Experimental studies of pressure/temperature dependence of protein adsorption equilibrium in reversed phase high performance liquid chromatography

The effect of the average pressure and temperature of the column on the adsorption equilibrium of insulin variants on a C8 bonded silica was studied in isocratic reversed-phase HPLC. Analytical injections of samples of four different insulins (bovine, porcine, Lys-Pro and human recombinant) were carried out at constant flow-rate but under increased average pressure. The temperature dependence of the retention parameters over the range 25-50 °C was studied under two different average column pressures (47 and 147 bar). Substantial increases of the retention time (up to 300%) were observed when the pressure and/or the temperature were increased. Similar adsorption-induced changes in the partial molar volume at constant temperature ( D Vm »102 ml/mol) were found for all the variants studied. Furthermore, D Vm was revealed to be practically independent of the temperature, which suggests that the temperature has no or very little influence on the mechanism of the pressure induced perturbations in the molecular structure of the solute. This conclusion was also derived from the observed temperature dependence of the logarithm of the retention factor ( k) measured under different pressures. The relation between the temperature and ln k was nonlinear with a parabolic shape. Moreover, the shapes of the plots corresponding to the low and high pressures were found to be exactly the same, except that the curves were vertically shifted, due to the difference between the two average column pressures. These results indicate that pressure and temperature affect the retention behavior of insulins in a different and separate way