Synthesis Of Titania Monolithic Column And Its Application In Separation Of Protein And Peptide

Titania, because of its wonderful chemical stability and surface properties, was deemed to be a kind of emerging and good HPLC packing material. Therefor, the research of titania has a significant meaning by developing titania monolithic column and applying it to the separation of large quantities of samples and macromolecular compound or to the food safety testing. In this paper, synthesis of titania monolithic material and the application of titania column in separation of casein hydrolysate were studied.The researches in this thesis included three aspects:(1) Using the sol-gel method to synthesize titania monolithic material, ascertained that the effect factors on formation and performance of titania monolithic material including that: the ratio of ingredients, the speed of adding water, the soaking process and the control of drying rate. Finally, we got a good titania monolithic material after the careful control of soaking and drying process with a ratio of ingredients was GT12,R=16,PEO0.4%.(2) The study of hydrolyzing conditions of casein by trypsin indicated the optimum condition was that: the substrate concentration was 5%, the hydrolysis temperature was 55℃, the pH value was 7.5, the enzyme concentration was 62.44 U/g and the hydrolysis time was 180 min. At that moment the hydrolyzing degree (DH) of casein reached the maximum value of 15.1%.(3) The research of HPLC and LC-MS/MS manifested that: on the basis of using titania column, an easy operation chromatographic condition was found: the mobile phase was 50% acetonitrile:50% water; the flow rate was 0.5ml/min; the injection volume was 2μL; the column temperature was room temperature. The constituents of casein hydrolysate was identified by MS/MS. There were eight constituents of casein hydrolysate, and five of them were polypeptide, respectively, Gln-Asp-Lys-Ile-His-Pro (β-CN,46-51), His-Leu-Pro-Pro-Leu-Leu (β-CN,134-139), Val-Leu (β-CN,162-163), Val-Leu-Pro-Val-Pro (β-CN,170-175), Gly-Pro-Phe-Pro-Ile (β-CN,203-207), the other three were amino acid, respectively, Phe, Tyr, and Leu.