Preparation And Characterization Of Adsorbent For Immobilized Metal-chelated Affinity Chromatography

The experiment systematic research the agarose microballoon preparation technique with emulsification-cooling method, agarose activation technique, technology of immobilization of IDA on surface of agarose bead with Cryogels method. Besides, characterization of IMAC (Immobilized metal-chelate affinity chromatography) and application in purification of biopolymers was tested. Thus, aiming to construct Immobilized metal-chelate affinity chromatography, progress of objective product filtration, purification, concentration was accomplished in a single step in a complex environment. Reducing the step of solid-liquid separation and primary separation, specific research as follows:Homogeneous and Controllable Agarose Micro-beads was prepared with emulsification-cooling method. A certain amount of agarose aqueous solution was selected as water phase, a certain mixture ratio of span-85 and paraffin was selected as oil phase. The related factors influencing the preparation of agarose microsphere were studied, through the observation of electronic microscope, particle size of microspheres was selected as index. The optimal process parameters was:agarose concentration was 4%, emulsifier concentration was 3%, agitating speed 1500-2000 r/min, stir time 2 min. In this condition, particle size was lowest and the particle size,less than 25?m, was comparatively centralized.Homemade agarose microspheres was selected as solid substrates, dimethyl sulfoxide, epoxy chloropropane and NaOH were selected as reaction system and activator, The related factors influencing the activation of the surface of agarose microsphere were studied. Epoxy group density of the surface of agarose beads was selected as index. The optimal process parameters was:DMSO 80%, NaOH 0.8 mol·L-1, epoxy chloropropane 10%, reaction time 3 h, reaction temperature 50?,shaker speed 170 r/min. In this condition, the microsphere epoxy group density of the surface of microsphere was the highest.IMAC medium was made by cryogels method. Homemade agarose microspheres, after activation, was selected as reaction materials, glutaraldehyde was selected as crosslinking agent, IDA was selected as ligands. The related factors influencing the density of ingrafting IDA of agarose microsphere were studied. The density of ingrafting IDA was selected as index. The experiment showed that, the ratio of agarose and IDA was 1:0.58; pH of the reaction to the environment was 10; the concentration of glutaraldehyde was 35%. under this condition, the density of grafting IDA reached the highest.IMAC was selected as experimental materials, its performance was characterized, its ability of separation and purification of biopolymers was studied. Research shows that:through infrared characterization of modified affinity chromatographic medium and immobilized IDA, the particle size was still uniform and under 25?m conform to the requirements of the size, besides, the preparation of medium with certain pore structure. Through the analysis of the IR image, characteristic peak of epoxy function group was found at 910 cm-1, which indicated that agarose beads was successful activated. Characteristic peak of CO double bond of aldehydes or ketones was found within the range of 1630-1700 cm-1,which indicated that characteristic peak of carboxyl and hydroxyl was generated in period of this peak. IDA was successful grafted on the agarose beads. Chelate ration of Cu2+ was 73.45% through copper standard curve. Ovum albumin crude product, obtained through the salting out method, was separated and purified with IMAC and electrophoresis. The adsorption capacity of IMAC was 91.95 mg and recovery rate of Ovum albumin was 85.37%, which indicated that IMAC possessed good purification ability.