FC 5C- Separation & Purification

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TechniqueStationary phaseMobile phaseBasis of separationNotes*Paper chromatographysolid (cellulose)liquidpolarity of moleculescompound spotted directly on a cellulose paper*Thin layer chromatography (TLC)solid (silica or alumina)liquidpolarity of moleculesglass is coated with thin layer of silica on which is spotted the compound*Liquid column chromatographysolid (silica or alumina)liquidpolarity of moleculesglass column is packed with slurry of silicaSize exclusion chromatographysolid (microporous beads of silica)liquidsize of moleculessmall molecules get trapped in the pores of the stationary phase, while large molecules flow through the gaps between the beads and have very small retention times. So larger molecules come out first. In this type of chromatography there isn’t any interaction, physical or chemical, between the analyte and the stationary phase.Ion-exchange chromatographysolid (cationic or anionic resin)liquidionic charge of the moleculesmolecules possessing the opposite charge as the resin will bind tightly to the resin, and molecules having the same charge as the resin will flow through the column and elute out first.Affinity chromatographysolid (agarose or porous glass beads on to which are immobilized molecules like enzymes and antibodies)liquidbinding affinity of the analyte molecule to the molecule immobilized on the stationary phaseif the molecule is a substrate for the enzyme, it will bind tightly to the enzyme and the unbound analytes will pass through in the mobile phase, and elute out of the column, leaving the substrate bound to the enzyme, which can then be detached from the stationary phase and eluted out of the column with an appropriate solvent.Gas chromatographyliquid or solid supportgas (inert gas like argon or helium)boiling point of the moleculessamples are volatilized and the molecule with lowest boiling point comes out of the column first. The molecule with the highest boiling point comes out of the column last.