Reverse-phase chromatography of proteins

Abstract

Abstract A glance through the literature shows that reverse-phase high-performance liquid chromatography (RP-HPLC) has now been assimilated into the canon of protein purification techniques: from the status of a novel, noteworthy technique when first applied it has now matured to that of being simply listed in the methods section along with high-performance size-exclusion, ion-exchange, or hydrophobic-interaction chromatography, no more noteworthy than any of the other techniques routinely employed or considered in any protein separation. The main way in which it differs from its companion techniques is in the necessary exposure of the sample proteins to high concentrations of organic solvents, and often low pH, both factors which can lead to the rapid denaturation of proteins, and which result in less denaturing methods being preferred, particularly where oligomeric proteins or enzymes are involved. Nevertheless, there are many circumstances where the risk of denaturation is not important; these include: most purely analytical work; separations for structural studies, or where structure and activity are retained or recovered despite the high concentrations of organic modifier; and where a rapid, simple, and powerful technique of unusual specificity like RP-HPLC is of great service.