毛细管电泳—电化学检测技术影响灵敏度因素及在自由基、多糖分析中的应用研究
摘要
毛细管电泳-电化学检测是近年来发展最快的分离分析技术之一。它具有仪器分析所要求的高效、快速、灵敏度高、进样量少、选择性好、仪器简单等优点。本论文围绕毛细管电泳-安培检测技术的原理及其应用做了一些工作。
第一章 概述
本章第一部分简单回顾了毛细管电泳的发展历史、现状及发展前景。第二部分对该技术的分离模式、检测技术及进样技术进行了较详细的总结。第三部分着重介绍了毛细管电泳技术在分子离子分析、单细胞分析、手性分离、药物分析、食品分析及糖类分析中的应用。第四部分简要介绍了本论文的研究工作。
第二章 稀释溶剂对毛细管电泳-安培检测灵敏度的影响
除了毛细管电泳缓冲溶液，缓冲溶液的PH值，分离电压和进样时间，本论文把稀释溶剂作为影响毛细管电泳-安培检测电动进样的一个重要影响因素进行了研究。盐酸可乐定、氢氯噻嗪和芦丁在水溶液中分别带正电荷，不带电荷、负电荷，它们可以通过毛细管电泳-电化学检测在25mmol·l~(-1) Na_2B_4O_7-50mmol·l~(-1)NaH_2PO_4的缓冲溶液中实现完全分离。实验时，分别用水、甲醇、甲酰胺、缓冲溶液、盐酸和氢氧化钠等做稀释溶剂稀释上述三种待测样品的储备液，然后研究它们对毛细管电泳-安培检测灵敏度的影响。结果显示在电动进样条件下，用不同稀释溶剂稀释待测样品严重影响待测样品的电流响应。复方降压药珍菊降压片中活性成分盐酸可乐定、氢氯噻嗪和芦丁的含量分别为每片0.03mg∶5mg∶20
摘要
mg，由于盐酸可乐定的含量比氢氯唾嗦和芦丁低很多，因此要实现三种待测样
品的同时测定比较困难，而我们通过选择合适的稀释溶剂调节三种待测样品的电
流响应，实现了这三种待测样品的同时分离，测定结果令人满意。
第三章轻基自由基的毛细管电泳一安培法检测
经基自由基是最具活性的活性氧自由基，当经基自由基过多或者细胞内的抗
氧化剂不足的时候就会导致细胞受损伤。因为经基自由基很活泼在体内存在时间
很短，所以对其检测具有一定的难度。我们将水杨酸加入到能产生轻基自由基的
Fenton反应体系中，它可以捕获轻基自由基生成具有电化学活性的2，3一二轻基
苯甲酸(2，3一DHBA)和2，5一二经基苯甲酸(2，5一DHBA)。在最佳条件下，水杨
酸、2，3一DHBA和2，5一DHBA可以在巧min内就达到完全分离和测定。在此基础上
研究了当归多糖对轻基自由基的消除能力。把当归多糖加入到Fenton反应体系
中后，检测到的2，3一OHBA和2，5一DHBA的峰高降低了，表明部分的经基自由基己
被消除。实验结果表明，我们提出的毛细管电泳一安培法(CZE一AD)方法在检测
轻基自由基和鉴定自由基清除剂的清除能力方面具有较高的灵敏度和较好的实
用性。
第四章毛细管电泳一安培检测法测定昆布中多糖成分
本论文首次用毛细管电泳一安培检测法测定了昆布多糖水解单糖的成分，实
现对昆布多糖的测定。在选定的最佳条件下，昆布多糖水解产物岩藻糖、半乳糖、
葡萄糖、甘露糖、木糖在20min内实现完全分离而且在铜电极上有着很好的电
流响应。通过对昆布多糖中水解单糖成分含量的计算显示该多糖中岩藻糖、半乳
糖、葡萄糖、甘露糖、木糖的摩尔比为10.5:2.8:1.0:7.3:3.4。与常见方法相
比，该方法有分析快速、样品用量少、操作仪器简单、灵敏高和重现性好等优点。
Capillary Electrophoresis (CE) has become one of the fastest technique in recent years because of its high efficiency, rapidness, good sensitivity and simple instrument, which are necessary for instrumental analyse. In the present paper, a series of investigation has been developed in principle and application on capillary zone electrophoresis with amperometric detection and the third is capillary zone electrophoresis with amperometric detection for composition analysis of Laminarin.
Chapter 1 Review
The first section has reviewed the history, the current situation on High Performance Capillary Electrophoresis (HPCE). Second one has introduced the fundamental theory of HPCE and its characters, detection technique and sample injection, individually. Third one is about the application of HPCE, which includes analyses of inorganic and organic small molecules, food, Chinese herb, chiral molecules, medicine, environmental, biomacromolecules etc. The last one is about the purpose and meanings of this dissertation.
