Types of Chromatography

Classification of chromatographyClassification of chromatography

• According to separation mode:

a) Adsorption chromatography

b) Partition chromatography

c) Ion-exchange chromatography

d) Size exclusion chromatography

e) Affinity chromatography

Classification of chrom. (cont.)Classification of chrom. (cont.)

• According to mobile phase: a) Gas Chromatography i- Gas solid chromatography ii- Gas liquid chromatography b) Liquid chromatography i- Paper chromatography ii- Thin-layer chromatography iii- Column chromatography

Classification of chrom. (cont.)Classification of chrom. (cont.)

• According to form of stationary phase

a) Planar chromatography

i- Paper chromatography

ii- Thin-layer chromatography

b) Column chromatography

i- Gas chromatography

ii- Liquid chromatography (LC/HPLC)

LIQUID-COLUMN CHROMATOGRAPHY

A sample mixture is passed through a column packed with solid particles which may or may not be coated with another liquid.

With the proper solvents, packing conditions, some components in the sample travel through the column more slowly than others resulting in the desired separation.

The basic liquid chromatography modes are named according to the mechanism involved:

 1. Liquid/Solid Chromatography (adsorption chromatography)

A. Normal Phase LSC

B. Reverse Phase LSC

 2. Liquid/Liquid Chromatography (partition chromatography)

A. Normal Phase LLC

B. Reverse Phase LLC

FOUR BASIC LIQUID CHROMATOGRAPHY

L C used for samples:L C used for samples:

containing large molecules/ioniccontaining substances with low vapor

pressure (non-volatile substances)Substances thermally unstableSubstances can’t be vaporized without

decomposing

LIQUID SOLID CHROMATOGRAPHY

Si - O - H

Normal phase LS Reverse phase LS

Silica Gel

The separation mechanism in LSC is based on the competition of the components of the mixture sample for the active sites on an absorbent such as Silica Gel.

LIQUID SOLID CHROMATOGRAPHY

Si - OH

HEXANE

OH

C-CH3

CH3

CH3- C

CH3

CH3

OH

OH

CH3

CH3

Adsorption chromatographyAdsorption chromatography

Stationery phase is solid and mobile phase is liquid

Distribution between two phases (adsorption and desorption)

Attractive forces (ionic, dipole-dipole, dipole induced dipole)

Good adsorbent has large surface area and more active sites

Equilibration occurs at: surface-solute, surface-solvent, solvent-solute

An adsorption isotherm is a plot of the concentration or amount of analyte on a surface as a function of its concentration in the bulk phase.

In liquid chromatography, the bulk phase is, of course, the mobile phase.

Adsorption isothermsAdsorption isotherms

Adsorption isothermsAdsorption isothermsLinear (k = Cs/Cm) = 1Convex (k = Cs/Cm1/n) where n >1Concave

AdsorbentsAdsorbents

Adsorbing power depends on

1- chemical nature of the surface

2- area available

3- pretreatment

Commonly used adsorbentsCommonly used adsorbents

Alumina, Al2O3 ,(Aluminum oxide). It may be acidic, basic and neutral in nature. Available in various grades

Silica gel (silicon dioxide). It is acidic in nature. Available in various grades.

CaCO3

SucroseStarch cellulose

Adsorbent should have uniform size and large surface area

Weight of the adsorbent should be 20-50 times more than the sample

Solvents & adsorbentsSolvents & adsorbentsSince adsorbents are polar, non-polar elute first.

Usually, the elusion order is as: alkyl halids < saturated hydrocarbons< unsaturated hydrocarbons <ethers < esters < ketones < amines < alcohols < phenols < acids and bases. Polymeric compounds and salts often don’t elute

The solvents in the order of polarity Hexane/Pet ether < CCl4 < toluene < dichloromethane < chloroform < diethyl ether < acetone < ethyl acetate < propanol < ethanol < methanol < acetic acid < water

Columns and packingColumns and packing

Various sizes are availableWet methodDry methodSample loading and running the column

WATER-SOLUBLE VITAMINS

1. Niacinamide 2. Pyridoxine

N

CONH2

N

CH2OH

CH2OH

HO

H3C

3. Riboflavin N

NNH

N

CH2

HOCH

HOCH

HOCH

CH2OH

O

OH3C

H3C

ClN

S

N

NH3C

CH2

NH2

CH3

CH2CH2OH

4. Thiamin

WATER-SOLUBLE VITAMINS

0 5 10 15 20

Column: u Bondapak C18 Solvent: MeOH Sample: Water-Soluble Vitamins

Inject1

2

3

4

LIQUID-LIQUID CHROMATOGRAPHY

ODPN(oxydipropionylnitrile)

Normal Phase LLC Reverse Phase LLC

NCCH3CH2OCH2CH2CN(Normal)CH3(CH2)16CH3 (Reverse)

The stationary solid surface is coated with a 2nd liquid (the Stationary Phase) which is immiscible in the solvent (Mobile) phase. Partitioning of the sample between 2 phases delays or retains some components more than others to effect separation.

