离子交换晶胶层析分离纯化中药多糖的研究-生物化工专业论文.docxVIP

万方数据 万方数据 摘要 超大孔连续床层析分离是一种新型的生物分离技术，其晶胶介质内部有几微米至上百微米的相 互连通的超大孔隙，因其具有良好的选择性、生物兼容性好、传质阻力小和吸附分离迅速等优点， 在生物分离方面有重要应用前景。 实验确定了多糖溶液的分析方法，选用苯酚-硫酸法测定多糖含量，并对该方法的加样回收率进 行了研究，发现其标准曲线线性关系良好，满足实验的要求。以甘草多糖为例，利用实验室自制的 离子交换超大孔晶胶介质层析对其进行纯化，考察得到多糖纯品的含量，研究超大孔晶胶介质在中 药多糖纯化中的应用。 在变温冷冻条件下制备了聚丙烯酰胺基超大孔连续床晶胶基质，通过孔内直接接枝法将甲基丙 烯酸二甲氨基乙酯接枝到晶胶基质制备成阴离子交换晶胶介质，通过测定时间停留分布、渗透率等 研究了其基础性能，并以牛血清白蛋白为模型蛋白进行层析。利用制得的阴离子交换晶胶介质对甘 草粗多糖溶液进行分离纯化。通过大量层析分离实验，结果表明：以去离子水进行层析，在 1.0 cm/min 流速下，用 1.0 M NaCl 一步洗脱得到甘草多糖纯品，其多糖含量为 55.4 %，晶胶吸附量达到 670 μg/mL 晶胶介质。经紫外、红外检测和比旋光度法检测纯度后鉴定得到的多糖为均一组分。 该方法简单快速，为从中药药材中提取纯化多糖提供了参考。实验中以去离子水做缓冲溶液提 取中药多糖，方法简便，成本低，安全无污染。 关键词：晶胶介质，离子交换，甘草多糖，分离纯化 I ABSTRACT Chromatography using supermacroporous monolithic cryogels is a novel method for separation and purification of bioproducts. Cryogels have interconnected supermacropores with pores of several microns to several hundreds of microns. Due to the high selectivity and biological compatibility, low mass transfer resistance, rapid adsorption and separation advantages, cryogels have potential applications in biotechnology and biological areas. In this thesis, the method for quantitatively determining the content of polysaccharides in a given solution was firstly established. The phenol-sulfuric acid method was used to determine the polysaccharide concentration. A good linear relationship on the standard curve, and the standard deviation and the regression coefficient were found to meet the requirements of the experiment. Glycyrrhiza polysaccharide was used as an example and the polyacrylamide-based monolithic cryogel beds were used to separate glycyrrhiza polysaccharide. The purities and recoveries of glycyrrhiza polysaccharide were studied in order to develop a simple and cost effective purification method. Polyacrylamide-based monolithic cryogels have been prepared under the variation freezing condition followed by graft polymerization using N,N’-diethlaminoethyl methacrylate as the grafting monomer. The obtained cryogels were used in the separa