| At present agarose gel microspheres have become one of the most widely used separation medium. Although there are more and more technology to ensure the quality, but the high cost still does not fall. In this study, agarose’s raw material agar was used to prepare agar gel microspheres. That greatly reduced the cost. Then scaled up the process step by step. We derived a kind of high strength, no charge agar-dextran composite microspheres as gel filtration chromatography packing, and explored its application in the separation and purification field. The thesis mainly included the following parts:(a) Preparation of agar gel microspheres. Using blade disc impeller, 6% agar solution, through emulsification-curing method to prepare microspheres. The conditions were emulsifier concentration 15%, emulsifying speed and time were 1200 rpm and20 min, curing speed and time were 800 rpm and 20 min, agar solution heating time 3h. The result showed the average particle size was 100.2 μm, particle size distribution coefficient was 1.66 and batch stability was good. Using 4%,10%,12% agar solution as water phase, separately need heating 2h,5h,6h respectively. The yield of microspheres was more than 85%. We found that agar powder’s viscosity and ash with different batches were the main factors to influence the quality of microspheres.(b) Scale up the preparation of agar gel microspheres.We scaled up step by step to 5L and 30L. The yield of 5L scale was over 90%, the average particle size distribution coefficient was 1.61, and had a good repeatability. Then enlarged to 30L scale. Through simulation found that there had cavitation in the reaction kettle. If add baffles the shear force is the largest. So we added baffle and feed tube. After that the preparation of microspheres was very good. Compared 1L,5L,30L scale preparation 30L scale had smaller particle size due to the stronger shear force. Calculated the cost of different concentration of agar microspheres, our cost only accounted for 4%-5% of the price of domestic same type product.(c) Preparation of agar-dextran composite gel microspheres. The main difference between agarose and agar microspheres is agar microspheres have negative charge. And they all have poor mechanical strength. So modification neutral dextran on agar microspheres, not only can cover up the negative charge on the surface of the microspheres, also can increase the mechanical strength. This paper studied the influence of various factors on the structure of composite microspheres. Ultimately determined the cross-linking 72 ml, dextran 5g, flow rate 0.4 ml/min. At this time the maximum dextran content was 0.23mg/mg, separating degree of standard material reached a maximum 0.822. Compared the characterization of agar and composite microspheres. Observed the morphology by scan electron microscope after freeze-drying. Composite microspheres showed good sphericity, and had bigger average particle size. The Zeta potential changed from-20.2mv to 0.659mv. S element content were reduced, mechanical stability were better, nonspecific adsorption were reduced greatly. Using the inverse size exclusion method to analysis microsphere pore structure found that with the increase of cross-linking dose, the porosity of composite microspheres increased, exclusion limit decreased, reduced from 534 kda to 304 kda.(d) The application of composite microspheres. The no charge composite microspheres especially suitable used as gel filtration packing. We used composite microspheres for protein desalination. The peak shapes were good. Also could use the composite microspheres to separate mixture with different molecular weight. Compared with commercial product, no specific adsorption and had longer peak time. |
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