离子液体溶解法制备纤维素层析介质及应用研究

发布时间：2019-07-05 15:49

【摘要】：层析是生物分离技术中最有效及最广泛应用的方法之一,具有分离效率高、选择性好、设备简单、便于自动化控制、分离过程温和及分离机理多样化等优势。层析分离广泛应用于蛋白质、基因工程药物等多种生物对象的分离纯化,其中层析介质是关键要素之一。本文采用离子液体直接溶解纤维素,探讨纤维素微球制备的新方法,并功能化成层析介质,应用于生物分离。主要包括以下三方面内容：第一,纤维素微球制备工艺的优化及孔道扩增。选取三种纤维素原料—精制棉、脱脂棉和微晶纤维素,利用离子液体BmimCl直接溶解纤维素制备黏胶,以环己烷或淀粉作为制孔剂,采用反相悬浮和降温固化法,制备得到多孔纤维素微球。考察了纤维素种类及浓度、油水比、搅拌转速、固化剂加入量、制孔剂种类及加入量等影响,优化了制备条件。所制纤维素微球的球形度好,粒径80～300μm,湿真密度约1.01g/ml,孔度95%左右,平均孔径约40nm,比表面积为90m2/ml,是一种生物大分子层析分离的良好基质。结果表明,环己烷扩孔效果较好,有利于生物大分子分离。第二,纤维素介质制备及蛋白吸附性能。将微晶纤维为原料制备的未扩孔、淀粉扩孔和环己烷扩孔微球分别偶联上阴离子交换配基DEAE,得到三种弱阴离子交换介质Cell-M-DEAE、Cell-S-DEAE和Cell-C-DEAE,考察了离子交换容量、静态吸附、动态吸附及穿透行为。离子交换容量均在250μmol/ml左右; Cell-C-DEAE的静态吸附容量较低,但有效扩散系数高,吸附速率快,10%穿透时动态吸附容量高。结果表明,扩孔微球的动态吸附性能得到改善,环己烷扩孔比淀粉扩孔效果好。将微晶纤维素-环己烷扩孔微球偶联上疏水性电荷诱导配基MMI,制备得到新型介质Cell-C-MMI,考察了卵黄抗体IgY的静态和动态吸附性能。发现pH中性条件下饱和吸附容量大,pH4时吸附能力显著减弱,加入适量硫酸铵有助于IgY吸附。第三,疏水性电荷诱导层析分离卵黄抗体。采用HCIC介质Cell-C-MMI,从蛋黄粉中分离卵黄抗体IgY,考察了上样和洗脱pH的影响,优化了分离条件。结果表明,合适的上样pH为8.0,洗脱pH为4.0,分离得到IgY纯度为70.1%,收率为75.6%,纯化倍数为2.7。新型HCIC介质对IgY有良好的分离性能,但IgY纯度有待进一步提高。本文围绕纤维素微球介质,开展了介质制备、功能化和应用研究。实现了离子液体直接溶解纤维素,制备纤维素微球,简化了黏胶制备工艺,过程绿色环保。所制备的纤维素新型介质,具有吸附容量高、分离性能优良等特点,为生物大分子分离提供了新型材料。
文内图片：

图片说明：图1.1层析原理示意图
[Abstract]:Chromatography is one of the most effective and widely used methods in the bioseparation technology, and has the advantages of high separation efficiency, good selectivity, simple equipment, convenient automatic control, mild separation process and diversified separation mechanism. The chromatographic separation is widely used in the separation and purification of various biological objects, such as protein, genetic engineering medicine and the like, wherein the chromatographic medium is one of the key elements. In this paper, the ion liquid is used to directly dissolve the cellulose, and a new method for the preparation of the cellulose microspheres is discussed, and the chromatographic medium is formed, and the method is applied to the biological separation. It mainly includes the following three aspects: Optimization of the preparation process of the first and the cellulose microspheres and the channel expansion The method comprises the following steps of: selecting three kinds of cellulose raw materials such as refined cotton, non-absorbent cotton and microcrystalline cellulose, directly dissolving the cellulose with ionic liquid BmciCl to prepare the adhesive, using cyclohexane or starch as a pore-forming agent, adopting an inverse suspension and temperature-reducing and curing method, and preparing the porous cellulose micro-emulsion. The effects of cellulose type and concentration, oil-water ratio, stirring speed, addition amount of curing agent, type of pore-forming agent and addition amount were studied, and the preparation strip was optimized. The prepared cellulose microsphere has the advantages of good sphericity,80-300 & mu; m, wet true density of about 1.01 g/ ml, porosity of about 95%, average pore diameter of about 40 nm, specific surface area of 90 m2/ ml, and is a good basis for chromatographic separation of biological macromolecules. The results show that the effect of the cyclohexane is good, which is beneficial to the separation of the biomacromolecule. Second, the preparation of cellulose medium and protein absorption The non-reamed, starch-reamed and cyclohexane-reamed microspheres prepared from the microcrystalline fiber as raw materials were respectively coupled with the anion-exchange ligand DEAE to obtain three kinds of weak anion-exchange media Cell-M-DEAE, Cell-S-DEAE and Cell-C-DEAE, and the ion exchange capacity, static adsorption, dynamic adsorption and wearing were investigated. The ion exchange capacity is about 250 & mu; mol/ ml; the static adsorption capacity of the Cell-C-DEAE is low, but the effective diffusion coefficient is high, the adsorption rate is high, and the dynamic adsorption at the time of 10% penetration The results show that the dynamic adsorption property of the microballoon is improved, and the expansion ratio of the cyclohexane is better than that of the starch. The novel medium Cell-C-MMI was prepared by coupling the hydrophobic charge-induced ligand (MMI) on the coupling of the microcrystalline cellulose-cyclohexane-reamer microspheres, and the static and dynamic absorption of the yolk antibody IgY was investigated. It was found that the adsorption capacity of the saturated adsorption capacity at the pH neutral condition was large, the adsorption capacity at pH 4 was significantly reduced, and a proper amount of sulfuric acid was added to help the Ig. Y adsorption. Third, hydrophobic charge-induced chromatography The yolk antibody IgY was isolated from the yolk powder by means of the HCIC medium Cell-C-MMI. The effects of loading and elution pH were investigated. The results showed that the appropriate loading pH was 8.0, the elution pH was 4.0, the purity of IgY was 70.1%, the yield was 75.6%, and the purification was the same. The number of IgY is 2.7. The new HCIC medium has good separation performance for IgY, but the purity of IgY is to be determined. In this paper, the preparation and work of the medium are carried out in the paper. The method has the advantages that the ion liquid is directly dissolved and the cellulose is prepared, the cellulose microsphere is prepared, and the preparation process of the adhesive is simplified. and the prepared cellulose novel medium has the characteristics of high adsorption capacity, excellent separation performance and the like,
【学位授予单位】：浙江大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：TQ352.7

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