| 其他摘要 | 色谱是一种广泛应用的下游分离技术，80%以上的分离过程——尤其是医药产品的分离过程——使用色谱技术。高效的色谱介质是色谱技术研究的核心内容。常用的色谱介质存在吸附容量小，传质速率慢等问题。近年来，接枝型色谱介质因其自身结构上的特点有效地提高了介质的吸附容量以及溶质的传质速率。目前，接枝型色谱介质——特别是葡聚糖接枝的琼脂糖介质已广泛用于生物大分子分离。然而，葡聚糖是线性多糖分子，一方面会显著降低介质的孔体积；另一方面，葡聚糖溶液较高的粘度会对接枝聚合反应造成困难。针对上述问题，本研究以琼脂糖介质Sepharose HP颗粒为基质材料，制备了接枝型聚蔗糖-琼脂糖介质。针对生物大分子分离的特点，以三(羟甲基)氨基甲烷 (Tris) 为配基，偶联于Sepharose HP和接枝型聚蔗糖10-Sepharose HP介质。通过研究吸附过程，对常规琼脂糖介质和接枝型聚蔗糖-琼脂糖介质进行了比较。主要结果如下：1. 首次制备了接枝型聚蔗糖-琼脂糖色谱介质并对其特性进行了详细探讨。考察了预混合时间、聚蔗糖初始浓度、NaOH加入量以及反应时间对聚蔗糖接枝量的影响。结果表明，聚蔗糖最大接枝量可达50 mg/mL以上，显著大于目前所报道的葡聚糖的接枝量。表征了所制备的接枝型聚蔗糖-琼脂糖介质，接枝聚蔗糖后，介质的比表面积最大提高约20%，羟基含量最大提高约40%；在聚蔗糖接枝量显著高于葡聚糖接枝量的情况下，孔径降低幅度略大于葡聚糖。2. Tris-Sepharose HP介质的制备与吸附机理研究。考察了反应体系pH值、温度、Tris溶液初始浓度以及反应时间对配基密度的影响，所得Tris-Sepharose HP介质的最大配基密度达63.5 μmol/mL。通过对吸附过程的研究发现Tris-Sepharose HP对牛血清白蛋白的吸附过程包括静电相互作用和氢键作用，pH 6.0条件下，以静电相互作用为主；pH 5.0条件下，以氢键作用为主。Tris-Sepharose HP的静电作用属于弱阴离子交换模式，可在较低盐浓度下洗脱，有利于保持蛋白质的结构和活性。3. 接枝型Tris-聚蔗糖10-Sepharose HP介质的制备。通过对吸附过程的研究，比较了常规琼脂糖介质和接枝型聚蔗糖-琼脂糖介质的吸附性能，结果表明接枝型聚蔗糖-琼脂糖介质在静态吸附容量、孔扩散系数以及动态吸附容量方面均显著提高，表明接枝型聚蔗糖-琼脂糖介质具有良好的色谱性能。4. 应用Tris-Sepharose HP和Tris-P10-Sepharose HP介质从坛紫菜中分离了R-藻红蛋白。比较了Tris-Sepharose HP、Tris-P10-Sepharose HP介质与常用离子交换色谱介质Q Sepharose HP对R-藻红蛋白的分离过程，结果表明Tris-P10-Sepharose HP介质对R-藻红蛋白的吸附量比Q Sepharose HP大约1倍，明显优于商品化Q Sepharose HP介质。; Chromatography is one of the most widely used separation technologies, which is involved in more than 80% separation processes, especially the separation process of pharmaceuticals. Recently, the research interest in chromatography focuses on the development of highly efficient chromatographic media. However, for most common media, the adsorption capacity is relatively low and the mass transfer is slow. The development of polymer grafted media has overcome these disadvantages mentioned for common media by introducing polymer to its surface. At present, more and more attentions have been paid to this kind of novel media, especially the dextran grafted agarose media in the separation process of macromolecules. However, the linear structure of dextran will occupy more pore volume of agarose gel and make it too viscous for grafting reaction.In this study, polysucrose, instead of dextran, is incorporated into Sepharose HP particles. Subsequently, tris(hydromethyl)aminomethane (Tris) is used as a ligand for functionalization of both Sepharose HP and polysucrose 10 grafted Sepharose HP. By comparing the adsorption processes of Tris-Sepharose HP and Tris-polysucrose 10-Sepharose HP, the performance of polysucrose grafted media is evaluated. In this study, the following conclusions have been drawn: 1. Polysucrose grafted agarose gel was prepared and characterized for the first time. The preparation has been optimized in terms of pre-mixing time, initial concentration of polysucrose, NaOH amount added and reaction duration. A maximum of over 50 mg/mL of polysucrose is grafted onto Sepharose HP, which is significantly higher than that of dextran reported. The prepared polysucrose grafted Sepharose HP particles have 20% larger specific surface area and 40% higher hydroxyl group content, as compared with Sepharose HP. While, the decrease of apparent pore radius of polysucrose grafted Sepharose HP is slightly more than that of dextran grafted media when the content of polysucrose grafted is significantly higher.2. The preparation of Tris-Sepharose HP has been optimized in terms of pH value, reaction temperature, initial concentration of Tris and reaction duration. The maximum ligand density obtained is 63.5 μmol/mL. By investigating the adsorption process, it has been found that both electrostatic interaction and hydrogen bond are involved. The electrostatic interaction is dominating at pH 6.0, while the hydrogen bond interaction at pH 5.0. The electrostatic interaction of Tris-Sepharose HP is functioned in a mode of weak anion exchange, indicating that molecules adsorbed can be eluted under low ion strength, which is favorable for the maintenance of structure and activity of proteins.3. Tris-polysucrose 10-Sepharose HP particles have been prepared. By comparing the adsorption process between Tris-polysucrose 10-Sepharose HP and Tris-Sepharose HP, it has been found that the static adsorption capacity, pore diffusion coefficient and dynamic binding capacity of Tris-polysucrose 10-Sepharose HP are all significantly improved, suggesting that Tris-polysucrose 10-Sepharose HP has excellent chromatographic performance.4. Both Tris-Sepharose HP and Tris-P10-Sepharose HP were employed for the separation of R-phycoerythrin. By comparing the separation results using Q Sepharose HP, Tris-Sepharose HP and Tris-P10-Sepharose HP, it has been found that Tris-P10-Sepharose HP has the largest adsorption capacity for R-phycoerythrin, which is much better than that of the commercial available media Q Sepharose HP. |
修改评论