Preparation and pore-size control of hydrophilic monodispersed polymer microspheres for size-exclusive separation of biomolecules by the SPG membrane emulsification technique

This paper describes an experiment directed toward the preparation of monodispersed porous polymer microspheres with a diameter of ca. 50 μm, which is applicable to the chromatographic separation of biomolecules such as proteins and peptides by size exclusion. Fairly monodispersed polymer microspheres were successfully prepared by suspension copolymerization of polyethylene glycol monomethacrylate and ethylene glycol dimethacrylate as monomer and cross-linker, respectively. Monodispersed O/W emulsion was prepared by the SPG membrane emulsification technique, and was used in the subsequent droplet-swelling process in which monodispersed seed droplets were swollen by adsorbing the secondary emulsion droplets. The effects of the organic diluent in suspension polymerization and comonomer on the porous structure of the polymer microspheres were investigated by scanning electron microscopy and mercury-intrusion porosimetry, and the separation performances of polystyrene, polyethylene glycol, and various biomolecules by size-exclusion chromatography. As a result, it was evident that benzene, 1-butanol, and butyl acetate worked as nonsolvents for the polymer prepared in this study, and that polymer microspheres prepared with these solvents had larger pores. Size-exclusion chromatography revealed that the exclusive limiting molecular weight was 1.9×10⁵ when polystyrene was used as a standard polymer, and 3.5×10⁴ when polyethylene glycol was used as a standard polymer. Furthermore, we confirmed that the monodispersed polymer microspheres with defined pores clearly separated the six representative kinds of biomolecules with molecular weights ranging from 75 to 6.4×10⁵.