| 毛细管电泳-蓝/紫光激光诱导荧光检测系统的构建及其分析应用 | |
| Alternative Title | Capillary Electrophoresis - Blue / Violet Laser-Induced Fluorescence Detection: System Construction and Its Applications in Analytical Chemistry |
| 吴成新 | |
| Subtype | 硕士 |
| Thesis Advisor | 周雷 |
| 2019-05-19 | |
| Degree Grantor | 兰州大学 |
| Place of Conferral | 兰州 |
| Degree Name | 硕士 |
| Degree Discipline | 分析化学 |
| Keyword | 毛细管电泳 激光诱导荧光检测 荧光衍生 姜黄素类化合物 香豆素 磺酰胺类化合物 醛类小分子 |
| Abstract | 毛细管电泳（CE）以其分离效率高、分析速度快、分离模式多、样品与试剂用量少等优点，已发展成为一种极为有效的分离技术，在生物医药、食品安全、环境监测等领域获得了广泛应用。然而，由于毛细管内径较小（25-100 μm），导致其进样体积小（纳升级），有效检测光程短（微米级），使得常规CE灵敏度较低。激光诱导荧光检测（LIF）是目前灵敏度最高的检测技术之一，毛细管电泳-激光诱导荧光检测技术（CE-LIF）结合了二者快速、高效、灵敏的优点，非常适合复杂样品中痕量物质的分析。但是，商用激光器波长较为单一，以及多数目标分析物自身没有荧光等因素极大地限制了LIF检测器的应用。近年来，随着激光技术的进步，市面上可选择的激光器种类日益繁多，其中二极管激光由于可选波长种类丰富，体积小，寿命长，且价格低廉，是LIF检测器的理想光源。但目前二极管激光诱导自身荧光的应用较少，且缺乏与之波长相匹配的荧光标记试剂和衍生方法，这在一定程度上限制了该类LIF检测器的推广应用。本论文旨在分别以蓝（445 nm）、紫（405 nm）光激光二极管作为光源构建激光诱导荧光检测器，并搭建完整的CE-LIF系统。在此基础上，以姜黄素类化合物为目标分析物建立445 nm 激光诱导自身荧光（LINF）的应用；以香豆素为母体开发适用于405 nm激光诱导荧光检测器的荧光标记试剂，并建立相应的荧光标记方法，来拓展蓝、紫光激光诱导荧光检测器的应用范围。全文分为四章：第一章：简要介绍了毛细管电泳-激光诱导荧光分析技术，包括毛细管电泳的分离模式、毛细管电泳主要的检测器、激光诱导荧光检测器的原理、构造及相关进展，并对目前激光诱导荧光检测器可用的激光光源进行了总结，对毛细管电泳-激光诱导自身荧光（CE-LINF）相关的应用进行了较为全面的综述，对用于LIF检测器的荧光标记试剂进行了简要的总结。第二章：以445 nm蓝光激光二极管为光源搭建共聚焦激光诱导荧光检测器，并构建了一套完整的CE-LIF分析系统。在此基础上建立了胶束电动色谱（MEKC）-LINF快速、灵敏分析姜黄素类化合物的方法。采用Triton X-100和SDS组成的混合胶束电动色谱分离体系，不仅可以极大提高分离效率，并且可以显著增敏姜黄素类化合物的自身荧光。荧光光谱显示，混合胶束诱导的荧光协同作用可使姜黄素、去甲氧基姜黄素（DMC）和双去甲氧基姜黄素（BDMC）三种姜黄素类化合物信号分别增强77、57、47倍。在最优条件下，可在10 min内实现3种姜黄素的基线分离，所建立的MEKC-LINF分析方法线性、重复性良好，姜黄素、DMC、BDMC的检出限分别为4.1 ng/mL、2.6 ng/mL、0.4 ng/mL。该方法已成功应用于中药姜黄、中药制剂姜黄消痤搽剂、调味料咖喱和人体尿样中三种姜黄素类化合物的分析。第三章：以405 nm紫光激光二极管为光源搭建共聚焦激光诱导荧光检测器，并构建了一套完整的CE-LIF分析系统。引入商业化的7-(二乙胺基)香豆素-3-甲酸（DEAC-C）作为405 nm LIF的衍生试剂，以氢氯噻嗪（HCTZ），氯噻嗪（CTZ），氯噻酮（CTD）三种磺酰胺类利尿剂为目标分析物，发展了一种磺酰胺类化合物的荧光衍生方法。最优的衍生反应条件为：DEAC-C与磺酰胺在三聚氯氰的存在下，以1.5%的三乙胺乙腈溶液为反应溶剂，烘箱加热50 ℃反应3 h。在此基础上，建立了DEAC-C标记的这三种利尿类降压药的MEKC-LIF灵敏分析方法。在最优条件下，可在15 min内实现3种磺胺类药物的基线分离，HCTZ、CTZ、CTD的检出限分别为0.24、0.29、0.23 nM。该方法成功应用于药片以及人体尿样中三种利尿剂的分析。第四章：在第三章的基础上合成了7-(二乙胺基)香豆素-3-甲酰肼（DEAC-H），并以此为405 nm LIF的衍生试剂，以乙二醛、甲基乙二醛等小分子醛为目标分析物，制定了醛类化合物的荧光衍生策略。衍生反应条件为：DEAC-H与醛类化合物以乙腈溶液为反应溶剂，在30 μM HCl提供的酸性环境下烘箱加热50 ℃反应1 h。相较于羧酸与磺酰胺的反应，酰肼与醛的反应更加高效，因此，结合高灵敏度的LIF检测，该方法有望应用于大气细颗粒物中醛类化合物的测定。 |
| Other Abstract | Capillary electrophoresis (CE), benefited from exceptional advantages such as high separation efficiency, short analysis time, versatile operation modes, low sample and reagent consumption, has developed into an effective separation technology which is widely applied in the fields of biomedicine, food, environment, and so on. Limited to small inner diameter (typically 25-100 μm) of capillary, the injection volume is often in nanoliters, and the effective optical path-length for detection is usually in micrometers, thus leading to a low sensitivity for CE analysis by using traditional absorbance detection. Laser-induced fluorescence detection (LIF) is one of the highest sensitive detection