Fluorescent Reagent Preparation And Their Applications In High Performance Liquid Chromatography

High performance liquid chromatography(HPLC),due to its powerful separation capacity,has been enthusiastically and widely adopted in many fields of industry and scientific research,such as biochemistry,chemical industry,toxicology,clinical sciences, clinical medicine,neurobiology,food hygiene,environmental science and quality control.It is also one of the fastest developing and the most widely used methods in analytical chemistry.However,There are many compounds not suitable to be directly analyzed by HPLC because of the limitation of the very low contents and the compound properties of the corresponding compounds.So it is very important to enhance the sensitivity of detection. Currently,fluorescence detection is recognized to be one of the most sensitive detection technologies.Therefore,derivatization of analytes with fluorescence labeling reagents has been widely adopted.In this paper,we designed and synthesized three novel fluorescence labeling reagents.The optimal derivatization and chromatographic separation conditions were investigated.At the same time,fluorescence and UV-Vis spectra of labeling reagents and the suitability of the developed method for the analysis of real samples were evaluated.Chapter one:The history and basic theory about HPLC were simply introduced.The development of the fluorescence derivation and basic theory were summarized.Chapter two:The syntheses of three fluorescence labeling reagents 2-[2-(7H-dibenzo [a,g]-carbazol-7-yl)-ethoxy]ethyl chloroformate(DBCEC-C1),2-[2-(7H-dibenzo[a,g]carbaz ol-7-yl)-ethoxy]ethyl carbonylhydrazine(DBCEC-NHNH₂) and 2-(7H-dibenzo[a,g]carbaz ol-7-yl)ethyl-p-toluenesulfonate(DBCES) were described,and followed by their structural characterization and spectral properties.Chapter three:Pre-column derivatization methods for the sensitive determination of fatty amines and estrogen using DBCEC-C1 as labeling reagents followed by HPLC-MS analysis have been developed.The optimal derivatization and chromatographic separation conditions using DBCEC-C1 as labeling reagent were evaluated.And the feasibility of this method has been identified by analyzing soil and root vole's urine samples.Chapter four:Pre-column derivatization methods for the sensitive determination of aliphatic aldehydes using DBCEC-NHNH₂as labeling reagents followed by HPLC-MS analysis have been developed.The optimal derivatization condition using DBCEC-NHNH₂ as labeling reagent was evaluated.And the feasibility of this method has been identified by analyzing wine sample. Chapter five:Pre-column derivatization methods for the sensitive determination of free fatty acids using DBCES as labeling reagents followed by HPLC-MS analysis have been developed.The optimal derivatization and chromatographic separation conditions using DBCES as labeling reagent were evaluated.And the feasibility of this method has been identified by analyzing Euphorbia sample.