CHROMATOGRAPHYMANISH DHAWANP. A. U.Panjab Agricultural University
HistoryMikhail Tswett, Russian, 1872-1919BotanistIn 1906 Tswett used to chromatographyto separate plant pigmentsHe called the new techniquechromatography because the resultof the analysis was written in coloralong the length of the adsorbentcolumnChroma means “color” and grapheinmeans to “write”
CHROMATOGRAPHY Chromatography basically involves theseparation of mixtures due to differencesin the distribution coefficient (equilibriumdistribution) of sample componentsbetween 2 different phases. One of these phases is a mobile phaseand the other is a stationary phase.
Definition:Different affinity of these 2 components to stationaryphase causes the separation.Concentration of component in stationary phaseConcentration of component in mobile phaseDistribution Coefficient (Equilibrium Distribution )
DUE DIFFERNCE IN DISTRIBUTION COEFFICIENT OFVARIOUS COMPONENT OF MIXTURE, THERE MOBILITY INTWO PHASES DIFFER HENCE, SEPARATIONMEASURE OF THIS MOBILITY IS CALLED RF VALUE I.E.RELATIVE FRACTION VALUEDistance traveled by substanceDistance traveled by solventRf=
Classification of chromatographyaccording to mobile phase:1- Liquid chromatography: mobile phaseis a liquid. (LLC, LSC).2- Gas chromatography : mobile phase isa gas. (GSC, GLC).
Classification according to the packing of thestationary phase:1- Thin layer chromatography (TLC): thestationary phase is a thin layer supported onglass, plastic or aluminium plates.2- Paper chromatography (PC): the stationaryphase is a thin film of paper supported on aninert support.3- Column chromatography (CC): stationaryphase is packed in a glass column.
Classification according to the force ofseparation:1- Adsorption chromatography.2- Partition chromatography.3- Ion exchange chromatography.4- Gel filtration chromatography.5- Affinity chromatography.
Thin Layer Chromatography (TLC) Stationary phase: Thin layerof adsorbent (silica gel oralumina coated on plate Mobile phase: Is adeveloping liquid whichtravels on the stationaryphase, carrying the sample.
Techniques of development with various flowdirectionsAscending developmentDescending development
Paper Chromatography Stationary phase: Cellulosepaper strips as carrier or inertsupport Mobile phase: organicsolvent-Butanol: Acetic acid: water[4: 1: 5 (v/v/v)]
Columnar Chromatography (CC)This includes chromatographic methods inwhich:The stationary phase is packed into a column.The mobile phase is a moving liquid or gas.According to the mechanism of separation ofsolutes, five major types of CC areditinguished. Usually, one mechanismpredominates but does not exclude the others
Different Types of chromatographyMode or type Stationary phase Mobile phase MechanismAdsorptionChromatographySolid that attractsthe solutesLiquid or gas Solutes move at different ratesaccording to the forces of attractionto the stationary phase.PartitionChromatographyThin film of liquidformed on thesurface of a solidinert supportLiquid or gas Solutes equilibrate between the 2phases according to their partitioncoefficientsIon ExchangeChromatographySolid resin thatcarries fixed ions& mobilecouterions ofopposite chargeattached bycovalent bondsLiquidcontainingelectrolytesSolute ions of charge opposite tothe fixed ions are attracted to theresin by electrostatic forces &replace the mobile counterions.Molecular ExclusionChromatographyPorous gel withno attractiveaction on solutemoleculesLiquid Molecules separate according totheir size:1.Smaller molecules enter the poresof the gel, and need a larger volumeof eluent.2.Larger molecules pass throughthe column at a faster rate.AffinityChromatographySolid on whichspecificmolecules areimmobilizedLiquid or gas Special kind of solute moleculesinteract with those immobilized onthe stationary phase
Adsorption Chromatography Very similar to partitionchromatography Adsorption just onsurface, partition into thinlayer Not used as widely aspartition used mainly inTLC & very smallparticles in LC
Partition Chromatography Used in GC & LC Molecules will partitioninto the stationary phasebased upon affinity forstationary phase &eventually partition intomobile phase again Thin layer is coated ontoinside of GC column oron small particles on LCcolumn
Ion Exchange Chromatography Separation of eithercations or anions Separtion based onrelative strength ofionic bond Anion exchange hascations on surface Used in LCexclusively
Molecular ExclusionChromatography Separation based onsize Small molecules gettrapped in pores &take longer to get out
Affinity Chromatography Very selective Specific binding site isused to concentrateanalyte on column Used a lot inbiological applications
HIGH PERFORMANCE LIQUID CHROMATOGRAPHY• High Performance Liquid Chromatography (HPLC) is one of themost widely used techniques for identification, quantification andpurification of mixtures of organic compounds.• In HPLC, as in all chromatographic methods, components of amixture are partitioned between an adsorbent (the stationaryphase) and a solvent (the mobile phase).• The stationary phase is made up of very small particles containedin a steel column. Due to the small particle size (3-5 um), pressureis required to force the mobile phase through the stationaryphase.
IMPORTANCE:Chromatography has application in every branchof the physical and biological sciences12 Nobel prizes were awarded between1937 and 1972 alone for work in whichchromatography played a vital role
ChromatographictechniqueUse in industrial microbiologyAdsorption Di-hydro-streptomycin can be extracted from filteratesusing activated charcoal column. It is then eluted withmethanolic hydrochloric acid and purified in furtherstages.Applications in small scale antibiotic purification.Ion exchange In the recovery of strepotomycin, the harvested filterateis fed on to a column of a weak acid cationic resin suchas Amberlite IRC 50 which is in the sodium form. Thestreptomycin is adsorbed on the column and sodiumions are displaced. The resin bed is now rinsed withwater and eluted with dilute HCL to release the boundstreptomycin.Gel permeation Purification of vaccines. This technique yields a fairlypure fraction which is then concentrated ten-fold bypressure dialysis to remove eluant buffer
Affinity It was used initially in protein isolation and purification,particularly enzymes. Since then many other large-scale applications have been developed for enzymeinhibitors, antibodies, interferon and recombinantproteins.HPLC Use in separation and purification of a wide range ofsolute species, including bio-molecules.