【作者】 喻彦林；

【导师】 夏之宁；

【作者基本信息】 重庆大学 ， 药物化学， 2005， 硕士

【摘要】 毛细管电泳在分析复杂体系（如环境体系、生物体系）中小分子时,颗粒物和生物大分子常会带来各种各样的麻烦,必须对样品进行烦琐的预处理。但目前的预处理技术多是离线方式,不仅过程复杂,还会引起严重的样品损失,给毛细管电泳在生命体系分析中的应用带来不便。因此,发展可在线（或在柱）滤除大分子和颗粒物干扰、简便易行的毛细管电泳方法,对进一步扩展毛细管电泳的应用有重要意义。本论文在分离毛细管进样端口原位制备透析膜,建立了一种在柱透析进样毛细管电泳方法。该方法可对复杂样品直接进样,有效避免基质中大分子和颗粒物干扰;采样量小,具有微透析特点。本研究详细考察了该方法的性能,并进行了其应用研究。主要研究内容如下: 1) 建立了透析进样毛细管电泳方法并考察了系统性能。在分离毛细管进样端端口采用相转化法原位制备了聚砜膜。直接用该毛细管对复杂样品中小分子采样,实现了透析与毛细管电泳在柱联用分析。膜在毛细管端口原位生成,与毛细管结合紧密,死体积小,能有效拦截大分子和颗粒物,pH 耐受范围宽,稳定性好,能连续使用12 h 以上。电泳重现性好,柱效损失小。既可以电迁移进样,也可以采用平衡透析进样。2) 对系统性能进行了优化。膜的截割分子量（样品净化能力）可以通过控制成膜液配方控制,提高了本方法的灵活性;进行了透析进样毛细管电泳电化学检测研究,提高了检测灵敏度,扩大了本方法的使用范围。3) 将本方法应用于咖啡牛奶中游离咖啡因的直接分析。咖啡牛奶没有经过任何预处理直接进样,测定步骤简单,蛋白等物质干扰小。测得其中游离咖啡因浓度为0.68 mmol·L^-1。4) 采用本方法研究了药物与蛋白的相互作用。通过测定与大分子作用后的小分子的游离浓度,可以求解它们之间的结合常数。测得盐酸异丙嗪与牛血清白蛋白的结合常数为:1.47×104 L·mol^-1。与常规毛细管电泳相互作用分析方法相比,本方法中不存在大分子的干扰,在小分子与大分子相互作用分析中有广泛的应用前景。5) 将本方法应用于内源性小分子的测定,测定了血糖浓度。采用柱端安培检测,以自制铜微电极为工作电极,通过优化电泳条件和检测条件,能较好的测定更多还原

【Abstract】 Macromolecules and particles are troublesome when small molecules in real samples such as biological samples and environmental samples are analyzed by capillary electrophoresis (CE), so it is really desirable to couple sample clean-up technologies with CE. Off-line coupled sample preparation, which is the most commonly used mode, may cause great sample loss and contamination; also it is tedious and time consuming. So on-line or in-line sample clean-up technologies are badly needed to further the application of CE in complex matrixes analysis. In this paper, a novel method, which is very simple, of in-line dialysis capillary electrophoresis was established. A polysulfone membrane was prepared in the inlet end of the separation capillary by phase-inversion process, which endued the capillary with an additional function of clean-up sampling. The capillary can be directly used for clean-up sampling and in-line electrophoresis separation of small molecules in complex sample. The preparation process and performance of the system was studied in detail and several applications with satisfactory results were performed. The main research and results are as below: 1. A novel CE method with dialysis sampling function was established and its performance was studied. A polysulfone membrane was prepared in the inlet end of the separation capillary by phase-inversion process, which endued the capillary with an additional function of clean-up sampling. The membrane cut off macromolecules and particles efficiently and persistently. As the membrane was formed in situ, there is almost no dead volume. Using this capillary in electrophoresis, column efficiency with little reduction was achieved and the RSDs of migration times and peak areas of chlorpheniramine were 0.8% and 4.6%, respectively. During continuous use of this capillary for 12 hours, the performance of the membrane changed little. Both electrokinetical injection and equilibrium dialysis injection was available. 2. The molecule weight cutoff of the membrane (sample cleanup ability) could be controlled by the control of the formula of the casting solution, which enhanced the flexibility of the method. In order to improve the detection sensibility, electrochemical detection method was developed, which extended the application field of this method. 3. This technology has been applied in the analysis of free caffeine in Coffee Milk Drink. Without any sample preparation, Coffee Milk was injected and analyzed. Little interference was observed, and the concentration was determined to be 0.68 mmol·L-1. 4. By applied this method into the evaluation of the molecule interaction, the binding constant of promethazine hydrochloride with BSA was obtained, that is 1.47×104 L·mol-1. In this method, the interfere of macromolecules which exist in common capillary electrophoresis interaction methods was eliminated, so the method is promising in the interaction analysis between macromolecules and small molecules. 5. This method was also applied into the determination of glucose in human blood. Electrochemical detection with copper microelectrode as working electrode was adopted. Whole blood was diluted and injected directly. The concentration of blood sugar was 5.53 mmol·L-1,and the limit of detection was 0.1 mmol·L-1. The technology may be further used to monitor the endogenous or exogenous small molecules and their metabolites, which may contribute to studies in life science and pharmaceutics. Furthermore, as the capillary could be inserted into the animal’s vein and equilibrium dialysis injection was available, this method may be used for in vivo analysis.更多还原