Development Of Novel Matrices For Protein Chromatography

The thesis focuses on the fabrication and characterization of novel matrices for protein chromatography. Chapter One is a review of previous studies. Following this review, rigid macroporous polymeric resins, novel macroporous agarose beads and dense pellicular agarose-glass composite beads are fabricated and characterized for protein chromatography. In addition, protein purification with model protein mixtures is carried out using the macroporous agarose gel derivatized to an ion-exchanger. Moreover, the expanded bed with the dense pellicular agarose-glass composite beads is characterized to find its flow hydrodynamics and solid phase classification. At first, a rigid macroporous polymeric resin is synthesized by a radical suspension copolymerization, in the presence of an inert phase (porogenic diluents), a mixture of toluene and n-heptane or cyclohexanol and dodecanol. The optimum synthesis recipes are obtained, and it is found that the molar ratios of toluene to n-heptane or cyclohexanol to dodecanol in the porogenic solvents affect the performances of macroporous adsorbents. Because of the presence of the macropores, both protein capacity and interior diffusive rate are improved. Second, a novel macroporous agarose matrix is successfully fabricated by a water-in-oil emulsification method with fine calcium carbonate powders as porogenic solid phase. Derivatized with diethylaminoethyl, an anion exchanger is obtained. The ion exchanger shows very high protein capacity and enhanced effective pore diffusivity. Moreover, the adsorbent exhibits good chromatographic properties in protein separation. Finally, dense pellicular composite solid matrices are prepared by coating agarose gel onto glass beads by a water-in-oil emulsification method. Two dense pellicular matrices are prepared by two different particle size glass beads. It is found that the apparent pore diffusion coefficient of protein in the pellicular beads is over two times higher than that in a homogeneous matrix due to their pellicular assembly. The two dense pellicular agarose-glass matrices of different sizes and densities are characterized for flow hydrodynamics and particle classifications in an expanded bed system. It is found that the composite matrices shows particle size as well as density classifications along the column axis. So their axial classifications are expressed by a correlation related to both the particle size and density as a function of the dimensionless axial bed height. The correlation may be considered as a general formulism for describing the solid phase classifications in expanded bed systems.