Chapter 2 Effect of diluting agent on sensitivity in capillary electrophoresis with amperometric detection
Besides the running buffer, pH of buffer, separation voltage and sampling time, the diluting agent was studied in this paper as one of the factors influencing the sensitivity in capillary electrophoresis (CE) with amperometric detection (AD). Clonidine hydrochloride, hydrochlorothiazide and rutin, which are positive charged, neutral and negatively charged respectively in aqueous solutions, could be perfectly
separated by CE with 20mmol-L-1Na2B4O7- 40mmol-L-1 NaH2PO4 as running buffer and detected by measuring their current responses with AD. Before CE running, water, methanol, formamide, running buffer, hydrochloric acid and sodium hydroxide were applied as diluting agents in this experiment to investigate their effects on the sensitivity of CE-AD. The results showed that the current responses of these three analytes were great affected in different appearance when the running buffer was constant but different diluting agents were used. This method was applied to simultaneously determine the active ingredients in one Chinese compound hypotensor named Zhen Ju Jiang Ya Pian, in which the contents of clonidine hydrochloride, hydrochlorothiazide and rutin is very different as 0.03mg : 5mg : 20mg per tablet, and satisfactory results were obtained by adjusting their sensitivity by selecting the suitable diluting agent. Chapter 3 Study on determination of hydroxyl radical by capillary zone
electrophoresis with amperometric detection
Hydroxyl radical (OH ) is the most aggressive one among these reactive oxygen radicals and cause cell injury when they are generated in excess or the cellular antioxidant defense is impaired. However, OH is very difficult to be detected for its very reactivity and very short half-time In this paper, the simple and high sensitive capillary zone electrophoresis with amperometric detection (CZE-AD) was introduced to determine OH- produced by Fenton system by determining its reaction products with salicylic acid (SAL): 2,3-dihydroxybenzoic acid (2,3-DHBA) and 2,5-dihydroxybenzoic acid (2,5-DHBA). Under the optimum conditions, SAL, 2,3-DHBA and 2,5-DHBA could be perfectly separated within 15 min. Their detection limits were as low as 2 10-8 mol l-1, which were much better than CE- UV method. The method was also applied to study the scavenging activity of angelica
polysaccharide. The experimental results showed that the introduced CZE-AD method was very sensitive and practical in both the determination of free OH and evaluation of the activities of OH scavengers. Chapter 4 Capillary Zone Electrophoresis with Amperometric Detection for
Composition Analysis of Laminarin
The composition of laminarin was firstly determined by analyzing its hydrolysis monosaccharides with capillary zone electrophoresis - amperometric detection (CZE-AD). Under the selected optimum conditions, fucose, galactose, glucose, mannose and xylose, which are hydrolysis products of laminarin, could
第一章 概述
本章第一部分简单回顾了毛细管电泳的发展历史、现状及发展前景。第二部分对该技术的分离模式、检测技术及进样技术进行了较详细的总结。第三部分着重介绍了毛细管电泳技术在分子离子分析、单细胞分析、手性分离、药物分析、食品分析及糖类分析中的应用。第四部分简要介绍了本论文的研究工作。
第二章 稀释溶剂对毛细管电泳-安培检测灵敏度的影响
除了毛细管电泳缓冲溶液，缓冲溶液的PH值，分离电压和进样时间，本论文把稀释溶剂作为影响毛细管电泳-安培检测电动进样的一个重要影响因素进行了研究。盐酸可乐定、氢氯噻嗪和芦丁在水溶液中分别带正电荷，不带电荷、负电荷，它们可以通过毛细管电泳-电化学检测在25mmol·l~(-1) Na_2B_4O_7-50mmol·l~(-1)NaH_2PO_4的缓冲溶液中实现完全分离。实验时，分别用水、甲醇、甲酰胺、缓冲溶液、盐酸和氢氧化钠等做稀释溶剂稀释上述三种待测样品的储备液，然后研究它们对毛细管电泳-安培检测灵敏度的影响。结果显示在电动进样条件下，用不同稀释溶剂稀释待测样品严重影响待测样品的电流响应。复方降压药珍菊降压片中活性成分盐酸可乐定、氢氯噻嗪和芦丁的含量分别为每片0.03mg∶5mg∶20
摘要
mg，由于盐酸可乐定的含量比氢氯唾嗦和芦丁低很多，因此要实现三种待测样
品的同时测定比较困难，而我们通过选择合适的稀释溶剂调节三种待测样品的电
流响应，实现了这三种待测样品的同时分离，测定结果令人满意。
第三章轻基自由基的毛细管电泳一安培法检测
经基自由基是最具活性的活性氧自由基，当经基自由基过多或者细胞内的抗
氧化剂不足的时候就会导致细胞受损伤。因为经基自由基很活泼在体内存在时间
很短，所以对其检测具有一定的难度。我们将水杨酸加入到能产生轻基自由基的
Fenton反应体系中，它可以捕获轻基自由基生成具有电化学活性的2，3一二轻基
苯甲酸(2，3一DHBA)和2，5一二经基苯甲酸(2，5一DHBA)。在最佳条件下，水杨
酸、2，3一DHBA和2，5一DHBA可以在巧min内就达到完全分离和测定。在此基础上
研究了当归多糖对轻基自由基的消除能力。把当归多糖加入到Fenton反应体系
中后，检测到的2，3一OHBA和2，5一DHBA的峰高降低了，表明部分的经基自由基己
被消除。实验结果表明，我们提出的毛细管电泳一安培法(CZE一AD)方法在检测
轻基自由基和鉴定自由基清除剂的清除能力方面具有较高的灵敏度和较好的实
用性。
第四章毛细管电泳一安培检测法测定昆布中多糖成分
本论文首次用毛细管电泳一安培检测法测定了昆布多糖水解单糖的成分，实
现对昆布多糖的测定。在选定的最佳条件下，昆布多糖水解产物岩藻糖、半乳糖、
葡萄糖、甘露糖、木糖在20min内实现完全分离而且在铜电极上有着很好的电
流响应。通过对昆布多糖中水解单糖成分含量的计算显示该多糖中岩藻糖、半乳
