新型荧光探针分子的制备及在液相色谱和电泳中的应用

【作者】 明永飞；

【导师】 尤进茂；

【作者基本信息】 曲阜师范大学 ， 物理化学， 2004， 硕士

【摘要】 液相色谱及电泳的分离分析被广泛应用于诸多领域，如生物学、化学、毒物学、临床化学、以及环境科学等。液相色谱是分析化学中发展最快、应用最为广泛的分析手段，其分离特性使之成为诸多领域中被广泛采用的一种有效的分离手段。继液相色谱之后，毛细管电泳也是一种近年来迅速发展的分离技术，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、甚至整个细胞和病毒颗粒。 毛细管电泳的分离机制与液相色谱不同，故两者之间可以提供正交的、互补分析，此外，电泳的方法开发较为简单，不仅样品用量很少，而且几乎没有有机废液。 随着现代分析技术的发展，人们对化合物的常规分析已转向痕量及超痕量分析。为了提高检测灵敏度，人们在放大信号和改进检测仪器方面进行着不懈的努力，如何使所测结果更加可靠和灵敏是所有科学工作者优先期盼的，但是在提高仪器灵敏度方面受到一定的限制，因此研制并开发新型高灵敏的荧光探针分子，提高检测灵敏度是一个行之有效的途径，也为克服常规的化学分析方法无法解决的困难开辟了新途径，使许多经典方法得以改进，使进行痕量和超痕量分析成为现实。为此，本研究采用化学衍生化方法，改变被测化合物特性，使之用于液相色谱和电泳的快速高灵敏的测定。论文中设计并合成了两种新型荧光探针分子，应用于氨基酸和多肽的高效液相色谱和毛细管电泳的分离分析，结果令人满意。论文分四章： 第一章： 对化学衍生化试剂及衍生化方法进行了简单介绍。对高效液相色谱和毛细管电泳的发展和相关基础知识进行了简述。综述了荧光试剂在氨基酸和多肽的高效液相色谱和毛细管电泳中的分离应用。 第二章： 两种新型荧光试剂咔哇一9一乙基氯甲酸酷和1，2一苯并一3，4一二氢咔哇一9一乙基氯甲酸酷的合成及结构表征。 第三章: 咔哇一9一乙基氯甲酸酷在高效液相色谱和毛细管电泳中的应用，包括 四部分。 3.1研究了咔哇一9一乙基氯甲酸酷用于氨基酸和多肤的高效液相色谱 分离分析。选择了衍生化条件，优化了洗脱程序，考察了氨基酸和 多肤衍生物的检测限、线性范围及回归方程。结果表明，在实验条件 下，氨基酸衍生物的线性范围宽，检测限达加ol，灵敏度高。在实 际应用中测定了四种苔鲜中的氨基酸含量，结果令人满意。 3.2研究了咔哇一9一乙基氯甲酸酷用于氨基酸的毛细管区带电泳分离。 通过对分离条件的考察，找到最佳的分离条件:18~ol几的醋酸 盐缓冲溶液(含28%的乙睛)，pH值为5.8，分离电压25kv，温度 为25℃。在选定的分离条件下，12种氨基酸衍生物在不足15min 内实现了基线分离，结果理想。 3.3采用毛细管胶束电动色谱在6min内实现了CEOC衍生的14种氨基 酸衍生物的完全分离。通过对分离条件的选择，得出了最佳的分离 条件:分离电压为25kV、柱温25℃、235lun DAD检测，采用硼酸 盐浓度为36~ol几，含有30~ol几SDS和3%乙睛的缓冲溶液 (pH=9.0)。考察了各种氨基酸的线性范围、检测限及回归方程， 从而为CEOC作衍生试剂，利用毛细管电泳对其它环境样品中的氨 基酸的分离测定奠定了基础。 3.4采用毛细管电泳在胶束电动色谱模式下实现了小肤CEOC衍生物的 完全基线分离。通过电泳条件的选择，得出最佳分离条件:分离电 压25kv、柱温300C、硼酸盐浓度为36mmol/L(pH=9.0)、sns 30~ol/L和3%的乙睛，235nm DAD检测。在选定的条件下，考察 了5种肤的线性、检测限和方法的重现性，结果表明，所建立的方 法简单快速.之第四章:研究了1，2一苯并一3，4一二氢咔哇一9一乙基氯甲酸酷在高效液相色谱和毛细管电泳中的应用，包括三部分。4.1研究了1，2一苯并一3，4一二氢咔哇一9一乙基氯甲酸酷用于氨基酸和多 肤的高效液相色谱分离分析。选择了衍生化条件，优化了洗脱程序， 考察了氨基酸和多肤衍生物的检测限、线性范围及回归方程。比较 了BCEOC和CEOC衍生物的荧光强度。结果表明，在实验条件下， 氨基酸和多肤衍生物的线性范围宽，检测限达fmol，灵敏度比CEOC 的高2一6倍。利用该方法测定了瓜子提取液中的氨基酸含量，结果 令人满意。4.2采用毛细管胶束电动色谱在18 min内实现了10种BCEOC氨基酸 衍生物的完全分离。通过对分离条件的选择，得出了最佳分离条件: 分离电压为ZOkV、柱温25℃、324nm DAD检测，采用硼酸盐浓度 为4Ommol/L，含有42rnmol几sns和4%乙睛的缓冲溶液(pH=9.4)。 并考察了各种氨基酸的线性范围、检测限以及回归方程，从而为 BCEOC作衍生试剂，利用毛细管电泳对氨基酸的分析奠定了基础。4.3利用胶束电动色谱在10mln内实现了五种小肤的BCEOC衍生物的 基线分离。通过电泳条件的选择，得出最佳分离条件:分离电压 19kv、柱温25oC、硼酸盐侬度为38mmol/L(pH=9.0)、Sns 32mmol/L更多还原

