substrare properties

&

• Applied surface bonding chemistry

• These forms the basis of retention and selectivity.

SubtratesSubtrates

• Inorganic oxides

• Polymers

• Carbons

• Have sufficient hydrophobic properties & used unmodified as RP stationary phases.

• Majority of presently available RPLC stationary phases are modified substrates.

SubtratesSubtrates

• Substrates & Stationary phases must posses physical and chemical properties to be suitable as Stationary phase.

• Mechanical strength

• No Shrinking & swelling properties

SilicaSilica

• Silica & silica base are the ideal materials.

• Synthesized in pure form & yield a large number

of substrates.

• Well defined physical properties.

• Possess sufficient mechanical strength

• No shrinking & swelling properties.

SilicaSilica Bonding chemistry of silica results into high

quality of RPLC-phase.

It covers a broad spectrum of different organic ligands attached to a variety of silicas & enables the separation of many different substances.

Eg. Neutral molecules in lower mol. Wt range.

Charge molecules by ion-pair

Silica based stationary phasesSilica based stationary phases

• Hydrogel from inorgnic silicates & alkoxy silicates Grinding and sieving yields irregular shape silica

substrate is obtained ( characterized as Xerogel)

Relatively high surface area High Porosity Variable wall thickness

SilGel

Silica based stationary phasesSilica based stationary phases

• Consolidation of silica particles by either

• oil emulsion or Coacervation

• Results in sphere shape particles

• Lower surface areas

• Lower porosities

• Regular shape having thicker wall• SolGel

Silica surfacesSilica surfaces

Silanol groups Siloxane bridgesAcidic reactive sites Hydrophobic unreactive

a)Single (geminal silanol) b)vicinal silanols SilanediolsMost reactive sites

•Responsible for residual silanol activity of bonded silicas for basic compounds.•Silanols cause peak tailing and excessive retention

Types of silanol on surface of silica gelTypes of silanol on surface of silica gel

Pretreatment stepsPretreatment steps

• Heating

• Rehydroxylation

• Homogenization

• In order to reduce the single silanols

• To obtain as many as bonded or

associated silanols of similar avctivity.

Analytical PurposeAnalytical Purpose• Silica usually produced of nominal 2,3,5 and 10 um

particle size• Surface area – 100-600 m2/gm• Particle porosity =0.6-0.7• Surface density 8 umol/m2• Equivalent to = ± 4.5 silanols/nm2• For large molecules = 30 -100nm• For unrestricted access to inner surface for smaller

molecules, the pore size is not less than 10 nm.

• Silica substrate uses alkoxysilane or chlorosilanes to attach organic ligands through siloxysilane linkages to the supports surface.

• To produce such covalently bonded organic stationary phases, the reactive alkoxy or chlorosilane reagent must contain atleast one leaving group which is able to reacts with silanol at substrate surface.

Surface hydrophobisation reactionSurface hydrophobisation reaction

• It is carried out under anhydrous condition.

• The is catalysed by base 2,6-lutidine or imidazole.

• It act as a scavenger base to neutralise acid

byproducts.

• Further step includes reflux, sonication, filtration,

rinsing and drying steps.

• Depending on the no. of leaving groups for synthesis of RPLC phase, three groups of organosilane reagents can be distinguished :-

• From the originally available no. of silanol groups, at a silica substrate, approximately only 50% can react.

• Due to the steric hindrances between ligands and side chains.

• Silanol concentration = 8umol/m2

• Ligand concentration = 4umol/m2

• The unreacted silanol concentration is equal to

ligand concetration.

• Residual silanols may strongly influence

• Retention

• Selectivity

• For ionic and polar compounds.

• Depending on activity and actual eluent pH silanols may influnce the chromatographic process by

• Hydrogen bonding

• Ion exchange

• Dipole interaction

May cause severe peak tailing and leads to irreproducible retention times.

• In order to suppress this residual silanol activity after bonding, secondary synthesis step to end cap or mask these group is performed.

• The endcapping is done by smallest possible silane

• EX-trimethyl (Most sensitive to hydrolyzation)

• Most common S.P in RPC are those in which a functional group is attached to a silica support

Synthesis of ODS (octadecylsilane, C18H37Si) the reagent used is octadecyl-chlorosilane (C18H37Si(CH3)2Cl)

--. --

Monofunctional S.P is prepared using the above procedure because the reagent C18H37Si(CH3)2Cl) used has one chloro group (only 8-12% carbon Loading)

-

-For steric reasons it is not possible for all silanol groups (-SiOH) groups on silica surface to react with functional group (only ~45% are bonded)

Endcapping is done tocover more silanol groups with di- and tr-chlorosilane reagent

Result in S.P which is more dense andand have 15-20% carbon loading

• Chemically bonding the hydroxy groups would generate a rugged s.p. and most importantly the thinnest possible (monolayer) coating.

