Chromatography of proteins using polybutadiene-coated zirconia

Polybutadiene-coated zirconia (PBD-ZrO2), when used as a stationary phase in conjunction with a mobile phase containing phosphates, constitutes a reversed-phase/cation-exchange mixed-mode chromatographic system. The separation of proteins on this phase can be achieved only through the use of mobile phases containing the correct combination of phosphoric acid, displacing salt, and organic cosolvent. We found that excessive Coulombic interactions between proteins and the stationary phase impair the system performance for the separation of proteins. The effects of mobile phase conditions on the separation of proteins using phosphate-adsorbed PBD-ZrO2 are studied in this work. Factors such as the presence of a multivalent cation, mobile phase pH, phosphate concentration, and salt concentration can be manipulated to reduce the net negative charge on the surface and thereby improve the performance of the system toward the separation of proteins.