【Abstract】 The enthusiastic adoption of high performance liquid chromatographic (HPLC) and capillary electrophoresis (CE) has been a great impact on chemical analysis, not only in chemistry and biochemistry but also in toxicology, clinical sciences, environmental science and many other fields.With the special separation characterization, HPLC is one of the fastest developed and the most widely used fields in analytical chemistry. After HPLC method, capillary electrophoresis (CE) is a rapidly growing separation technique with many advantages including high performance, high speed and small sample consumption. It has proved to be powerful tool for the separation of compounds such as amino acids, chiral drugs, vitamins, pesticides, inorganic ions, organic acids, dyes, peptides and proteins, and even whole cells and virus particles.The responsible mechanisms for the separation of components in CE are different from those in liquid chromatography. In view of this, two methods can each other offer orthogonal, complementary analyses. In addition, CE may offer simpler method development, minimal sample volume requirements with almost no organic waste.With the development of modern analytical technology, the method of traditionally conventional analysis has been converted to the trace analysis and even the extreme trace analysis. In order to improve the sensitivity of detection, people have been devoting to amplify signals and renovate the detectors. All the scientific researchers have been expected to obtain the reliable and sensitive results, and this is also a question which claims precedence over all others. However, there is a limit in improving the sensitivity of instrument. So it is an accessible approach to design and synthesize novel high sensitive fluorescent probe to improve detection sensitivity that it is difficulty to achieve the goal withthe conventional analysis. With the development of these novel sensitive probes, the classical analytical method has been developed and made the trace and extreme trace analysis true. Therefore, labeling of the analytes with fluorescent probe has been widely adopted to alter the chemical physical properties of target compounds to make them be analyzed easier with HPLC or CE.In this thesis, the method for the sensitive determination of amino acids and peptides using the synthesized tagging reagents with different chromophores by HPLC and CE with fluorescence or DAD detector has been developed. The thesis consists of four chapters.Chapter one:The development of the labeling reagents and the method of derivatization have been simply introduced. At the same time, the basic knowledge and the further improvement of HPLC and CE are briefly introduced. Then the applications of fluorescence reagents to analyze amino acids and peptides by HPLC or CE are reviewed.Chapter two:9-(2-Carbazole)ethyl chloroformate (CEOC) andl,2-benzo-3,4-dihydrocarbazole-9-ethyl chloroformate (BCEOC) are synthesized and their structures are characterized.Chapter three:The applications of 9-(2-carbazole)ethyl chloroformate (CEOC) for the analysis of different components by HPLC and CE are investigated. The principal contents of this chapter consist of four parts.3.1 The separation and determination of amino acids and oligopeptides using CEOC as labeling reagent by HPLC with fluorescence detection are studied. The derivatization conditions in conjunction with the gradient elution program are optimized. At the same time, the detection limit and linearity for derivatizedamino acids and oligopeptides are obtained under the optimum conditions. The results indicate that the detection limits for tagged amino acids and oligopeptidesis are at fmol levels (S/N=3) with relatively wide linear range. The established method for the determination of amino acids from extracted four bryphytes is evaluated with satisfactory results.3.2 The separation of derivatized amino acids using CEOC as derivatization agent by capillary zone electrophoresis (CZE) with DAD detectio更多还原