糖、葡萄糖、甘露糖、木糖的摩尔比为10.5:2.8:1.0:7.3:3.4。与常见方法相
比，该方法有分析快速、样品用量少、操作仪器简单、灵敏高和重现性好等优点。
Capillary Electrophoresis (CE) has become one of the fastest technique in recent years because of its high efficiency, rapidness, good sensitivity and simple instrument, which are necessary for instrumental analyse. In the present paper, a series of investigation has been developed in principle and application on capillary zone electrophoresis with amperometric detection and the third is capillary zone electrophoresis with amperometric detection for composition analysis of Laminarin.
Chapter 1 Review
The first section has reviewed the history, the current situation on High Performance Capillary Electrophoresis (HPCE). Second one has introduced the fundamental theory of HPCE and its characters, detection technique and sample injection, individually. Third one is about the application of HPCE, which includes analyses of inorganic and organic small molecules, food, Chinese herb, chiral molecules, medicine, environmental, biomacromolecules etc. The last one is about the purpose and meanings of this dissertation.
Chapter 2 Effect of diluting agent on sensitivity in capillary electrophoresis with amperometric detection
Besides the running buffer, pH of buffer, separation voltage and sampling time, the diluting agent was studied in this paper as one of the factors influencing the sensitivity in capillary electrophoresis (CE) with amperometric detection (AD). Clonidine hydrochloride, hydrochlorothiazide and rutin, which are positive charged, neutral and negatively charged respectively in aqueous solutions, could be perfectly
separated by CE with 20mmol-L-1Na2B4O7- 40mmol-L-1 NaH2PO4 as running buffer and detected by measuring their current responses with AD. Before CE running, water, methanol, formamide, running buffer, hydrochloric acid and sodium hydroxide were applied as diluting agents in this experiment to investigate their effects on the sensitivity of CE-AD. The results showed that the current responses of these three analytes were great affected in different appearance when the running buffer was constant but different diluting agents were used. This method was applied to simultaneously determine the active ingredients in one Chinese compound hypotensor named Zhen Ju Jiang Ya Pian, in which the contents of clonidine hydrochloride, hydrochlorothiazide and rutin is very different as 0.03mg : 5mg : 20mg per tablet, and satisfactory results were obtained by adjusting their sensitivity by selecting the suitable diluting agent. Chapter 3 Study on determination of hydroxyl radical by capillary zone
electrophoresis with amperometric detection
Hydroxyl radical (OH ) is the most aggressive one among these reactive oxygen radicals and cause cell injury when they are generated in excess or the cellular antioxidant defense is impaired. However, OH is very difficult to be detected for its very reactivity and very short half-time In this paper, the simple and high sensitive capillary zone electrophoresis with amperometric detection (CZE-AD) was introduced to determine OH- produced by Fenton system by determining its reaction products with salicylic acid (SAL): 2,3-dihydroxybenzoic acid (2,3-DHBA) and 2,5-dihydroxybenzoic acid (2,5-DHBA). Under the optimum conditions, SAL, 2,3-DHBA and 2,5-DHBA could be perfectly separated within 15 min. Their detection limits were as low as 2 10-8 mol l-1, which were much better than CE- UV method. The method was also applied to study the scavenging activity of angelica
polysaccharide. The experimental results showed that the introduced CZE-AD method was very sensitive and practical in both the determination of free OH and evaluation of the activities of OH scavengers. Chapter 4 Capillary Zone Electrophoresis with Amperometric Detection for
Composition Analysis of Laminarin
The composition of laminarin was firstly determined by analyzing its hydrolysis monosaccharides with capillary zone electrophoresis - amperometric detection (CZE-AD). Under the selected optimum conditions, fucose, galactose, glucose, mannose and xylose, which are hydrolysis products of laminarin, could
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126. Q. Wang, F. Ding, H. Li, R He, Y. Fang., Biochem. Chromatogr. 2003,17(7):483