The Effect of Salt Ions on Responsive Membrane Based Hydrophobic Interaction Chromatography

Recent efforts have been devoted to develop responsive membrane based hydrophobic interaction chromatography (HIC) to achieve high recovery and resolution for protein purification at preparative and analytical scale respectively. Our group has designed novel responsive membrane based HIC by grafting the thermo-responsive poly(N-vinylcaprolactam) (PVCL) from the surfaces of micro-porous regenerated cellulose membranes (0.45μm) via atom transfer radical polymerization. It is known that the lower critical solution temperature (LCST) of PVCL is sensitive to both salt type and salt concentration in solution. In order to optimize the binding capacity and recovery using responsive membrane based HIC, systematic investigations have been conducted to understand the effects of salt ions on the HIC behavior based on this responsive membrane system. Dynamic binding capacities and recoveries for immunoglobulin G₄ (IgG₄) and bovine serum albumin (BSA) were determined varying solution pH, salt ion type and salt concentration at room temperature. In addition, the cationic effects were studied through static binding tests using various monovalent (Na⁺, NH₄⁺), divalent (Mg²⁺, Zn²⁺) and trivalent (Al³⁺) sulfate salt solutions. BSA adsorption isotherm as well as adsorption kinetics were obtained. Moreover, the adsorption mechanism was elucidated. Finally, the effect of salt on the PVCL ligand was investigated and correlated with the performance of this responsive membrane based HIC system.