SOLUTE RETENTION OF LYSOZYME IN HYDROPHOBIC INTERACTION PERFUSION CHROMATOGRAPHY

Perfusion chromatography is a rather new HPLC concept using packing ma terials having large through-pores, which were developed to overcome t he problems of stagnant mobile mass transfer without loss of separatio n speed and column capacity. The perfusion media have been developed f or both analytical and preparative separations in a number of distinct HPLC modes including hydrophobic interaction chromatography (HIC) whe re the separation principle is based on the surface hydrophobicity of the solute, which is adsorbed to the mildly hydrophobic stationary pha se 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 perfus ion chromatography is examined using the BioCAD Workstation. Retention data of lysozyme are measured on four perfusive HIC media at differen t 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 t he 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 mobil e phase, modelled by a modified Debye-Huckel equation, and the protein activity coefficient on the stationary phase, expressed by a simple n on-linear term. The model is capable of correlating ln k' from zero to high ionic strength.