This paper is a review of the thermodynamics of retention in hydrophobic interaction chromatography (HIC) with mildly hydrophobic stationary phases and aqueous salt solutions usually employed in protein purification. Since the role of salt in HIC has been well documented, our focus was to investigate the temperature effect on the retention behavior in HIC and to compare the results with those obtained for other processes driven by the hydrophobic effect. Using nonpolar dansyl amino acids as model compounds, retention data obtained on three stationary phases yielded nonlinear van't Hoff plots in the temperature range from 5 to 50°C. Thermodynamic analysis of the data revealed significant heat capacity effects. The enthalpy and entropy changes were large and positive at low temperatures, decreased with increasing temperature, and became negative at high temperatures. The results parallel those 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 measurements also confirmed the existence of certain exothermodynamic relationships, such as enthalpy-entropy compensation and molecular area correlations. In order to examine at the molecular level the energetics of HIC retention as well as the dissolution of nonpolar gases in water, the pertinent thermodynamic parameters were expressed in terms of nonpolar molecular area and interfacial tensions by employing the solvophobic theory. It was found that these expressions from HIC and dissolution data are nearly identical, thus confirming the mechanistic identity of the two processes.

Exothermodynamic relationships between thermodynamic quantities and molecular structure are employed to facilitate a molecular interpretation of enthalpy−entropy compensation (EEC). For hydrophobic interactions the compensation temperature T C is expressed in terms of the enthalpy and entropy change, both per unit nonpolar surface area of the molecules, and it is concluded that the utility of T C as a diagnostic tool for the mechanistic identity of processes rests on this simple dependence of T C on molecular parameters. Whereas classical EEC is observed only with processes involving no heat capacity change and T C is evaluated from the slopes of linear enthalpy versus entropy plots of data measured at any temperature, this investigation shows that even when the heat capacity change is finite and constant or varies linearly with the temperature, EEC can occur with processes if they are subject to the same mechanism at a fixed temperature. In turn, the compensation temperature changes with the experimental temperature, reflecting mechanistic changes as expected with processes such as hydrophobic interaction chromatography that are governed by hydrophobic interactions and driven by entropy or enthalpy change at low or high temperatures. These compensating processes exhibit at least one isoenergetic temperature , which marks the intersection point of curved van't Hoff plots, where all species have the same free energy change in the same way as at T C in the case of linear van't Hoff plots. In turn, the isoenthalpic and isoentropic temperatures mark the intersection points of the respective plots of enthalpy and entropy versus temperature as described in the literature. The triad of isothermodynamic temperatures is characteristic for processes which can be represented by constant heat capacity change and evince compensation behavior.