High efficiency and fast separation of active proteins by HIC chromatographic pie with sub-2 μm polymer packings

This paper reports the development of hydrophobic interaction chromatography (HIC) by synthesizing sub-2 μm polymer packings which was packed into a chromatographic pie for fast separation of native proteins at low pressures demonstrating high efficiency. Using styrene as monomer and ethylene dimethacrylate (EDMA)as swelling agent, the polystyrene seeds with an average particle size of 0.8 μm and monodisperse polymeric microspheres with a particle size of 1.5-5.0 μm were synthesized through dispersion polymerization and one-step swelling method, respectively. In order to separate active proteins, the microspheres were modified to hydrophobic chromatographic packings through covalent bonding with benzene methanol. Compared with the traditional column chromatography, the sub-2 μm polymer packings in chromatographic pie exhibited higher column efficiency for protein separation at lower column pressures, even at higher flow rates. The van Deemter curve showed that the flow rate had insignificant effect on column efficiency of chromatographic pie. Seven example proteins were clearly separated within 3 min at a flow rate of 10 mL/min. The applicability of this method was further demonstrated by the separation of human serum samples. The results indicated that this chromatographic mode can be potentially applied for the fast separation of complex active proteins, such as protein drugs from natural products.