【作者】 龚波林；

【导师】 耿信笃；

【作者基本信息】 西北大学 ， 分析化学， 2003， 博士

【摘要】 高分子聚合物基质高效液相色谱固定相具有选择性高、使用pH范围宽、亲水性好、稳定性好以及负载能力高等优点，更适合于生物工程产品的分离纯化和制备，是固定相的发展趋势。所以本文将种子溶胀技术与高分子溶液致孔技术相结合，采用一步种子溶胀技术制备了颗粒单分散并且具有大孔结构的交联甲基丙烯酸环氧丙酯微球，且以此微球为基质进一步用新的化学改性方法制备了一系列新型聚合物基质固定相，将其用于生物大分子的分离和纯化。 论文包括以下八个部分： 1．文献综述：系统地综述了聚合物基质固定相的研究进展，主要综述了聚合物基质类型、制备方法和化学改性方法。对分离生物大分子的有机聚合物基质色谱固定相的发展趋势进行了展望。共引用文献150篇。 2．采用分散聚合方法，制备了单分散的聚苯乙烯微球，系统地研究了单体浓度、引发剂用量、稳定剂用量、反应温度和时间等各种聚合参数，对聚合产物粒度及其分散性的影响。在优化反应条件的基础上，制备出低分子量粒径在1.5～7.0μm范围内的单分散聚苯乙烯微球。 3．以3.4μm单分散线性PSt为种子，将种子溶胀聚合技术与高分子溶液致孔技术相结合，采用一步种子溶胀聚合法成功地制备了颗粒单分散并具有大孔结构的交联聚甲基丙烯酸环氧丙酯微球(P_GMA-EDMA)。首次用凝胶渗透色谱法较深入地研究了交联度、惰性小分子致孔剂和高分子溶液致孔剂浓度等因素对P_GMA-EDMA微球孔径、孔径分布的影响，筛选出了一种孔径分布范围较宽的微球作为体积排阻色谱介质用于五种标准聚苯乙烯样品的分离。 4．首次以孔径分布范围合适的P_GMA／EDMA微球为基质，键合甘油、季戊四醇、二乙醇胺、δ-葡萄糖酸内酯等含有多羟基一类小分子化合物进行亲水化改性反应。选择出亲水性良好的改性树脂，用于三种蛋白质和一种肽混合样的体积排阻色谱（SEC）分离。 5．以大孔单分散交联P_GMA／EDMA为基质，采用新的化学改性方法制备了的弱阳离子交换（WCX）、中强阳离子交换（MCX）和强阳离子交换（SCX）三种类型新型性能优异的离子交换色谱填料。详细考察了每种填料的离子交换特征、标准蛋白的分离性能、酸碱稳定性、表面亲水性能、盐浓度、盐种类、负载量以 及pH值对保留的影响，结果表明所合成的填料完全符合蛋白在阳离子交换色 谱上的保留规律。将三种填料应用于生物工程产品的分离纯化，结果满意。6.以单分散交联PGM、EoMA树脂为基质，采用新的改性方法设计合成了三种弱阴 离一r交换（场认X）、两种强阴离子交换（SAX）色谱填料。依据侮种改性方法 的亲水性程度及蛋白的质量回收率，初步解释了填料的分离性能差异。选择性 能优异的场人X和SAX填料用于基因工程产品重组人粒细胞一巨噬细胞集落刺 激因r（rhG一CSF）和重组人干细胞因子（rhSCF）的分离纯化，取得较满意 的效果。7.以单分散大孔交联PGM、DMA树脂为基质，采用新的化学改性方法合成了邻二 醇配基和苯基配基两种新型疏水色谱填料。两种填料分离性能优良，动力学吸 附容量高于TSK gel和连续棒状的HIC填料。首次以邻二醇疏水填料为介质， 可一步复性及同时纯化7mol几欲酸肌提取的重组人干扰素一Y，千扰素一Y的 纯度高达到90%以上，比活达5.3 x 107Iu/mg。8.首次以单分散大孔交联PGM、EDMA树脂为基质，以氨基葡萄糖为配基，制备了 亲水性好，耐压性高，吸附容量大的氨基葡萄糖高效聚合物亲合色谱填料，将 其应用于粗品Con一A的分离纯化，从纯度仅为15%提高到95%。更多还原

