【作者】 封顺；

【导师】 王吉德； 邹汉法；

【作者基本信息】 新疆大学 ， 应用化学， 2002， 硕士

【摘要】 本文分别将牛血清白蛋白和人血清白蛋白键合到用1,1’-羰基咪唑活化的氨丙基硅胶表面，制备了两种白蛋白键合液相手性固定相。同时在这两种手性固定相上拆分了7种和5种旋光异构体。并对手性固定相的热力学和动力学参数进行了表征。本论文第二章阐述了以1,1’-羰基咪唑活化的氨丙基硅胶合成牛血清白蛋白键合手性固定相的方法，比较了柱上衍生与柱外衍生两种键合蛋白方法的优缺点，发现柱上衍生法要优于柱外衍生法。用牛血清白蛋白键合手性固定相成功拆分了色氨酸、华法令等7种旋光异构体，并考察了流动相的pH值以及溶液中正丙醇浓度对其拆分色氨酸的保留行为及分离度的影响。发现流动相为50 mM pH=7.0 的磷酸氢钠缓冲溶液，室温下拆分效果最好，(=2.21。第三章介绍了采用所合成的牛血清白蛋白键合手性柱拆分实际样品--亚叶酸钙的方法，考察了不同色谱条件对其拆分的影响，并对其色谱条件进行了优化。优化结果为以pH 6.0的50 mmol/L磷酸氢钠缓冲溶液为流动相，流速1 ml/min时，[6S]-、[6R]-LV分别在6.45 min和12.00 min时出峰,全过程可在16 min内实现基线分离，其分离度α = 2.19，Rs = 3.88。并做了热力学上的探讨，推测出亚叶酸钙与BSA之间可能属于单个位点作用。另外，将所合成的牛血清白蛋白键合手性柱作为分子生物色谱，快速筛选中药川芎、当归中的有效成分。第四章陈述了以1,1’-羰基咪唑活化的氨丙基硅胶合成人血清白蛋白键合手性固定相的方法，键合量达到57.5 mg/g。用人血清白蛋白键合手性固定相成功拆分了色氨酸、华法令等5种旋光异构体，并考察了流动相的pH值，溶液中正丙醇浓度，盐浓度对拆分色氨酸的影响。发现流动相为50 mM pH=7.0 的<WP=3>磷酸氢钠缓冲溶液，25℃时拆分效果最好，(=10.87。第五章考察了温度对色氨酸保留值的影响，ΔΔH= -2.64 kJ/mol，ΔΔS= 8.382 J/mol·K；利用前沿分析法，分别测得了色氨酸在HSA手性柱上的吸附常数为3.23×104 L/mol，活性位点数是250 nmol/g，当L-色氨酸浓度由203 μmol/L 上升至 324 μmol/L时，饱和吸附量从244 ng 上升至 2092 ng .更多还原

【Abstract】 In this paper, two protein chiral stationary phases (CSP) have been synthesized by immobilizing Bovine serum albumin (BSA) and human serum albumin (HSA) on aminopropyl silica gel which had been further activated with 1,1’- carbonyldiimidaole. Good separations of several racemic compounds were obtained on the above CSPs. Furthermore, character of thermodynamics and dynamics was studied. In chapter two, synthesis of BSA-CSP was developed on the CDI-activated silica gel; the methods used in this study provide numerous advantages over traditional methods, such as easy preparation, low cost, as well as timesaving. BSA was immobilized on the CDI-activated silica gel after packing column and before packing column. The results indicated that the former method is superior to the latter. Good separations of 7 racemic compounds were obtained on the BSA-CSP. The effects of the mobile phase, including the pH and the concentration of 1-propranol in the mobile phase, on the retention behavior of D-, L-tryptophan on BSA-CSP were investigated in detail. The ( of D-, L-tryptophan is 2.21 under the optimal chromatographic condition, mobile phase is 50mM Na2HPO4-NaH2PO4(pH=7.0).In chapter three, [6S]- and [6R]-calcium folinate(LV) were successfully separated on the BSA-CSP prepared in our lab. The effects of pH, the salt concentration, and column temperature on the separation were investigated in detail. Baseline separation was achieved in 10 min: 4.46 min and 7.59 min, α = 2.10，Rs = 2.78 under the optimal chromatographic condition, 50mM Na2HPO4-NaH2PO4(pH=6.5). Character of thermodynamics was studied, and we presume that LV has a single type of binding site on BSA. <WP=5>In chapter four, HSA-CSP was synthesized on the CDI-activated silica gel, 5 racemic compounds were successfully separated on HSA-CSP. The effects of the compsition of the mobile phase, the column temperature on the retention behavior of D-, L-tryptophan on HSA-CSP were also investigated. The ( of D-, L-tryptophan is 10.87 under the optimal chromatographic condition, temperature is 25℃, mobile phase is 50mM Na2HPO4-NaH2PO4(pH=7.0).In chapter five, the effect of column temperature on D-, L-tryptophan was investigated, and ΔΔH= -2.64 kJ/mol, ΔΔS= 8.382 J/mol·K respectively. Frontal analysis was used to determine the association constant and the active binding sites of L-tryptophan on HSA, Ka=3.23×104 L/mol, mL=250 nmol/g, adsorption capacity ranged from 244 ng to 2092 ng when the concentration of L-tryptophan ranged from 203 μmol/L to 324 μmol/L.更多还原