| Chondroitin sulfate ,relative molecular mass from 10,000D to 50,000D,is an acidic Polysaccharide composed of disaccharide units of glucuronic acid and aminohexose. Chondroitin sulfate distributing in various animal tissues has been proven to posses various biological activities such as adjusting immunity and fat metabolism, preventing infection, reducing the risk of heart diseases. Therefore, chondroitin sulfate has great application potentials in fields of food, medicine and cosmetic.Molecular imprinting is a new and potential separation technique for preparing specialized recognition chromatography media. The process of the preparation consists of: (1) mixing the template molecules with monomers with appropriate functional groups,(2)low temperature and photo polymerization, and (3) extracting the template molecules from the polymer, which leaves cavities inside the polymers that are complementary in terms of size, shape, and functional group orientation to those of the template molecules. The technique has been applied widely in enantioselective separation for a variety of molecules, such as amino acids and their derivatives, free sugars, and racemic drugs. In this thesis, chiral separation of drugs and preparation of pueraria using molecular imprinting and imprinting of protein were studied. Molecular imprinting affinity model was established, and utilized to descript quantificationally imprinted separation.A microwave-HPLC method was established for extract and determination the Chs-C in compound chondroitin sulfate tablets, taken the Chs-C extraction rate as the inspection target, carried on the orthogonal test based on single factor experiments, and definited extraction optimum condition. The optimal extraction conditions were : microwave power 595W,extracting time 60s,the ratio of solid and liquid 1∶75. In this condition the extraction rate of chondroitin sulfate C was 4.1%. A C18 column was used ,with the mobile phase of, acetonitrile-0.5%phosphoric acid-isopropyl alcohol (15∶85∶1) ,at the determination wave length of 210nm,and column temperature 40℃.Molecular imprinting technique and ethanol precipitation were applied to purify chondrointin sulfate. The results indicated that the method had higher extraction rate at pH 6.4, the NaCl density was 3% (w/v), solid content density wasl8% (w/v) with 70%(v/v)ethanol solution. The results showd that the yield of chondroitin sulfate was 45% (w/w) and the glucuronic acid content in the extract was 44.3% (w/w) .The imprinted polymer was prepared by bulk polymerization using Chs as template, AM as the functional monomer. The molar ratio of template, functional monomer and cross-linker was l/n:4:20(n=60).EGDMA was the cross-linking monomer and AIBN was the initiator. The pre-polymerization mixture was dissolved in acetonitrile /water (85:15). The mixture was put into a water bath at 60℃for 24 h and finally turned into bulk MIP. Bulk MIP were ground and sieved through 100um sieves, and washed with methanol/water(l:4) to remove template molecular completely and dried at 60℃.According to the IR and UV spectrum, the best molar ratio of template and functional monomer was 1/n:4(n=60). Different solvents were examined, which showed that mechanism capability of MIP prepared by acetonitrile /water (85:15) was better than that prepared by methanol/water.The recognition of MIP was estimated by Scatchard equation, which was usually used showed that MIP had two kinds of different enery binding sites. According to the slope and intercept of the straight line, calculated the dissociation constants (Kd)and Qmax of every binding sites. Kd1 and Qmax1 were 9.58×10-4 mol/L and 6.67μmol/g respectively as to highest binding site .While Kd1 and Qmax1 were 5.38×10-3 mol/L and 25.87μmol/g respectively as to lowest binding site .Compared with ethanol precipitation ,the high selectivity and the endophilicity of the MIPs may decide its selectivity in advance. The preparation of MIPs was simple, fast and necessary,. It can reduced the pretreatment time,the consume and the exposition of the organic solvent greatly, and conform to the green chemistry principle;it can use with other technical association and realize the automated online analysis . |
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