Solute retention of lysozyme in hydrophobic interaction perfusion chromatography

Perfusion chromatography is a rather new HPLC concept using packing materials having large through-pores, which were developed to overcome the problems of stagnant mobile mass transfer without loss of separation speed and column capacity. The perfusion media have been developed for both analytical and preparative separations in a number of distinct HPLC modes including hydrophobic interaction chromatography (HIC) where the separation principle is based on the surface hydrophobicity of the solute, which is adsorbed to the mildly hydrophobic stationary phase in an aqueous mobile phase at high salt concentration and eluted at a lower salt concentration. In this study, the retention behaviour of egg white lysozyme in semi-preparative hydrophobic interaction perfusion chromatography is examined using the BioCAD Workstation. Retention data of lysozyme are measured on four perfusive HIC media at different ammonium sulphate concentrations in the eluent in both isocratic and gradient mode at three different pH values representing more than 400 chromatographic runs. The solute retention behaviour of lysozyme on the HIC columns is described by a model of the capacity factor, k′, as a function of the mobile phase pH and the ionic strength. The capacity factor is a function of the protein activity coefficient in the mobile phase, modelled by a modified Debye-Huckel equation, and the protein activity coefficient on the stationary phase, expressed by a simple non-linear term. The model is capable of correlating ln k′ from zero to high ionic strength.