【作者】 王晓葵；

【导师】 何友昭；

【作者基本信息】 中国科学技术大学 ， 分析化学， 2007， 博士

【摘要】 本论文综述了毛细管电泳（capillary electrophoresis，CE）的分离模式、化学发光（chemiluminescence，CL）分析的原理、发光体系分类，以及与毛细管电泳联用的研究现状和最新进展。指出将CE的高分离性能和CL的高灵敏检测性能结合，CE-CL联用技术已经成为一种较理想的分离分析技术，也是分析化学近十几年来的研究热点之一。但是，由于CE和CL联用需要使用引入发光辅助试剂的接口，接口的设计影响到发光的灵敏度和分离性能。本文设计了一种狭缝式CE-CL联用接口，并应用于实际样品多酚物质的分离检测。为了更进一步分离多酚物质，本文还用Tween 20—胶束电动毛细管色谱（micellarelectrokinetic capillary chromatograph，MEKC）方法分离了其他方法难以分离的两种多酚。最后，本文探讨了一种制备型的微柱电泳。具体研究工作如下：1，研制了新式的狭缝式CE-CL联用接口，实现了分离毛细管和反应毛细管在显微镜下快速、精准对接。用内径1mm的石英管作为发光试剂流路，又作为检测光信号窗口。石英管的应用使引入发光试剂所需要的接口制作简便。由于分离毛细管和反应毛细管内径接近，所以样品从分离毛细管进入反应毛细管时扩散小，对峰宽的影响小，是一种较理想的CE-CL联用接口。制作玻璃砂芯片将高电位电极池分隔成两部分：一部分含鲁米诺的缓冲溶液，放置毛细管入口端；另一部分仅含缓冲溶液，放置电极。隔离池有效阻止了鲁米诺的电解，解决了支持缓冲液中鲁米诺电解造成的发光信号漂移问题，避免发光试剂的损耗，并延长缓冲溶液的使用时间，也解决了鲁米诺电解造成的电流增大问题。2，将狭缝式CE-CL联用接口用于烟草中多酚物质的测定。实验测得芦丁、绿原酸、槲皮素和咖啡酸的检出限分别为：3.7×10^-9、6.4×10^-8、8.5×10^-8、1.3×10^-10mol／L，比套式接口检测多酚物质的检出限(10^-5mol／L)降低了二、三个数量级。3，分别用SDS、Tween 20、Tween 80三种表面活性剂作为胶束和混合胶束，用毛细管胶束电动色谱分离分析莨菪亭、芸香苷、七叶亭、咖啡酸和绿原酸，发现只有采用Tweeen 20的MEKC法可以使芸香苷和七叶亭分离。对分离条件，包括分离电压、pH、Tween 20、甲醇以及Na₂B₄O₇浓度进行系统研究，最后对三种秦皮中草药实际样品进行了五种多酚物质的定量测定。鉴于莨菪亭、芸香苷、七叶亭在SDS、Tween 80胶束电动色谱中难以分离，用示差分光光度法测定了Tween 20与芸香苷和七叶亭的反应平衡常数和结合比，反应平衡常数分别为4.53×10⁵和2.23×10⁵（L／mol）²，结合数均为n=2。结果表明吐温20-MEKC分离三种多酚是基于多酚与吐温20结合作用和电泳淌度的差异。4，制作内径为2.2mm的电泳微柱，用100-125μm石英砂填充进石英玻璃管，两端用柱塞防漏。比较了多孔尼龙、水玻璃、玻璃砂芯柱塞，实验结果证明水玻璃和玻璃烧砂芯柱塞吸附严重，而尼龙柱塞吸附小、不产生气泡。在1500V电压下分离氨基酸样品，结果峰型对称。色氨酸、组氨酸检出限分别为0.9，2.8μmol/L（0.30，0.27mg／L），较毛细管电泳有明显改善。2.2mm内径石英砂填充微柱电泳限制了热效应，柱容量大，检出限低，可使用常规分析仪器检测，是一种制备型的微柱电泳。更多还原

