Physicochemical Basis of Hydrophobic Interaction Chromatography

Abstract

In hydrophobic interaction chromatography (HIC), which is widely used for protein separation, a mildly hydrophobic stationary phase is employed with an aqueous salt solution as the mobile phase, and the magnitude of retention is modulated by the concentration of the salt. Retention is governed by the hydrophobic effect, which is attributed to the strongly ordered structure and high cohesive energy of water. Various theoretical approaches are used to treat chromatographic retention in HIC. The most comprehensive is the solvophobic theory that was first adapted to provide a theoretical framework for reversed-phase chromatography, a technique fundamentally similar to HIC. According to this approach the role of salt as the primary retention modulator in HIC is primarily due to the increase in the surface tension of the mobile phase with the salt concentration. However, when specific salt binding occurs the appropriate preferential interaction parameters have to be considered. The effect of temperature on the retention in HIC is yet to be elucidated. Recent advances in exploring the temperature dependence of the hydrophobic effect are expected to provide a framework for the interpretation of experimental data in HIC and to facilitate further understanding of the role of temperature in hydrophobic interactions.