High-Performance Hydrophobic Interaction Chromatography of Proteins

Abstract

Hydrophobic interaction chromatography was introduced in 1957 under the name salting-out chromatography as a technique for separating substances, according to their hydrophobicities, by gradient elution with a decreasing salt concentration, which reduces the solubility of substances such as ammonium and sodium sulfates. The effects of the type of ligand of support have been studied by many workers and it has been found that retention and selectivity depend substantially on the type of ligand. Proteins are generally more retained on the supports with more hydrophobic ligands. If the ligand is too hydrophobic, it is difficult to elute proteins in native states from the column. In contrast, if the ligand is very hydrophilic, very high concentrations of salt are required to retain proteins. Supports with hydrophilic ligands can provide satisfactory results even for unstable and rather hydrophobic proteins. The usefulness of high-performance hydrophobic interaction chromatography has been demonstrated for purifications and analyses of various proteins.