The effect of temperature from 5°C to 50°C on the retention of dansyl derivatives of amino acids in hydrophobic interaction chromatography (HIC) was investigated by HPLC on three stationary phases. Plots of the logarithmic retention factor against the reciprocal temperature in a wide range were nonlinear, indicative of a large negative heat capacity change associated with retention. By using Kirchoff's relations, the enthalpy, entropy and heat capacity changes were evaluated from the logarithmic retention factor at various temperatures by fitting the data to a logarithmic equation and a quadratic equation that are based on the invariance and on an inverse square dependence of the heat capacity on temperature, respectively. In the experimental temperature interval, the heat capacity change was found to increase with temperature and could be approximated by the arithmetic average. For HIC retention of a set of dansylamino acids, both enthalpy and entropy changes were positive at low temperatures but negative at high temperatures as described in the literature for other processes based on the hydrophobic effect. The approach presented here shows that chromatographic measurements can be not only a useful adjunct to calorimetry but also an alternative means for the evaluation of thermodynamic parameters.Hydrophobic interaction chromatography (HIC) is widely used for the separation and purification of proteins in their native state (1, 2). The technique employs weakly hydrophobic stationary phases and the retention is modulated by varying the salt concentration in the aqueous mobile phase (3, 4). The effect of salt on protein interactions in aqueous solutions (5-9) as well as on protein adsorption in HIC (5, 10, 11) has been extensively investigated, and the salt effect on HIC retention has been treated within the hermeneutics of the solvophobic theory (12-14) and Wyman's linked functions (15)(16)(17)(18).Less progress has been made in elucidating the effect of temperature on retention in HIC. The observed increase in protein retention with temperature, for instance, has been ascribed to enhancement of hydrophobic interactions with increasing temperature due to temperature-induced conformational changes of proteins and concomitant increase in hydrophobic contact area upon binding to the chromatographic surface (16). So far the only thermodynamic study on the effect of temperature in HIC was carried out with aliphatic alcohols and carboxylic acids on octyl-agarose stationary phase with neat aqueous phosphate buffers (19) and hydro-organic mobile phases containing methanol or ethylene glycol (20). Since the salt concentration was very low in these studies, the conditions differed from those employed for protein separation in HIC. In order to shed light on the HIC retention behavior of proteins, it is necessary to understand first the physicochemical basis of HIC with simple molecules that undergo no significant conformational changes and to useThe publication costs of this article were defrayed in part by page c...

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