糖类、氨基酸的毛细管电泳分离研究

【作者】 盛筱；

【导师】 尤进茂；

【作者基本信息】 曲阜师范大学 ， 有机化学， 2008， 硕士

【摘要】 毛细管电泳（CE）又称高效毛细管电泳（HPCE）或毛细管电分离法（CESM）,是一类以毛细管为分离通道、以高压直流电场为驱动力、依据样品各组分之间淌度和分配行为上的差异而实现分离的高效、高选择性的新型液相分离分析技术。该方法具有高效、快速、自动化程度高、试剂消耗少、环境污染小等优点,在十几年的时间中,特别是在最近几年,受到分离分析科学家的极大关注,已成为生物化学和分析化学中最受瞩目,发展最快的分离分析新技术之一。糖是自然界和生物体中广泛存在的物质。长期以来人们认为糖的主要功能是提供能量,而新兴的糖生物化学研究发现糖类物质除了提供能量外还有其它多方面的生物活性与功能。氨基酸是在生物体内构成蛋白质分子的基本单位,与生物的生命活动有着密切的关系,它在抗体内具有特殊的生理功能,是生物体内不可缺少的营养成分之一。氨基酸、糖类是生命有机体的重要组成部分,研究其毛细管电泳分离对生物、医药、食品、卫生等领域都具有重要意义。在糖类和氨基酸的研究领域,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。目前广为应用的糖类衍生试剂主要有酰肼类、氨基吡啶类、苯胺类等,氨基酸衍生化试剂主要有邻苯二甲醛（OPA）、9-芴甲氧羰基氯（FMOC-Cl）和5-二甲氨基萘-1-磺酰氯（DNS-Cl）等,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,本文采用实验室自行合成的新型衍生化试剂,改变被测化合物的特性,进行了糖类物质和氨基酸的毛细管电泳分离,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药物分析、生物分子分离、基因工程研究、临床医学等各方面的应用,详细介绍了其在糖类、氨基酸分离方面的应用,并对毛细管电泳分离被分析物的策略及其相关的基础知识进行了阐述。第二章新型衍生试剂1-萘基-3-甲基-5-吡唑啉酮（NMP）用于单糖的毛细管电泳分离,分为四部分。2.1研究了毛细管区带电泳模式下单糖NMP衍生物的分离情况。考察并优化了单糖NMP衍生物的分离条件,在18min内实现了九种单糖衍生物的基线分离,并在此基础上进行了白刺和蜂花粉实际样品的测定,获得的结果令人满意。2.2以1-苯基-3-甲基-5-吡唑啉酮（PMP）作为柱前衍生试剂,在毛细管区带电泳模式下,考察并优化了单糖衍生物的分离条件,实现了九种单糖衍生物的基线分离。2.3对毛细管区带电泳模式下单糖NMP衍生物和PMP衍生物的分离进行了比较与总结。通过比较两种试剂的异同及两种试剂衍生单糖分离效果的异同得出结论:NMP衍生单糖在毛细管电泳分离中具有更高的检测灵敏度。2.4研究了胶束电动毛细管色谱模式下单糖NMP衍生物的分离情况。考察并优化了单糖NMP衍生物的分离条件,在10分钟内实现了八种单糖衍生物的基线分离。分离快速、简单,结果令人满意。第三章2-（11H-苯[a]咔唑）乙基氯甲酸酯（BCEC-Cl）作为柱前衍生试剂用于氨基酸的毛细管电泳分离,分为两部分。3.1以2-（11H-苯[a]咔唑）乙基氯甲酸酯（BCEC-Cl）作为柱前衍生试剂,在甲酰胺为溶剂,醋酸-醋酸铵为缓冲体系,毛细管柱长58.5 cm（有效长度50 cm）,18℃,30 kV,压力进样12s,279 nm二极管阵列检测（DAD）条件下,采用非水毛细管电泳模式考察并优化了氨基酸衍生物的分离条件,在不加任何添加剂的情况下实现了16种氨基酸的基线分离。3.2以2-（11H-苯[a]咔唑）乙基氯甲酸酯（BCEC-Cl）作为柱前衍生试剂,在β-环糊精（β-CD）作为手性拆分试剂,Tris-H₃PO₄为缓冲体系,十二烷基硫酸钠（SDS）为表面活性剂,毛细管柱长58.5 cm（有效长度50 cm）,20℃,20 kV,压力进样10s,279 nm二极管阵列检测（DAD）条件下,采用胶束电动毛细管色谱模式考察并优化了氨基酸对映体的分离条件,实现了15种氨基酸的手性拆分。更多还原

