Pressure-induced retention of the lysozyme on reversed-phase liquid chromatography

This study investigated the effect of pressure on the retention behavior of a model protein, lysozyme, on octadecylsilica (C18) stationary phase under various equilibrium conditions. It is demonstrated that the retention time of the lysozyme was increased by as much as two to three times as the absolute pressure on the viewing window was increased from 23 to 318 bar. This pressure-induced retention was likely to be reversible and the corresponding volume change (ΔV=Vsta−Vmob) was found to be on the order of minus tens to hundreds of mL/mol. Moreover, the pressure-induced retention was also observed for a homologous series of hydrophobic poly-l-phenylalanine, which do not have the secondary structure, and the volume change was determined to be around minus 10 mL/mol per phenylalanine. Perturbations in solute ionization and conformational change are predicted to have a minor impact under the investigated conditions. It is believed that the pressure-induced shift of the equilibria regarding hydrophobic ad–desorption is the major cause of the observed increase of protein retention. About ten phenylalanine-equivalent residues on the lysozyme surface were involved in the hydrophobic association with the chromatographic ligands.