糖类物质的衍生化分离分析方法研究

【作者】 徐瑾；

【导师】 王风云； 李彤；

【作者基本信息】 南京理工大学 ， 物理化学， 2003， 硕士

【摘要】 本文是国家“十五”攻关重大项目“科学仪器研制与开发”中“色谱仪与专用部件的研制与开发”子课题的部分工作。 近年来，随着人们对糖类物质生物功能的不断深入了解，糖类方法学研究已成为当今化学家和生物学家的研究“热点”，而高效液相色谱技术又是糖类方法学研究的重要组成部分。该论文选择1-苯基-3-甲基-5-吡唑啉酮(PMP)作为柱前衍生化试剂进行糖类物质分离分析的研究。在前人的工作基础上，采用简化的柱前衍生化方法，使反应时间缩短近1／3，减少了衍生产物的损失。优化选择反应温度，反应时间，衍生试剂PMP和NaOH用量等衍生化条件，最终确立的衍生化方法条件温和，衍生化反应产率高。使用质谱扫描确定反应生成1：2结构形式的糖-PMP衍生产物，无立体异构体，并对衍生产物稳定性加以评价，结果表明PMP柱前衍生化方法能够满足实际样品常规分析的要求。结合建立的衍生化方法与高效液相色谱HPLC，优化分离条件，并选择紫外(UV)和电喷雾—离子阱质谱(ESI-MS)两种检测模式对实际样品中所含的单糖进行分离分析，建立了5种单糖的常规定性定量方法。也采用毛细管胶束电动力学色谱(MEKC)分离单糖衍生物，与HPLC分离的结果加以比较，说明了MEKC和HPLC应用于单糖分离分析的特征，确立了分离模式选择的基本框架。PMP柱前衍生法用于还原性双糖的衍生，同样能取得较好的结果。实验表明，建立的PMP柱前衍生—HPLC分离—ESI-MS检测方法可以作为聚糖研究方法，能够很好的说明其“结构单元”—摘要单糖的组成。更多还原

【Abstract】 This thesis is a part of "The research and development of chromatography instruments and their special fittings", which is a subresearch of mega-projects of science research-"The research and development of chromatography instruments" supported by Chinese Tenth Five-year Plan.The important roles of carbohydrates in many biological processes has become more and more recognized over the past recent year, thus, there are considerable interests in carbohydrate research, of which high-performance liquid chromatography (HPLC) is an essential analytical tool. In this paper, l-phenyl-3-methyl-5-pyrazolone (PMP), a precolumn derivatization reagent, was used to study the analysis of carbohydrates. A simplified derivatization method using PMP was developed. With the simplification, one-third of the derivatization time was saved, meanwhile, the losing of derivatives was decreased without any influence on property of derivatives. A systematic research was carried out on the reaction time, reaction temperature, amount of derivative and catalyst to establish optimum mild derivatization conditions, under which higher production rate was achieved. By Mass spectrometry, it was demonstrated that two groups of PMP are introduced to each reducing carbohydrate to give a bis-PMP derivative, without causing isomery. In addition, the stability of derivatives was evaluated. The result indicated that the precolumn derivatization method with PMP is quite suitable for routine analyses of real samples. So far this method has been successfully applied to HPLC analysis of simple monosaccharides. A baseline separation of five monosaccharides was obtained under optimized isocratic condition. Both UV detector and electrospray ionization-Mass spectrometry (ESI-MS) were selected to determine the monosaccharide composition in real samples. Then a routine identification and quantification method has been established.Further, the PMP method was also well-suited for micellar electrokinetic chromatography (MEKC), whose results were compared with those of HPLC to illustrate the characteristics of these two methods, therefore, to help researchers to choose appropriate separation mode. The use of PMP method in reducing disaccharides gave good results. The present method, which combined PMP derivatization-HPLC with ESI-MS, could be used as a research method to study the structural units-specific monosaccharides of polysaccharides.更多还原