【Abstract】 Capillary electrophoresis （CE） coupled with chemiluminescence （CL） detection have certain advantages of high separation efficiency and sensitivity. In this dissertation, the instrumental development and application of CL detection in CE were reviewed. In the last 10 years, the extensive work of CE-CL was studied. However, one of the most formidable challenges in applying CL detection to CE is the on-line introduction of CL reagents into the separation system. Suitable interfaces are very important to establish a successful CE-CL system. In this paper, a novel interface for CE combined with CL was developed and utilized to separate polyphenols. A kind of micellar electrokinetic capillary chromatography （MEKC） using polyoxyethylene sorbitan monolaurate （Tween 20） was introduced for the simultaneous determination of five polyphenols also. At last, a microcolumn was prepared by filling with quartz sand of 120-150μm in a 2.2 mm i. d. quartz tube and adopted to separate amino acids. The research topics are discussed in detailed as follows:1.A novel on-line chemiluminescence detector was designed for capillary electrophoresis. A gap interface with height injection was made by two capillaries, which were placed in close proximity by careful micropositioning, and the distance between the capillaries was 50μm approximately. The preparation processes of the gap interface were easy with an oriented capillary, which was eliminated after the gap preparation. The gap interface was carefully inserted into a 1.0 mm i. d. quartz tube, as a detection window situated just in front of a photomultiplier tube （PMT）, and a reagent tube surrounded by CL reagent solution.A high potential buffer reservoir was constructed from a running buffer cell and an electrode buffer one, which were jointed with a frit, in order to avoid the luminol electrolysis, the excursion of chemiluminescence baseline, and the increase of electric current in CE-CL.2.The CE-CL system was employed for the determination of four polyphenols in cigarette samples. The detection limits （S/N=3） of rutin, chlorogenic acid, quercetin and caffeic acid were 3.7×10^-9, 6.4×10^-8, 8.5×10^-8 and 1.3×10^-10mol/L, respectively, two order magnitude lower than those reported in the previous work.3.A simple and convenient method of MEKC using Tween 20 as single micelle and methanol as buffer additive was introduced for simultaneous determination of five polyphenols, including scopoletin, rutin, esculetin, chlorogenic acid and caffeic acid. A running buffer solution of pH 9.3, 20 mmol/L sodium tetraborate containing 64 mmol/L Tween 20 and 9% （v/v） methanol was adopted in the separation. For rutin and esculetin were difficult to be separated by capillary zone electrophoresis （CZE） and SDS-based MEKC, Tween 20-based MEKC was used and two polyphenols were separated satisfactorily.The separation mechanism of Tween 20-based MEKC for the polyphenols was discussed preliminarily. The equilibrium constant K of Tween 20 and rutin （or esculetin） is calculated to be 4.53 ×10⁵ （2.23 ×105） L²/mol² by a differential spectrophotometry, respectively. And both of the binding numbers were 2. It shows that the satisfied separation of the polyphenols was achieved by the different interaction between Tween 20 and rutin （or esculetin） and the different mobility.The proposed method was used to determine the polyphenol components in the herbal medicine of Cortex fraxini. The average recoveries of the polyphenols ranged from 95% to 112%. It means that the proposed method is suitable for the simultaneous determination of the polyphenols in the herbal samples.4.To overcome the disadvantages of CE due to its small inner diameter, such as poor detection sensitivity and low sample loadability, a microcolumn was prepared by filling with 100-125 μm quartz sand in a 2.2 mm i. d. quartz tube. The effect of electric field strength and quartz sand size on electroosmotic flow rate was studied. Nylon films were used firstly as frits. In this dissertation, the separation results of two amino acids were satisfying with the microcolumn and the detection limits （S/N=3） were improved, of which tryptophan and histidine were 0.30 and 0.27 mg/L, respectively. The electrophoretic separation can be developed as a complementary method for CE to improve the sample capacity and concentration sensitivity.更多还原