Investigation Of The β-cyclodextrin Bonded Stationary Phase In The Application Of The Enantioseparation And Renaturation Of The Denatured Lysozyme

The thesis includes four parts as following:1. Preparation and enantioseparation properties of the naphthyl-carbamate-substituted β-cydodextrin bonded stationary phaseThe naphthyl-carbamate-substituted P-cyclodextrin bonded chiral stationary phase (CSP) was prepared, and the enantioseparation properties of the CSP were investigated by high-performance liquid chromatography under normal and reverse phase condition, respectively. Eight chiral compounds were enantioseparated under these two chromatographic modes;the relation between the ennatioseparation abilities of the CSP and the structure of the compounds was analyzed.2. Preparation and enantioseparation properties of the 3, 5- dimethylphenyl-carbamate-substituted β-cyclodextrin bonded stationary phaseThe 3, 5-dimethyl phenylcarbamate-substituted P-cyclodextrin chiral stationary phase was prepared by a modified method. Twelve chiral compounds were enantioseparated under normal phase condition, and seven compounds under reverse phase condition. The effects of the composition of the mobile phase and column temperature on the retention behavior of four chiral compounds with hydroxyl groups in their structure were investigated by using six kinds of mobile phase composed with hexane and alcohols. Capacity factors of the solutes were found to decrease with carbon number of alcohols except pentanol, and there was a good linear relation between capacity factor and the concentration of the alcohol, which was accordantwith the stoichiometric displacement model for retention ( SDM-R). Also, the enantioselectivity of the solutes was found to increase with the column temperature, and the relation between them was described with the Van't Hoff equation. The thermodynamic parameters indicated that the process of the enantioseparation was mainly derived by the entropy, and the four compounds had the similar enthalpy-entropy compensation temperature of 333K.3. Preparation and enantioseparation Application of the L-phenylalanine -substituted /?-cyclodextrin bonded stationary phaseUsing L-phenyl alanine as a modified reagent , an amino acid-substituted P-cyclodextrin stationary phase was designed and prepared. Eight chiral compounds were enantioseparated under normal phase condition and five compounds under reverse phase condition. The stationary phase had particular enantioseparation ability for the compounds with amino acid or compounds with carboxyl in their structure.4. Preparation of the y-aminopropyl-P-cycIodextrin bondedstationary phase and its application in renaturation of the denatured lysozymeThe ?y-aminopropyl-P-cyclodextrin bonded stationary phase was prepared. On the obtained stationary phase, the denatured Lys and reduced-denatured Lys were successfully renatured with size-exclusion liquid chromatography (SEC) and hydrophobic interaction chromatography (HIC). The renaturation efficiency was significantly improved with P-CD as an additive in the mobile phase simultaneously. In addition, the mass and bioactivity recovery were also improved at lower concentration of Lys, the mobile phase with the 4 mol/L urea, or the proper flow rate.