Preparation And Performance Evaluation Of Novel Solid Phases For Affinity Chromatography

With the development of science and technology, especially the rapidly development of life sciences, people made higher requests on the technology of separation and analysis, the affinity chromatography was required to have higher resolution ,faster separation velocity. When the traditional soft gels were used as the matrix in affinity chromatography, the question of "stagnant mobile phase mass transfer" appeared, the peak shape of the chromatograms was wide, the separation time was long. To solve this problem, investigators brought forward the assumption to use nonporous matrix in affinity chromatography. Due to the fact that there were no porous on the surface of the matrix, the mass transfer time between biomacromolecule and the solid phase was shorten ,the analysis time was greatly shorten; also the mechanical strength of the matrix was greatly enhanced, which made it appropriate to be used under high pressure.In order to comprehend the development of nonporous matrix, enough literatures at home and abroad were collected. Based on these documents, Urea-Formaldehyde resin -Zirconia(UF-ZrO₂) composite microspheres were chosen as the affinity matrix, based on learning the preparation experience of Lei Shulei, the microspheres were prepared and used as the solid phase in Immobilized Metal Ion Affinity Chromatography (IMAC) and Dye Ligand Affinity Chromatography. The work done was as follows:1. Iminodiacetic acid bonded UF-ZrO₂ composite microspheres were synthesized. After using epichlorohydrin to activate the matrix, IDA was bonded under alkalescence condition. After backfilling the solid phase in chromatographic column, the unchelated solid phase was obtained; zincum ion and copper ion were chosen to chelate with IDA, two kinds of immobilized metal ion solid phases was prepared. Bovine Serum Albumin and Lysozyme was used as testing samples. The change of retention time of the two proteins on three different solid phases under mobile phase with different pH value, and the infection of the concentration of salt in the mobile phase on the retention time of the two proteins on the unchelated solid phase and the solid phase chelated with copper ion were examined. The result obtained indicated that after chelating the metallic ion, the solid phase maintained the characteristic of unchelated solid phase, and also it showed the different performance. To the unchelated solid phase, the retention of protein depended on the static electrification between protein and the ligand, while to the chelated solid phase, the retention of protein depended on the chorus of the static electrification between protein and the ligand and the coordination between protein and the metallic ion; When salt was added to the mobile phase, the retention time of proteins reduced with the thicken of the salt, which could be represented with empirical formula.2. Reactive blue X-BR bonded UF-ZrO₂ composite microspheres were synthesized. The solid phase was got by the reaction of the acylamino on the surface of the microspheres and the active chlorine ion in the dye under alkalescence condition. The dynamics performance of the static adsorption for the solid phase on BSA was studied. The results obtained indicated that the adsorption quantity of BSA reached the maximum at its isoelectric point, and its saturation adsorption amount was 30.3mg·g^-1; the adsorption quantity reduced with the strength of ion strength; the adsorption quantity reached the maximum when the experiment was carried out for 2 hours, the adsorption and desorption was quicker than traditional porous fillings, which strongly embodied the characteristic of nonporous fillings. Based on the results obtained from the static adsorption experiment, the desorption of BSA by changing the pH value of the mobile phase and changing the ion strength of the mobile phase were carried out. The results indicated that the desorption by changing the ion strength of the mobile phase was more effective.3. The steric mass-action model of the dye affinity adsorption of BSA was established. By fitting the data got in the experiment with the model, the three parameters in the model were obtained: the adsorption equilibrium constant Ka; the binding number n of BSA and the ligand; the steric factor s. By this model the fact that the dye ligand was mild was corroborated, the adsorpted protein could be eluted by general method, by the way, the result showed the characteristic of the nonporous fillings.