毛细管电泳中氨基酸、二肽类BCEOC衍生物的手性拆分

【作者】 白敏；

【导师】 尤进茂；

【作者基本信息】 曲阜师范大学 ， 物理化学， 2006， 硕士

【摘要】 毛细管电泳是近年来迅速发展起来的一类分离分析技术,它具有高效、快速、样品用量少等特点,能用于多种化合物的分离,如氨基酸、手性药物、维生素、杀虫剂、无机离子、有机酸、染料、肽和蛋白质、甚至整个细胞和病毒颗粒。被广泛应用于生命科学、生物技术、医学药物、环境科学、法医科学等诸多领域。 在手性拆分方面;毛细管电泳作为一种强有力的手性分离技术,已经成功的分离了多种类的手性物质,尤其在氨基酸、肽的衍生物以及药物的手性分离领域应用最广,显示了其独特的优势。此外,电泳的方法开发较为简单,具有多种灵活变换的分离模式,样品用量少,而且几乎没有有机废液。 在氨基酸和二肽类物质的手性拆分研究领域,由于这些生物分子没有或者仅有微弱的紫外吸收,直接紫外检测满足不了低含量组分测定的要求,化学衍生化法应运而生。目前广为应用的衍生化试剂主要有邻苯二甲醛(OPA)、9-芴甲氧羰基氯(FMOC-Cl)和6—氨基喹啉基琥珀酰亚胺碳酸酯(AQC)。但是这些衍生试剂本身的一些特性决定了其应用的局限性。为此,本研究采用新型衍生化试剂,改变被测化合物特性,进行了毛细管电泳的氨基酸和二肽对映体的手性拆分,结果令人满意。论文分三章: 第一章: 简单介绍了毛细管电泳的发展及其在医学药物、生命科学、生物技术、法医科学等领域的应用情况。对手性拆分的意义和毛细管电泳手性拆分相关的基础知识进行了简述。综述了毛细管电泳用于手性拆分的研究现状。 第二章: 1,2-苯并-3,4-二氢咔唑-9-乙基氯甲酸酯(BCEOC)用于氨基酸毛细管电泳手性拆分,包括三部分。 2.1 研究了毛细管区带电泳模式下氨基酸BCEOC衍生物的手性拆分情况。在毛细管区带电泳模式下,考察并优化了氨基酸BCEOC衍生物的手性分离条件,在15min内实现了七种单一氨基酸衍生物的对映体的基线分离,分离度均在1.2以上。所建立的方法简单、快速,拆分环境友好,获得的结果较为满意。更多还原

【Abstract】 Capillary electrophoresis (CE) is a kind of separation technique, which has been rapidly developed in recent years. It has many advantages including high performance, high speed and small sample consumption, and can be used to separate many compounds such as amino acids, chiral drugs, vitamins, pesticides, inorganic ions, organic acids, dyes, peptides and proteins, and even whole cells and virus particles. It has been widely used not only in chemistry and biochemistry but also in life science, medical drugs, environmental science and many other fields.CE has been proved to be a powerful tool for the chiral separation, and has been used successfully for separating many types of chiral enantiomers. It has demonstrated its particular advantages, especially in the enantioseparation of amino acids, peptides and pharmaceutical drugs. In addition, CE has many varieties of flexible separation modes and can offer simple method, minimal sample volume requirements with almost no organic waste.Chemical derivation has been developed in the chiral separation of aminoacids and dipeptides, as these biological molecules have no or weak ultraviolet absorption and the low contents cannot be detected directly by UV detection. At present, the widely used derivatizations are o-phthalaldehyde (OPA) 9-fluorenylmethyl chloroformate (FMOC-Cl) 6-Aminoquinolyl-N-hydroxy-succinimidylcarbamate (AQC), but these reagents have also been reported some various shortcomings in their application, such as short detection wavelengths, poor stability of reagents and serious interference in the determination of biological samples.In this thesis, a new labeling reagent has been synthesized and applied to the chiral separation of amino acids and dipeptides by capillary electrophoresis. The thesis consists of three chapters.Chapter one:The developments of capillary electrophoresis (CE) and the applications in life science, medical drugs, environmental science and many other fields have been simply introduced. At the same time, the significances of chiral separation and the basic knowledge of chiral separation by capillary electrophoresis are briefly introduced. Then the applications of CE to the chiral separation of amino acids and dipeptides are reviewed.Chapter two:The applications of l,2-benzo-3-4-dihydrocarbazole-9-ethyl chloraformate (BCEOC) for the chiral separation of amino acids by CE are investigated. The principal contents of this chapter consist of three parts.2.1 A simple and rapid method for the chiral separation of derivatized amino acids (AAs) using BCEOC as derivatization reagent by capillary zone electrophoresis (CZE) has been developed. Under the CZE mode, the chiral separation conditions have been studied and optimized. In optimal conditions, seven pairs of AAs derivatives were separated within 15 min with a resolution of > 1.2.2.2 Under the micellar electrokinetic chromatography (MEKC) mode, thechiral separation of BCEOC-AAs derivatives haas been studied. On the basis of optimization of some main factors for the chiral separation such as the concentration of running buffer, pH value, the concentration of surfactant reagents and organic additives etc. 13 pairs of AAs derivatives are separated within 30 min with a good baseline resolution of > 1.0. The method is simple and rapid.2.3 The chiral separation difference of BCEOC-AAs derivatives between capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) has been compared and summarized. The results indicate that micellar electrokinetic chromatography (MEKC) mode is superior to capillary zoneelectrophoresis (CZE) for enantioseparation of amino acids. Chapter three:The applications of l,2-benzo-3-4-dihydrocarbazole-9-ethyl chloraformate (BCEOC) for the chiral separation of dipeptides by CE are investigated. The principal contents of this chapter consist of three parts.3.1 Under the capillary zone electrophoresis (CZE) mode, the chiralseparation of derivatized dipeptids using BCEOC as labeling reagent with DAD detection has been studied. On the basis of optimization for CZE separation, the optimum separation conditions are followed: the separation voltage 15kV, column temperature at 25 °C, 60mmol/L tris- (hydroxymethyl) aminomethane-phosphate (tris-H3PO4, pH 9.0), 20mmol/L sodium dodecyl sulphate (SDS), 3% acetonitrile(v/v), 1.8 mmol/L /?-CD and 2.4 mmol/L SDC. Under the optimized conditions, 8 pairs of dipeptide enantiomers were separated with a good baseline resolution.3.2 Under the micellar electrokinetic chromatography (MEKC) mode, the chiral separation of BCEOC-dipeptide derivatives has been studied. Under the optimized separation conditions, 15 pairs of dipeptide enantiomers were separated using BCEOC as derivatization reagent within 25 min with the resolution between 2.85 ( Giy-Trp ) ~ 35.29 ( Gly-Ser ). This method is simple and rapid, and likely to find numerous applications in CE system for peptide chiral separation.3.3 The chiral separation difference of BCEOC-dipetide derivatives between capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) has been compared and summarized. The elution orders of enantiomers are also evaluated. The results indicate that micellar electrokinetic chromatography (MEKC) mode is much superior to capillary zone electrophoresis (CZE) for dipeptid enantioseparation with higher resolution andperformance.更多还原