Fabrication Of Flowthrough Matrices For Chromatographic Separation Of Biomacromolecles

The thesis focuses on the fabrication of bipores beads for high-speed flow- through chromatography and its application in the purification of biomacromolecules.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 42.8μm was characterized to possess two types of pores, i.e., micropores (20-100 nm) and superpores (300-4000 nm). Comparing with the traditional beads (micropores beads, MiPB), BiPB have the similar static adsorption capacity. However, frontal analysis demonstrated that the dynamic binding capacity of the BiPB column was 1.6-2.4 times higher than that of the MiPB at high flow rates ranging from 1200 to 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.To obtain more stable emulsion, we choose the calcium carbonate suspension and cyclohexanol / dodecanol to create superpores and micropores. Derivatized with diethyl amine (DEA), the biporous beads were changed into a kind of anion-exchange matrix (denoted as DEA-B). The static adsorption capacity of the DEA-B was close to that of the DEA-M for BSA (bovine serum albumin). However, frontal analysis demonstrated that the dynamic binding capacity of the DEA-B column was two times higher than that of the DEA-M at a flow rate of 1800 cm/h. The results showed that the velocity of mobile phase have a little effect on the dynamic adsorption capacity of DEA-B.To test the property of our customized bipores beads, we use it to purify the biomacromolecules from the E. coli broth. After fermentation, ultrasonication and centrifugation of E. coli, the supernatant containing the GroEL was obtained. After some experiment for purification of GroEL, electrophoresis purity of GroEL was got at mobile phase velocities of 150 cm/h and 1500 cm/h. To obtain the pcDNA3 plasmid DNA (5.4 kb) solution, the E. coli was lysed by alkalinelysate method. Purified by customized biporous beads (DEA-B) column with gradient elution, the electrophoresis purity of plasmid DNA was obtained. And the effects of velocity of mobile phase and salt concentration of sample on the purification of plasmid DNA were studied. Finally, we breakthrough the biporous beads column using alkaline lysate at 150 cm/h and 1500 cm/h, and got electrophoresis purity 0.014 and 0.113 mg plasmid DNA/min·mL bed after step elution at 63% buffer B, respectively.All the results indicate that the bipores bead contains interconnected flow-through pores is promising for high-speed flow-through chromatography.