techniques for CE at present. Capillary electrophoresis-laser-induced fluorescence detection (CE-LIF) combined the advantages of high separation efficiency and excellent sensitivity is very suitable for the analysis of trace substances in complex samples. However, the available wavelengths of commercial lasers for LIF detector are still insufficient, and many analytes of interest show no native fluorescence, which greatly limit the development and application of LIF detectors. Recently, the advances of laser technology have provided more possibilities for LIF detection. Diode lasers have received increasing attention and developed as the main excitation source in (miniaturized) LIF detector because of their advantages of rich wavelength varieties, low-cost, long-life, and small-size. However, the application of diode laser-induced native fluorescence is relatively rare at present, and there is a lack of fluorescence labeling reagents and derivatization strategies matching these wavelengths, which to some extent limits the promotion and application of such diode laser-induced fluorescence detectors.The purpose of this paper is to construct the confocal blue / violet light-excited LIF detector with 445 nm / 405 nm diode laser as excitation source and then establish a complete CE-LIF system coupled with LIF detection. And to expand the application of blue and violet laser-induced fluorescence detector, the application of 445 nm LINF was established with curcuminoids as the target analytes; and the fluorescence labeling reagents and corresponding fluorescence derivatization methods suitable for the 405-nm LIF were developed using coumarin fluorophore as the parent. This dissertation can be divided into four chapters:Chapter one: This chapter briefly introduces the capillary electrophoresis - laser induced-fluorescence detection (CE-LIF) technique, including the separation modes and detectors for CE, and the principle, structure and related progress of LIF detector. This chapter focused on the available laser sources for LIF detector, and systematically summarized the applications of capillary electrophoresis-laser induced native fluorescence (CE-LINF). In addition, the fluorescence labeling reagents for LIF detectors were also summarized briefly.Chapter two: A commercial blue laser diode (445 nm) was used as an excitation source to construct a confocal laser-induced fluorescence detector and then a complete capillary electrophoresis system coupled with LIF detection was established. With three major curcuminoids, curcumin, demethoxy curcumin (DMC) and bisdemethoxy curcumin (BDMC) as target analytes, a micellar electrokinetic chromatography (MEKC) method was proposed using mixed micelles consisting of Triton X-100 and SDS to sensitize the native fluorescence of curcuminoids and enhance their separation efficiency. Fluorescence spectra revealed that the mixed micelles induced fluorescence synergism