True and apparent temperature dependence of protein adsorption equilibrium in reversed-phase HPLC.

The adsorption behavior of bovine insulin on a C(8)-bonded silica stationary phase was investigated at different column pressures and temperatures in isocratic reversed-phase HPLC. Changes in the molar volume of insulin (deltaV(m)) upon adsorption were derived from the pressure dependence of the isothermal retention factor (k'). The values of deltaV(m) were found to be practically independent of the temperature between 25 and 50 degrees C at -96 mL/mol and to increase with increasing temperature, up to -108 mL/mol reached at 50 degrees C. This trend was confirmed by two separate series of measurements of the thermal dependence of ln(k'). In the first series the average column pressure was kept constant. The second series involved measurements of ln(k') under constant mobile-phase flow rate, the average column pressure varying with the temperature. In both cases, a parabolic shape relationship was observed between ln(k') and the temperature, but the values obtained for ln k' were higher in the first than in the second case. The relative difference in ln(k'), caused by the change in pressure drop induced by the temperature, is equivalent to a systematic error in the estimate of the Gibbs free energy of 12%. Thus, a substantial error is made in the estimates of the enthalpy and entropy of adsorption when neglecting the pressure effects associated with the change in the molar volume of insulin. This work proves that the average column pressure must be kept constant during thermodynamic measurements of protein adsorption constants, especially in RPLC and HIC. Our results show also that there is a critical temperature, T(c) approximately equals 53 degrees C, at which ln(k') is maximum and the insulin adsorption process changes from an exothermic to an endothermic one. This temperature determines also the transition point in the molecular mechanism of insulin adsorption that involves successive unfolding of the protein chain.