Zwitterionic stationary phase with covalently bonded phosphorylcholine type polymer grafts and its applicability to separation of peptides in the hydrophilic interaction liquid chromatography mode

A novel phosphorylcholine type zwitterionic stationary phase was synthesized by graft polymerization of 2-methacryloyloxyethyl phosphorylcholine onto the surface of porous silica particles. The resulting material possesses both negatively charged phosphoric acid and positively charged quaternary ammonium groups, which renders it a low net charge over a wide pH range. The composition of the surface grafts were determined by elemental analysis and solid state NMR, and the surface charge (zeta-potential) in different buffer solutions were measured using photon correlation spectroscopy. Separation of several peptides was investigated on packed columns in the hydrophilic interaction liquid chromatography (HILIC) separation mode. It was shown that small peptides can be separated based on hydrophilic interaction and ionic interaction between the stationary phase and analyte. The organic solvent composition, the pH and the salt concentration of the eluent have strong effects on the retention time. Compared to native silica before grafting, the newly synthesized zwitterionic material gave more stable retention times for basic peptides over pH range 3-7 due to elimination of the dissociation of silanol groups.