Hydrophilic-interaction chromatography for the separation of peptides, nucleic acids and other polar compounds

Abstract

When a hydrophilic chromatography column is eluted with a hydrophobic (mostly organic) mobile phase, retention increases with hydrophilicity of solutes. The term hydrophilic-interaction chromatography is proposed for this variant of normal-phase chromatography. This mode of chromatography is of general utility. Mixtures of proteins, peptides, amino acids, oligonucleotides, and carbohydrates are all resolved, with selectivity complementary to those of other modes. Typically, the order of elution is the opposite of that obtained with reversed-phase chromatography. A hydrophilic, neutral packing was developed for use in high-performance hydrophilic-interaction chromatography. Hydrophilic-interaction chromatography is particularly promising for such troublesome solutes as histones, membrane proteins, and phosphorylated amino acids and peptides. Hydrophilic-interaction chromatography fractionations resemble those obtained through partitioning mechanisms. The chromatography of DNA, in particular, resembles the partitioning observed with aqueous two-phase systems based on polyethylene glycol and dextran solutions.