Advantages of Partition Advantages of Partition chromatographychromatography

– Advantage over adsorption chromatographyMore reproducible and predictableDistribution coefficient is constant over a much

greater range of concentration yielding sharper and symmetrical peaks

It is also of two types1-Normal phase2-Reversed phase

Supports for stationary phaseSupports for stationary phase

Silica gelKieselguhr/CeliteCellulose

stepssteps

Sample preparationSample loadingElutionDetection: chemical methods; diverse types

of detectors can be used

ION-EXCHANGE CHROMATOGRAPHY SO3

-Na

+

Separation is based on the competition of different ionic compounds of the sample for the active sites on the ion-exchange resin (column-packing).

Ion exchange chromatographyIon exchange chromatography

A process where ions held by solid matrix are exchanged for counter ions in the solution

Synthetic ion exchange resins are used for water purification and separation of ions

MECHANISM OF ION-EXCHANGE CHROMATOGRAPHY OF AMINO ACIDS

SO3-

SO3-

Na+

COO-

H3N+

Na+

COOHH3N

+

pH2

pH4.5

Ion-exchange Resin

H3N

+

SO3-

SO3-

SO3-

SO3-

SO3-

SO3-

H3N+

COOH

OH

COOH

COOH

H3N+

H3N+

OH

COO-

Na+

H3N+

COO-

Na+

Na+

H+ OH

- = H2O

H+ OH

- = H2O

Na+

Na+

pH3.5

Mobile PhaseStationary Phase

Exchange Resin

pH4.5

Chromatography of Amino AcidsChromatography of Amino Acids

Ions exchange resinsIons exchange resinsConsist of three dimensional polymeric

chains, cross linked by short chains, which carry ionisable functional groups.

Based on ions these are of two types– Cation exchangers (weak or strong) – Anion exchangers (weak or strong)

Formation of resinFormation of resinStyrene and divinylbenzeneThe number of cross linkers

determine by the ratio of Styrene : divinylbenzene

Increasing cross linkers increases the rigidity and reduces swelling

Ion-exchange chromatography can be used to perform preparative separation of amino acids

Negatively charged resin binds selectively to positively charged amino acids

Behavior of resinBehavior of resin

Important properties which determine behavior of resin are:

1- size of particles

2- degree of cross linking

3- nature of functional groups

4- number of functional groups

Theoretical principlesTheoretical principlesIon exchange equilibriumdistribution coefficient (KD) indicates affinity of

the resin for ions relative to hydrogenGenerally, if KD is large, resin will incline to

attract the ionPolyvalent ions are more attracted to the resin

compared to mono-valent

In groups where charges are same, the difference between KD is related to the size of the ion

Uses of ion exchange Uses of ion exchange chromatographychromatography

Ion separationConcentration of trace mattersSeparation of alkali and alkali earth metalsSeparation of amino acids, proteins,

peptides, nucleic acid and nucleotidesimmunoglobulin

StepsSteps

Same to that of the othersDetection: conduction measuring

Size exclusion chromatographySize exclusion chromatography

Molecular gel chromatography, gel permeation, molecular sieving or molecular exclusion

Stationary phase serves as molecular sieveSeparate molecules based on size via

sieving or filtrationAdsorption and electrical charge play role

in separation

GelsGelsOpen three dimensional network formed by

cross linking large ploymeric chainsPolar groups absorb water and swellHave an exclusion limit i.e critical size of a

molecule that can just penetrate the interior

Theoretical principlesTheoretical principles

There are 2 kinds of solvent in the gel

Vi = Volume within gel

Vo = volume outside the beads of the gel

Large molecules will not be able to enter or penetrate the pores of the gel, hence their elution volume (Ve ) will be

Ve = Vo

Whereas, smaller molecules must be swept through Vo plus some additional volume which is a fraction of Vi

Ve = Vo + KDVi, where KD = distribution coefficient

KD = Average concentration of solute in gel/ Average concentration of solute outside gel

KD value should be between 0 and 1 If sieving action is the only mechanism of

separation (KD=1) thenVe = Vo + Vi

If K<1, It indicates solvent interacts with the gel (adsorption, hydrogen bonding)

