Preparation Of Monolithic Material And Its Application In Separation And Analysis Of Biomacromolecules

The monolithic stationary phases have become a rapidly burgeoning field in recent years. The advantages of good hydrodynamic characteristic, low flow resistance as well as easy preparation make monolithic material an ideal support for the purification of biomacromolecules.Reactive continuous rods of macroporous poly(glycidyl methacrylate-co-ethylene dimethacrylate) have been prepared by "in-situ" copolymerization of the monomers in the presence of porogenic diluents. Protein A and pseudo-biospecific ligands were immobilized on the continuous rods respectively. These two affinity columns were used for the purification of immunoglobulin G from human serum. The effects of the nature and the pH of the buffer system on the human IgG adsorption on pseudo-biospecific affinity column were also investigated.The epoxide groups of the monolithic rod were modified by a reaction with iminodiacetic acid (IDA) that affords active site to form metal IDA chelates used for the immobilized metal affinity chromatography (IMAC). The efficiency of coupling of IDA to the epoxide-contained matrix was studied as a function of reaction time and temperature. High-performance separation of proteins, based on immobilized different metals on the column, were described. The influence of pH on the adsorption capacity of BSA on the Cu2+-IDA continuous rod column was investigated. Purification of lysozyme from egg white and human serum albumin (HSA) from the commercially available HSA solution were performed on the naked IDA and Cu2+-IDA continuous rod columns, respectively.Diethylamine was covalently coupled on the rod for ion-exchange chromatography of proteins. The flow rate effects on frontal analysis, adsorption isotherm, and protein separations were investigated, respectively. The mobile phase effects, including buffer system, pH, and organic additives, on the protein retentions were studied in detail. The mass recovery of five model proteins on the DEAE monolithic column was also investigated.Papain was immobilized on the monoliths directly or through a spacer arm. Anew method for studying the digestion of human immunoglobulin G (IgG) with papain by protein A affinity column was developed. The apparent Michaelis-Menten kinetics constant of free and immoblized papain, Km and Fmat, were determined, respectively. The digestion conditions of human IgG with free and immobilized papain were optimized.