| β-cyclodextron (β-CD) is torus-shaped oligosacchrides made up of seven cyclic-arrangedα-1,4-linked D-glycopyranose units. The outside of the cyclic molecule is hydrophilic and the central cavity is hydrophobic.β-CD has the ability to form specific inclusion complexes with some compounds, and the stability of inclusion complex is depend on how the guest molecule fits into the cavity of theβ-CD. This property has been widely employed in many separation techniques including classical liquid chromatography. In order to develop new media with improved selectivity,β-CD and its derivatives are used as a ligand in liquid chromatography.In this study,β-CD was coupled on polyvinyl acetate-based carrier. The novel stationary phases were used for separation puerarin from pueraria flavones, overcoming the deficiency of gel beads in mechanical rigidity and high cost. The experimental results showed that the coupled stationary phases had high selectivity for flavonoids of natural products and the separation efficiency was enhanced. This thesis optimized the process of activation and coupling conditions and investigated the adsorption mechanism. The main studies were divided into three sections as following:Firstly, poly(vinyl acetate-triallyl isocyanurate-divinylbenzene) coupled with (β-cyclodextrin was prepared by suspension polymerization method. The porous surface morphology and porous structure were investigated through scanning electron microscope and mercury porosimetry, respectively. After alcoholysis and epoxy reaction, the beads were activated with plenty of epoxide group. The optimal activation conditions were found to be as follows: after the alcoholysis was carried out for 15 h, with the alkalinity 3%, the hydroxyl content was up to 22 mmol/g; the epichlorohydrin was added before alkaline solution in the epoxy reaction, then the epoxide group content reached 40μmol/g. The optimal coupling conditions were also found to be as follows:in the solvent system comprising of DMF and NaCl solution, the mass ratio of polymer beads/(β-CD was 1:2, the coupling reaction was carried out at 70℃for 20 h, the content of (β-CD could reach 10μmol/g.Secondly, the chromatographic separation characteristics of puerarin on the the novel coupled medium were evaluated. By controlling the mobile phase composition, the stationary phase could provide efficient separation of puerarin from the mixture of three isoflavone homologues. The optimal mobile phase was comprised of 7% acetic acid and methanol. The separation of three isoflavone homologues, puerarin, daidzin and daidzein, was achieved by increasing the proportion of methanol from 40% to 85%. In a single column pass on P(VAC-TAIC-DVB)-β-CD, the purity and recovery of puerarin were in the range of 55-90% and 30-40%, respectively.Finally, the sorption mechanism onβ-cyclodextrin coupled polyvinyl acetate-based matrix has been investigated using the isosteric heat approach and molecular dynamics (MD) simulation. Isotherms of Freundlich equation with characteristic parameters for the media were well fitted to the equilibrium adsorption data. Both TAIC andβ-CD were contributed to the increase of Gibbs free energy. It was found in the MD simulation that the benzene ring of DVB and the benzene ring A of puerarin were in two parallel planes, resulting fromπ-πinteraction; the hexatomic ring C of puerarin was the active binding site for TAIC, while the hydrogen bonding between the carbonyl group of TAIC and the hydroxyl group with flexible carbochain in the glucose moiety could enhance the bonding force. The interaction between the polymeric medium and the puerarin was studied by static adsorption and retention research, the result showed that the adsorption efficiency on P(VAC-TAIC-DVB)-CD was driven by both hydrogen bonding interaction and hydrophobicity.
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