【Abstract】 Polymeric separation media having a of high selectivity, chemical stability in the entire pH range, good hydrophilicity, high column loading, are more suitable for biopolymer separation, and will be the tendency for the development of stationary phase in the future. With the combining dispersion polymerization with swelling polymerization and polymeric solution porogens, this thesis presents monodisperse poly(glycidylmethacrylate-ethylenedimethacrylate) (POMA/EDMA) particles with macroporous synthesized by one-step swelling and polymerization method. Based on this media, a series of new stationary phase for HPLC were synthesized by new chemically modified methods and can be applied for biopolymer separation. The thesis includes the following eight parts:1. Chapter 1 provides a comprehensive review on the recent development of the polymeric matrix stationary phases in HPLC, mainly on polymeric matrix types, preparation methods and chemically modified methods. The tendency for separating biopolymer by polymeric matrix was also predicted. Totally 150 references were listed.2. Adopting dispersion polymerization method, monodisperse polystryrene seed beads were prepared. The effects of the concentration of monomer, amount of initiator, amount of stabilizer, reaction temperature and time on particle distribution and diameter were systematically investigated. Monodisperse, polystryrene seed beads with low molecular weight in the range of 1.5-7.0 m were prepared under optimized reaction conditions.3. Taking polystryrene beads of 3.4 m as seed, mondisperse PGMA/EDMA beads with macroporous in the range of 8.0~12.0 m were prepared by a single-step swelling and polymerization method. The effects of the concentration of monomer, small molecular and linear polystyrene on pore size distribution in uniformly sized porous PGMA/EDMAbeads were firstly investigated in details by gel permeation chromatographic method, and a kind of PGMA/EDMA bead with suitable pore size distribution range was picked out for separation of five standard polystyrenesamples by size exclusion chromatography(SEC).4. Using PGMA/EDMA beads with suitable pore size distribution range as matrix, a series of hydrophilic modified reactions were carried out for the first time by using small molecular compounds, such as glycerol et al. A kind of modified resin with better hydrophilicity was used for the separation of three proteins and a peptide by SEC. It was found that it followed SEC mechanism and a satisfactory result was obtained.5. Based on PGMA/EDMA beads with macroporous PGMA/EDMA as matrix, three kinds of resins, as weak cation exchange (WCX), strong cation exchange (SCX), middle-strong cation exchange (MCX) chromatographic stationary phase were synthesized by new chemically modified methods. Each kind of stationary phases was evaluated with the chromatographic behaviour, isolation, reproducibility, hydrophilicity, effects of salt concentration, salt type, column loading and pH on the separation and retention of proteins. It was found that the three kinds of stationary phases follow ion exchange chromatographic (IEC) retention mechanism, and can be used for rapid separation of biotechnology production with satisfactory results.6. Using PGMA/EDMA beads with macroporous PGMA/EDMA as matrix, three kinds of weak anion exchange (WAX) packings and two kinds of strong anion exchange (SAX) chromatographic packings were synthesized by new chemically modified methods. The difference in terms of resolution of the packings were explained according to hydrophilically modified methods and mass recoveries of proteins. The chromatographic behaviours of WAX and SAX packings were proved to be excellent and were applied for the separation of recombinant human stem cell factor (rhSCF) and granulocyte colony stimulating factor (rhG-CFS).7. Applying PGMA/EDMA beads with macroporous PGMA/EDMA as matrix, two new kinds of hydrophobic interaction chromatographic (HIC) stationary phases with diol and更多还原