Development of Rigid Bidisperse Porous Microspheres for High-Speed Protein Chromatography

Abstract

Development of a high-performance stationary phase is an essential demand for high-speed separation of proteins by liquid chromatography. Based on a novel porogenic mode, that is, using superfine granules of calcium carbonate as solid porogen and a mixture of cyclohexanol and dodecanol as liquid porogen, a rigid spherical biporous poly(glycidyl methacrylate-co-ethylene dimethacrylate) matrix has been prepared by radical suspension-polymerization. The epoxide groups of the matrix were modified with diethylamine to afford the ionizable weak base 1-N,N-diethylamino-2-hydeoxypropy functionalities that are required for ion exchange chromatography. Results from scanning electron microscopy and mercury intrusion porosimetry measurements revealed that the matrix contained two families of pores, that is, micropores (10-90 nm) and macropores (180-4000 nm). Furthermore, the biporous medium possesses specific surface area as high as 91.3 m(2)/g. Because of the presence of the macropores that provided convective flow channels for the mobile phase, the dynamic adsorption capacity was found to be as high as 54.6 mg/g wet bead at 300 cm/h, approximately 63.2% of its static capacity. In addition, the column efficiency and dynamic binding capacity decreased only slightly with mobile-phase flow rate in the range of 300-3000 cm/h. These properties made the packed bed with the bidisperse porous matrix suitable for high-speed protein chromatography.

Porous polymer particles—A comprehensive guide to synthesis, characterization, functionalization and applications

“…After removal of CaCO 3 via HCl etching, the beads exhibited pores as large as 10 µm and a surface area value of 79 m 2 /g. In another report [43], a mixture of solid (CaCO 3 ), nonsolvent (dodecanol) and good solvent (cyclohexanol) porogens are utilized all together for the suspension polymerization of the same EGDMA-GMA monomer mixture. Together with a total surface area of 91 m 2 /g, the formation of a bimodal distribution of micropores (10-90 nm) and macropores (180-4000 nm) is observed.…”

Porous microsphere and its applications

“…[52][53][54][55][56][57][58][59][60][61] High-speed protein chromatography Due to the huge specific surface area, porous microspheres are fit for adsorption and desorption substances. Thus a separation of substances can be realized.…”

“…On the other hand, the micropores connecting the macropores or superpores provide a large surface area for solute binding. 52 Employing novel porous microspheres, an efficient low-pressure preparative chromatography for purification of icariin from crude extract has been developed. The microspheres column even demonstrated a higher resolution and better selectivity than the C18 column.…”

A Novel Method to Prepare PolyGMA and its Application to Immobilization of β-Galactosidase

“…This is because free enzyme, as a biocatalyst, is lack of long-term stability under processing conditions and difficult to be recovered and recycled from the reaction mixture, making the reuse of the enzyme impossible. Hence the idea of immobilizing the enzyme on a rigid solid support enabling easy separation and the possibility of operation in a packed bed or fluidized-bed reactor has been of great industrial interest for many years 1,2 .…”