Types of the gelsTypes of the gelsSephadex, dextran gel (classified by the

amount of water regain)Biogel, polyacrylamide gel, inert series of

gels, insoluble in water and common organic solvents

Styragel rigid cross linked polystyrene gel

useful at temperature > 150oC with organic solvents, it can be used under high pressure

Gel Filtration

ApplicationsApplications

Desalting (removal of salts and small molecules from macromolecules)

Concentrating (concentration of dilute solutions of macromolecules with MW> exclusion limit

Fractionation (separation of mixture of closely related molecules having small difference in KD values namely proteins, peptides, nucleic acids, polysaccharides, enzymes and hormones)

MOBILE PHASE LIQUID

Liquid-LiquidChromatography (Partition)

Liquid-SolidChromatography (Adsorption)

Liquid Solid

Normal Phase Reverse Phase Normal Phase Reverse Phase

Mobile Phase - NonpolarStationary phase - Polar

Mobile Phase - PolarStationary phase - Nonpolar

FORMAT

STATIONARYPHASE

Types of ChromatographyTypes of Chromatography

1.1. Ultraviolet DetectorUltraviolet Detector

200-400nm 200-400nm 254 nm254 nm

2.2. Reflective Index DetectorReflective Index Detector

Universal DetectorUniversal Detector

DetectorsDetectors

Affinity chromatographyAffinity chromatography

Separation where surface of inert phase has been modified to selectively bind compounds having specific functional group

Binding force should be strong enough to effect separation but weak enough to get the compound when desired

Properties of Inert matrixProperties of Inert matrix

Mechanically and chemically stableLarge surface areaEasily derivatizedGood flow characteristicsExamples (agarose, controlled pore glass,

cellulose)

Spacer Spacer An arm to move active group away from the bead

so that steric hindrances are at minimumEffectiveness depend on their

– length– stability of the attachment to the bead– hydrophobic nature– presence of fixed charges and their concentration

Affi-gel has spacer arm –O- (CH2)3 NH2 [Oxypropylamine]

Affinity Chromatography

Affinity Chromatography

Selected ligands and their affinity Selected ligands and their affinity compoundscompounds

Ligands

Diazo-NAD-

AMP analogues

Blue dextran 2000

Methotrexate

B12

Affinity compounds

dehydrogenases

NADP- binding proteins

Yeast phosphofructokinase

Dihydrofolate reductase

Transcobalamin I and II

Chromatographic techniquesChromatographic techniques

Classical LC TLC/ paper chromatography Modern LC

Classical chromatography Classical chromatography technoquestechnoques

Glass or plastic columns Need skill Solvent flow (gravity, suction) and individual

samples collected manually Detected using different detectors Detection and quantification achieved by manual

analysis of fractions Results are recorded in the form of chromatogram

(sample concentration vs fraction number)

DisadvantagesDisadvantages

Column packing procedure tediousLow column efficiency, long analysis timeTechnique depends on user Detection of solutes is labor intensive and

takes a lot of time

Plane chromatographyPlane chromatography

Plane surface rather than column2 dimensionalSelective properties (use of two solvents)Include

– Paper chromatography and – thin layer chromatography

PrinciplesPrinciples

Principles are similar to columnSuccessive equilibrations of the analyte

between two phasesNon ideal processes may cause zone

spreadingDegree of retention is Rf

– Ratio between distance traveled by solute/distance traveled by solvent

Relation between RRelation between Rff and K and K

Rf = number of moles of solute in mobile phase/total moles in both phases

= Cm Am/CmAm+ CsAs

Am and As are the cross sectional areas of two phases. By dividing Cm

Rf = Am/Am+AsCs/Cm = Am/Am+ KAs

Paper chromatographyPaper chromatography

Mainly qualitative and semi quantiativeEasy to performMechanisms

1- liquid liquid

2- adsorption

3- hydrogen bonding

4- ion exchange

Nature of the paperNature of the paper

Highly purified celluloseGreat affinity for water and polar solventsPaper may be impregnated with alumina,

silica or ion exchange resin

ProcedureProcedure

Sample application Development

1- ascending Simple and popular Solvent flow through capillary action Slow development Slow rate enhances partition, separation

2- descending2- descending

Flow is downwardPaper folded U shapeSolvent flow capillary and gravityMuch faster

detectiondetection Visible Application of Reagents UV absorbance Florescence IR Radioactivity Chemical tests Bioautography

qualitativequalitative

Based on Rf values

Semi-quantitativeExtraction and spectroscopydensitometry