Carbohydrate Derivative Separation And Analysis Methods

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.