Preparation And Characterization Of HPAC Resin Dye-P(GMA-EDMA)

The monodisperse poly(glycidylmethacrylate-co-ethylenedimethacrylate) beads are particularly used for biopolymer separation, since their have good hydrophilicity, which was abbreviated as P(GMA-EDMA). The hydrolization of the epoxide groups existed on the surface of P(GMA-EDMA) can brought a lot of hydroxyl group which is good for chemical modification. Affinity chromatographic column based on porous particles of 8μm P(GMA-EDMA) is applicable for the analysis and micropreparative separation of proteins. The resin has advantages for rapid analysis, high column efficiency, low column backpressure, high protein mass recovery and good resolution for protein. In this paper, we prepared dye-affinity resin in which P(GMA-EDMA) microspheres were used as the support, and two dye ligands were incorporated into these microbeads for the separation and purification of proteins. The main work follows:1. Porous particles, P(GMA-EDMA) micropheres with diameter of 7μm were prepared by a single-step swelling and polymerization method, using polystyrene micropheres prepared by dispersion polymation as seed particles and GMA,EDMA as monomers. The effect factors of preparation were studied in detail, and the favorable operation conditions were found at: the swelling multiple is 30, the concentrations of crosslinking reagent in monomers is 45%, the ratios of monomer to porogen is 1.25. The synthesized monodisperse P(GMA-EDMA) resin possesses macroporous and high mechanic intensity.2. Procion blue MX-R and Cibacron blue F3GA were covalently coupled with P(GMA-EDMA) micropheres, thus two novel dye-affinity resins PB MX-R-P(GMA-EDMA) and CB F3GA-P(GMA-EDMA) were prepared. The effect factors of preparation were studied. It was found that these dye-attached micropheres were stable in acidic and alkaline solution while the ligand leakage was very low.3. The dye-immobilized P(GMA-EDMA) beads were slurry packed into stainless steel column. The affinity columns were investigated in column efficiency , selectivity, stablility, column pressure, protein loading capacity and recovery et. al., using lysozyme as standard protein.On the PB MX-R-P(GMA-EDMA) column(10×0.46cm I.D.), lysozyme was well eluted with mobile phase which containing 1.0mol/LNaCl, 15% ethylene alcohol in 0.05 mol/L Tris-Hcl buffer(pH7.8), flow at 0.5ml/min. The column has low column backpressure, good stability, high protein mass recovery, and was factual used for the purification of lysozyme from egg white.On the CB F3GA-P-(GMA-EDMA) column (25 X 0.46cm I.D.), proteins of lysozyme and bovine serum albumin(BSA) were well separated and the lysozyme mass dynamic loading capacity of it was 45.3mg/ml. In factual using, the resin was used for the purification of lysozyme from egg white and the separation of glucoprotein abstracted from rape pollen.