HPLC Separation Modes

Chromatographic ModeAbrv.Column TypeMobile PhaseApplication
Reverse-phase chromatographyRPCNon-polar (e.g. C18)Polar mixture of water and organic solvent (e.g. acetonitrile)Water-soluble samples
Normal-phase chromatographyNPCPolar ( e.g. unbonded silica)Less-polar (than stationary phase) mixture of organic solvents (e.g. hexane, ethyl ether, chloroform, methylene chloride) Water-insoluble samples, isomer separation, and preparative HPLC
Non-aqueous reverse-phase chromatographyNARPNon-polar (e.g. C18)Mixture of organic solvents (e.g. ACN + methylene chloride)Very hydrophobic samples
Hydrophilic interaction chromatographyHILICPolar (e.g. silica or amide-bonded)Mixture of water organic solvents (e.g. ACN + H2OHighly polar samples that are poorly retained by reverse-phase mode
Ion-exchange chromatographyIECUsually an organic resin that has charged groups able to bind ions of opposite chargeAqueous solution of a salt with buffer.Separating ionizable samples, large biomolecules (e.g. proteins, carbohydrates)
Ion-pair chromatographyIPCNon-polar (e.g. C18)Polar mixture of water and organic solvent (e.g. ACN) w/ an ion-pairing reagent (e.g. alkylsulfonates, trifluoroacetic acid) that interacts w/ sample ions of oposite chargeAcids and bases that are weekly retained by reverse-phase
Size-exclusion chromatographySECInertAqueous or organicLarge biomolecules, polymers. Separates by molecular weight.

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