| Bile acids are the main active ingredient contained in animals (cholic acid is one of the essential components),belong to highly hydrophobic steroids. Bile acid play an important role in the digestion and absorption of lipids and cholesterol. The various components(such as cholic acid, chenodeoxycholic acid, ursodeoxycholic acid, etc)extracted from animals have different pharmacological activities. In addition, bile acids in the human body can also be diagnosed hepatobiliary by measuring the concentration of each component. In this study, we set up a new type of surface molecular imprinting technique to polymer microspheres as a substrate, the molecular recognition mechanism of cholic acid was studied, a similar study has not been reported in other literatures.Firstly, in this paper, the crosslinked copolymer microspheres CPVAc were prepared with suspension polymerization method by using vinyl acetate as a main monomer, divinyl benzene as crosslinker and polyvinyl alcohol as disperser. The effects of various factors, such as the used amount of the disperser, the stirring rate, the ratio of oil phase to water phase and the used amount of NaCl, on the sphericity and size of the crosslinked microspheres were mainly examined. The alcoholysis of the microspheres CPVAc were performed, and the crosslinked polyvinyl alcohol microspheres were obtained. The experimental results show that the physical aspect of the microspheres CPVA is depending on the morphology and grain size of the precursor microspheres CPVAc. In the suspension polymerization, the main factors are the used amount of the disperser PVA, the stirring rate and the ratio of oil phase to water phase. The balling process can not occur as the amount of PVA is smaller than 0.3%, the stirring rate is lower than 200rpm and the ratio of oil phase to water phase is greater than 1:4 (V:V). As long as controlling the various conditions of the suspension copolymerization, the microspheres CPVA with excellent sphericity and controllable particle diameter can be gained. The key factor effecting on the alcoholysis degree of CPVA is reaction temperature. The fitting temperature is 40℃and an alcoholysis degree of 92 % in 15 h can be reached.Graft polymerization of acrylamide (AM) on the surface of crosslinked polyvinyl alcohol (CPVA) microspheres was realized by using cerium ammonium sulfate as initiator in an acidic aqueous solution (sulfuric acid), resulting in the grafted microspheres PAM/CPVA. The effects of various factors on the graft polymerization reaction and the mechanism were emphatically studied. The experimental results show that by the oxidation action of Ce (Ⅳ) ion, free radicals can be produced on the surface of CPVA microspheres, on which there are a great deal of hydroxyl groups, and the radical graft polymerization of acrylamide (AM) can be favorably carried out. The grafting degree of PAM on PAM/CPVA microspheres is firstly decided by the acid concentration. The grafting degree initially increases with increasing the acid concentration, and then gradually decreases with the further increase of the acid concentration. As the concentration of H+ ion is 0.36mol/L, the grafting degree has a maximum. The dependence of the grafting degree on the acid concentration reflects the special mechanism of the initiation-graft polymerization. As the concentration of the initiator, cerium ammonium sulfate, is too great, the grafting degree will be decreased owing to the accelerating of the oxidation termination step, leading to the decrease of the grafting degree. The optimal concentration of cerium ammonium sulfate is 5.98×10-3mol/L. During the graft polymerization process, the concentration of the monomer AM, and the reaction temperature also affect the graft polymerization reaction to a certain extent.The grafted polyacrylamide (PAM) of grafted microspheres PAM/CPVA was transformed to polyvinylamine (PVAm) via Hofmann degradation reaction, resulting the grafted microspheres PVAm/CPVA. The grafted microspheres PVAm/CPVA was characterized by infrared (FTIR). The amine value of PVAm/CPVA was determined by treating with excess HCl and back titration. The effects of various factors, such as the concentration of NaOH and NaOCl, the reaction temperature, the reaction time, on the Hofmann degradation reaction were mainly examined. The experimental results show that the grafted microspheres PVAm/CPVA can successfully be realized via the Hofmann degradation reaction, the optimal reaction condition for Hofmann degradation is: temperature is -3℃, n(NaOH)/n(PAm)=30:1,n(NaOCl)/n(PAm)=1.4:1,reaction time is 11h, and the amine value can reach 0.002 mol/g.Finally, the adsorption capacity and adsorption mechanism of PVAm/CPVA for cholic acid in water/ethanol mixture solution is further studied. The experimental results show that the grafted microspheres PVAm/CPVA have strong adsorption ability for cholic acid. The influence of various factors on adsorption capacity is studied, and the conclusion about the adsorption mechanism is: the adsorption driving force of PVAm/CPVA towards cholic acid in the mixture of ethanol and water should be the synergy of electrostatic interaction and hydrogen bonding, the electrostatic interaction is the main driving force, also the hydrogen bonding is important.The molecular imprinted polyvinylamine on the crosslinked polyvinyl alcohol microspheres (MIP-PVAm/CPVA) is prepared with cholic acid as a template molecule and glutaraldehyde as crosslinking agent, and by right of the intermolecular hydrogen bond and electrostatic interactions between the grafted PVAm macromolecules and cholic acid molecules. The binding character of MIP-PVAm/CPVA towards cholic acid molecules was studied in depth with both batch and column methods and using cholesterol as a contrast compound whose chemical structure is similar with cholic acid to a certain extent. The experimental results show that the surface-imprinted material MIP-PVAm/CPVA has excellent binding affinity and recognition selectivity for the template molecule, cholic acid. The selectivity coefficient of PVAm/CPVA microspheres (non-imprinting material) for cholic acid relative to cholesterol is only 1.314, and very poor recognition selectivity for cholic acid is displayed. However, after imprinting, the selectivity coefficient of MIP-PVAm/CPVA in respect to cholesterol is remarkably enhanced to 11.231, displaying the excellent recognition selectivity and binding affinity of the surface imprinting material MIP-PVAm/CPVA towards cholic acid molecules. Besides, MIP-PVAm/CPVA microspheres have fine desorption property.
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