寡聚β-环糊精键合聚醋酸乙烯酯树脂的制备及在天然活性成分分离中的应用
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摘要
β-环糊精内疏水外亲水的性质以及特殊的分子结构,使其具有对客体分子凭借尺寸、几何形状和性质等差异进行识别和选择的能力。利用这种与特定客体分子形成包结络合物的能力,可将β-环糊精键合到合适的载体上,用于制备液相色谱固定相,提高介质的分离能力。
本文将β-环糊精键合到聚醋酸乙烯酯聚合物吸附树脂上,提高了它对天然产物活性成分的分离效率,克服了β-环糊精键合天然多糖凝胶介质机械强度不高、不能耐受某些有机溶剂的缺点,得到的新型色谱分离介质,用于分离纯化葛根素等植物天然产物活性成分,取得了良好的分离效果。本文优化了制备β-环糊精键合聚合物的活化和键合条件,对它们吸附保留葛根素的机理进行了探讨。具体包括以下三部分内容：
第一部分是介质的合成和表征。采用悬浮聚合法自主合成了树脂P(VAC-TAIC-DVB)-β-环糊精,通过扫描电镜、红外光谱和压汞仪对介质的表面形态和孔结构进行表征；通过醇解反应和环氧反应两步活化以使树脂在接枝环糊精前具有较大的环氧基含量,醇解反应的最优条件为：碱度3%,活化15h,羟基含量达到22mmol/g,环氧反应投料模式为先加环氧氯丙烷后加入碱液,环氧基团含量达到40μmol/g；确定了P(VAC-TAIC-DVB)树脂键合β-环糊精的最优条件：键合溶剂体系为DMF／NaCl溶液,键合时间20h,在70℃、投料质量比1：2的反应条件(树脂/β-CD)下,固载β-CD量达到10μmol/g。
第二部分为键合介质在色谱分离中的应用。以P(VAC-TAIC-DVB)-β-CD为层析介质分离葛根素、大豆苷和大豆苷元的混合物,流动相组成为甲醇/7%HAc溶液。在甲醇浓度40%的条件下,葛根素和大豆苷能够得到分离,纯度达到98%以上,收率在40-50%范围内；在甲醇浓度80%的条件下,大豆苷和大豆苷元能够得到分离；采用甲醇与7%HAc溶液的梯度洗脱后,三种物质能够得到分离,分离度达到0.894和2.787,纯度范围55-90%,收率约30-40%。
第三部分中,作者结合吸附动力学与分子动力学模拟对实验现象进行机理研究。聚醋酸乙烯酯微球对葛根素的静态吸附符合Freundlich模型,在交联剂中添加TAIC或是在树脂上键合β-环糊精后,能够增大系统的吉布斯自由能,从而使吸附量增大；通过分子动力学模拟发现,TAIC六元环与葛根素的C环结合,且TAIC的羰基与葛根素中葡萄糖单元上的羟基形成氢键,DVB上苯环与葛根素的A环结合,无氢键作用；通过在静态实验中添加十二烷基磺酸钠和尿素后测定吸附性能,说明吸附过程是氢键和疏水作用的共同作用；通过考察柱分离中保留时间随温度的变化,证实了氢键作用是确实存在的。
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.
本文将β-环糊精键合到聚醋酸乙烯酯聚合物吸附树脂上,提高了它对天然产物活性成分的分离效率,克服了β-环糊精键合天然多糖凝胶介质机械强度不高、不能耐受某些有机溶剂的缺点,得到的新型色谱分离介质,用于分离纯化葛根素等植物天然产物活性成分,取得了良好的分离效果。本文优化了制备β-环糊精键合聚合物的活化和键合条件,对它们吸附保留葛根素的机理进行了探讨。具体包括以下三部分内容：
第一部分是介质的合成和表征。采用悬浮聚合法自主合成了树脂P(VAC-TAIC-DVB)-β-环糊精,通过扫描电镜、红外光谱和压汞仪对介质的表面形态和孔结构进行表征；通过醇解反应和环氧反应两步活化以使树脂在接枝环糊精前具有较大的环氧基含量,醇解反应的最优条件为：碱度3%,活化15h,羟基含量达到22mmol/g,环氧反应投料模式为先加环氧氯丙烷后加入碱液,环氧基团含量达到40μmol/g；确定了P(VAC-TAIC-DVB)树脂键合β-环糊精的最优条件：键合溶剂体系为DMF／NaCl溶液,键合时间20h,在70℃、投料质量比1：2的反应条件(树脂/β-CD)下,固载β-CD量达到10μmol/g。
第二部分为键合介质在色谱分离中的应用。以P(VAC-TAIC-DVB)-β-CD为层析介质分离葛根素、大豆苷和大豆苷元的混合物,流动相组成为甲醇/7%HAc溶液。在甲醇浓度40%的条件下,葛根素和大豆苷能够得到分离,纯度达到98%以上,收率在40-50%范围内；在甲醇浓度80%的条件下,大豆苷和大豆苷元能够得到分离；采用甲醇与7%HAc溶液的梯度洗脱后,三种物质能够得到分离,分离度达到0.894和2.787,纯度范围55-90%,收率约30-40%。
第三部分中,作者结合吸附动力学与分子动力学模拟对实验现象进行机理研究。聚醋酸乙烯酯微球对葛根素的静态吸附符合Freundlich模型,在交联剂中添加TAIC或是在树脂上键合β-环糊精后,能够增大系统的吉布斯自由能,从而使吸附量增大；通过分子动力学模拟发现,TAIC六元环与葛根素的C环结合,且TAIC的羰基与葛根素中葡萄糖单元上的羟基形成氢键,DVB上苯环与葛根素的A环结合,无氢键作用；通过在静态实验中添加十二烷基磺酸钠和尿素后测定吸附性能,说明吸附过程是氢键和疏水作用的共同作用；通过考察柱分离中保留时间随温度的变化,证实了氢键作用是确实存在的。
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.
引文
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