Novel biporous polymeric stationary phase for high-speed protein chromatography

A novel rigid biporous bead (BiPB) had been fabricated by double emulsification to prepare a (w/o)/w emulsion and a subsequent polymerization. The polymerization of monomers, glycidyl methacrylate and ethylene glycol dimethacrylate, was initiated with benzoin ethyl ether by ultraviolet irradiation. The BiPB with an average diameter of 42.8 μm was characterized to possess two types of pores, i.e., micropores (20–100 nm) and superpores (300–4000 nm). Its specific surface area was determined to be 41.9 m2/g, about 20% smaller than that of a microporous bead (MiPB) (52.1 m2/g). Flow hydrodynamic experiments showed that the BiPB column had smaller backpressure and plate height than those of the MiPB column at a given flow rate. Derivatized with diethylamine (DEA), the static adsorption capacity of the DEA-BiPB was about 7% smaller than that of the DEA-MiPB for BSA (bovine serum albumin). However, frontal analysis demonstrated that the dynamic binding capacity of the DEA-BiPB column was 1.6–2.4 times higher than that of the DEA-MiPB at high flow rate range of 1200–2400 cm/h. Moreover, separation of a model protein mixture (myoglobin and BSA) was conducted at mobile phase velocities up to 3000 cm/h to compare the performance of the two stationary phases. All the results indicate that the BiPB contains interconnected flowthrough pores and the BiPB column is promising for high-speed protein chromatography.