• Bonded carbon chains forms the primary s.p. material C4 – C18 (RP).

• Derivatization of the terminal carbons allows us to ‘tailor’ s.p.’s with different polarities.

• Such derivatized s.p. are used in the reverse phase

• Isopropyl instead of methyl group or modification by alkyl ligands carrying a polar function near the silane group

– Octadecyl

– Octyl

– Hexyl

– Cyclohexyl

– Phenyl

– alkyl phenyl

• The most popular column is a octadecyl carbon chain (C18) bonded silica (USP classification L1) with 297 columns commercially available C8 bonded silica (L7 - 166 columns),

• pure silica (L3 - 88 columns),

• cyano bonded silica (L10 - 73 columns)

• phenyl bonded silica (L11 - 72 columns).

• Note that C18, C8 and phenyl are dedicated reversed phase packings ..

• Cyano columns can be used in a reversed phase mode depending on analyte and mobile phase conditions.

• It should be noted at this point that not all C18 columns have identical retention properties.

RPLC phases inorganic oxidesRPLC phases inorganic oxides

• Alumina

• Titania

• Zirconia

• Higher stability 0 to13

• Interact with anlytes with ligands exchange interaction

• These are strong secondary interaction and usally unwanted

• The high activity of the surfaces of these oxides

• Lack of straight forward synthesis procedure

• Surface modification is done by deposition of polymers layer on substrate.

Polymer based RPLC –SPPolymer based RPLC –SP • Styrene divenyl benzene 0 to 14

• Methacrylate or

• Polyvinyl alcohol based phase 2 to 12

• Hydrolytical stability over wide pH range

• Have found appication in aqueous size exclusion and ion exchange chromatography.

Carbon RPLC-SPCarbon RPLC-SP• High chemical stability over wide pH range

• 0 to 14

• Show ultimate hydrophobic properties.

• Sufficient hardness

• Well define pore structure

• Do not suffer from swelling and shrinking

• Porous graphitized carbon

• Black carbon

Applications of Reversed Phase Chromatography (RPC)

-RPC is the most widely used separation mode in HPLC-Cover ~ 75 % of HPLC separations.Applicable to most non-polar analytes & to many ionizable & ionic compounds.

-Best suitable for the separation of neutral solutes that are soluble in water or relatively polar solvents and with molecular weights less than 2000-3000

--

The following table lists a few examples of the multitude of uses of RPC in various fields

Samples• A) Regular Sample a) ionic ex. Acids,bases, organic salts b) neutral• B) Special Sample very hydrophilic or hydrophobic compounds Eg:- achiral isomers, chiral isomers, enantiomers,

biomolecules, inorganic ions, synthetic polymers

Retention and selectivity in RPLC-SPRetention and selectivity in RPLC-SP• Classical measures of retention

– capacity factors

– partition coefficients

– Van’t Hoff Plots

• Give bulk properties only - do not give molecular view of separation process

Solvophobic TheorySolvophobic Theory

• Considers retention and selectivity mainly as function of-

• Surface tension

• Dipole-dipole interaction

• The interaction is between polar groups of a compound and mobile phase.

• Solvent cavities are created by the hydrophobic part of compound.

• The assumption is the principal shortcoming.

• RPLC-phase is considered as passive part of system.

• In many studies , it is shown that specially for non-polar and ionic substances, this is unrealistic.

Partitioning TheoryPartitioning Theory

• It is supported by the good correlation by octanol-1/water partition coefficient.

• RPLC retention data not found for very polar compounds.

• This theory insufficiently explains shape selectivity.

Combining solvophobic and Partitioning theoryCombining solvophobic and Partitioning theory

• Solvent–stationary interphase layer is formed.

• Depending upon the composition of eluent and nature of the stationary phase, enrichment by the organic modifier in that phase takes place.

• Partition of solutes between interphase and mobile phase is assumed to take place by displacement of solvent molecules.

• Together with column efficiency retention, selectivity determines the finally achievable chromatographic peak resolution.

None of these theories can completely explain all of the observed retention in reversed phase HPLC.

Thank You….