could enhance the signals of three curcuminoids by 77-, 57-, and 47-fold for curcumin, DMC, and BDMC. Under the optimal conditions, a baseline separation of three curcuminoids was achieved within 10 min and the detection limits were found to be 4.1, 2.6, and 0.4 ng/mL for curcumin, DMC, and BDMC, respectively. Furthermore, the developed MEKC-LINF method was validated in terms of precision, linearity, accuracy and successfully applied for the determination of three curcuminoids in turmeric, medicinal turmeric liniment, curry seasoning, and human urine samples.Chapter three: Based on the previous chapter, a set of violet light-excited CE-LIF system was constructed using 405 nm diode laser as the excitation source. And then, a commercial fluorescent dye, 7-(diethylamino)coumarin -3-carboxylic acid (DEAC-C) was introduced as derivatization reagent and corresponding derivatization strategy for DEAC-C labeling sulfonamides was developed for 405 nm LIF detection. After systematic optimization, three sulfonamides including hydrochlorothiazide (HCTZ), chlorothiazide (CTZ) and chlortalidone (CTD) could be efficiently labeled by DEAC-C in the presence of cyanuric chloride and triethylamine (1.5%) in acetonitrile at 50 ºC for 180 min. Based on the laboratory-built CE-LIF system, a robust method was then proposed for the separation of DEAC-C labeled sulfonamides by micellar electrokinetic chromatography (MEKC). Under the optimized conditions, a baseline separation of three sulfonamides was achieved within 15 min, and the detection limits were determined to be 0.24, 0.29, and 0.23 nM for HCTZ, CTZ, and CTD, respectively. Furthermore, the developed MEKC-LIF method was successfully applied for the analysis of three sulfonamides in complex pharmaceutical and physiological samples.Chapter four: Based on the previous chapter, 7-(Diethylamino)coumarin-3-carbohydrazide (DEAC-H) was synthesized as derivatization reagent for 405 nm LIF detection. And corresponding derivatization strategy was proposed for aldehydes using glyoxal (Gly) and methylglyoxal (Mgly) as target analytes. After optimization, aldehydes could be efficiently labeled by DEAC-H in the presence of 30 μM HCl in acetonitrile at 50 ºC for 60 min. Compared with the reaction of carboxylic acid with sulfonamide, the reaction of hydrazide with aldehyde is more efficient. Therefore, combined with the high sensitivity of LIF detection, the developed method is expected to be applied to the determination of such compounds in atmospheric fine particulate matter. |
| Pages | 82 |
| URL | 查看原文 |
| Language | 中文 |
| Document Type | 学位论文 |
| Identifier | https://ir.lzu.edu.cn/handle/262010/340694 |
| Collection | 化学化工学院 |
| Affiliation | 化学化工学院 |
| First Author Affilication | College of Chemistry and Chemical Engineering |
| Recommended Citation GB/T 7714 | 吴成新. 毛细管电泳-蓝/紫光激光诱导荧光检测系统的构建及其分析应用[D]. 兰州. 兰州大学,2019. |
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