RETENTION THERMODYNAMICS IN HYDROPHOBIC INTERACTION CHROMATOGRAPHY

This paper is a review of the thermodynamics of retention in hydrophob ic interaction chromatography (HIC) with mildly hydrophobic stationary phases and aqueous salt solutions usually employed in protein purific ation. Since the role of salt in HIC has been well documented, our foc us was to investigate the temperature effect on the retention behavior in HIC and to compare the results with those obtained for other proce sses driven by the hydrophobic effect. Using nonpolar dansyl amino aci ds as model compounds, retention data obtained on three stationary pha ses yielded nonlinear van't Hoff plots in the temperature range from 5 to 50 degrees C. Thermodynamic analysis of the data revealed signific ant heat capacity effects. The enthalpy and entropy changes were large and positive at low temperatures, decreased with increasing temperatu re, and became negative at high temperatures. The results parallel tho se of calorimetric studies on other processes based on the hydrophobic effect, such as dissolution in water of nonpolar liquids, gases, and solids as well as protein folding. Thermodynamic parameters from HIC m easurements also confirmed the existence of certain exothermodynamic r elationships, such as enthalpy-entropy compensation and molecular area correlations. In order to examine at the molecular level the energeti cs of HIC retention as well as the dissolution of nonpolar gases in wa ter, the pertinent thermodynamic parameters were expressed in terms of nonpolar molecular area and interfacial tensions by employing the sol vophobic theory. It was found that these expressions from HIC and diss olution data are nearly identical, thus confirming the mechanistic ide ntity of the two processes.