【Abstract】 Capillary electrophoresis(CE),also known as high-performance capillary electrophoresis (HPCE)or capillary separation(CESM),is a kind of New Liquid Separation Technology with high efficiency and selectivity,which carries out separation by taking capillary tubes for separation passage and high voltage electricity for motive force and in accordance with the difference of mobility and distribution behaviors of all the components of samples.This method has some advantages of high efficiency,fast analysis,high automaticity,little sample consumption and little contamination.Being given great concerns by separation and analysis scientists,it has been one of the most-watched and most advanced technologies in biochemistry and analytical chemistry in last ten years,especially in recent years.Carbohydrate is a kind of substance which widely exists in nature and in organism.Over a long period of time,it has been considered that the main function of carbohydrate is to provide energy.But the newly emerging research on carbohydrate biochemistry shows that it has many other biological activities and functions besides energy-providing.Being the basic unit of protein and having close relationship with vital activity,amino acid has special physiological function in antibody and is one of the indispensable nutrition component in organism.Carbohydrate and amino acid are both the important conponents of life organism,so that it has great value in biology,medicine,food,health and other fields to do research on them by Capillary Eleetrophoresis.Chemical derivation has been developed in the separation of amino acids and carbohydrates, 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 of carbohydrate are acylhydrazine, amino-pyridine,and aniline ect.And the widely used derivatizations of amino acids are o-phthalaldehyde(OPA),9-fluorenylmethyl chloroformate(FMOC-C1)and Dansyl Chloride (DNS-C1),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,new labeling reagents have been synthesized and applied to the separation of amino acids and carbohydrates by capillary electrophoresis,the results are satisfactory.The thesis consists of three chapters.Chapter OneThe developments of capillary electrophoresis(CE)and the its applications in pharmaceutical analysis,biomolecular separation,genetic engineering,clinical medicine and many other fields have been simply introduced.And the application of capillary electrophoresis on carbohydrate seperation and amino acid seperation are introduced in detail.At the same time, the separation strategy of analyte by capillary electrophoresis and the related basic knowledge are reviewed,too.Chapter TwoThe applications of 1-naphthyl-3-methyl-5-pyrazolone(NMP)for the separation of monosaccharides by CE have been investigated.The principal contents of this chapter consist of four parts.2.1 A simple and rapid method for the separation of derivatized monosaccharides using 1-naphthyl-3-methyl-5-pyrazolone(NMP)as derivatization reagent by capillary zone electrophoresis(CZE)has been developed.Under the CZE mode,the separation conditions have been studied and optimized.In optimal conditions,9 monosaccharide derivatives are separated within 18 min.The proposed method has been applied to the determination of monosaccharides in Nitraria and bee-pollen samples with satisfactory results.2.2 Under the capillary zone electrophoresis(CZE)the separation of derivatized monosaccharides using 1-phenyl-3-methyl-5-pyrazolone(PMP)as derivatization reagent has been studied.The separation conditions have been optimized,and under the optimal conditions,9 monosaccharide derivatives are separated within 16 min.The separation is efficient and rapid,the results are satisfactory.2.3 The separation differences between NMP-derivatized monosaccharides and PMP-derivatized monosaccharides by capillary zone electrophoresis(CZE)has been compared and summarized.The results indicate that the NMP-derivatized monosaccharides have higher detective sensitivity.2.4 Under the micellar electrokinetic chromatography(MEKC)mode the separation of derivatized monosaccharides using 1-naphthyl-3-methyl-5-pyrazolone(NMP)as derivatization reagent has been studied.8 NMP-derivatized monosaccharides achieve baseline resolution within 10 min under the proposed conditions.Chapter ThreeThe applications of 2-(11H-benzo[a]carbazol- 11 -yl)ethyl carbonochloridate(BCEC-C1)for the separation of amino acids have been investigated.The principal contents of this chapter consist of two parts.3.1 A simple and rapid method for the separation of derivatized amino acids using 2-(11H-benzo[a]carbazol-11-yl)ethyl carbonochloridate(BCEC-Cl)as derivatization reagent by non-aqueous capillary electrophoresis has been proposed.The separation conditions have been studied and optimized,the optimum conditions are as follows: 0.7mol/L acetic acid,60 mmol/L ammonium acetate buffer,voltage 30kV,column temperature at 18℃and DAD detection at 279 nm.The samples are introduced atmospherically with an injection time of 12 s.The results indicate that 16 derivatized amino acids achieve baseline resolution under the proposed conditions.3.2 Under the micellar electrokinetic chromatography(MEKC)mode,the chiral separation of BCEC-Cl-derivatized amino acids has been studied.To optimize the conditions,the background electrolyte concentration,buffer pH,column temperature,voltage and other factors have been studied.It has been found that the buffer concentration and pH value have a greater impact on the separation,but the influences of temperature,voltage and additives on resolution are not obvious.On the basis of the optimization,15 pairs of amino acid enantiomers are all separated within 20 min with the resolution between 1.41(Phe)～12.04(Asp).This method is simple and rapid,the rusult is satisfactory.更多还原