Synthesis Of New Sensitive Fluorescent Reagent And Its Application In High Performance Liquid Chromatography

The high-performance liquid chromatographic method is considered as one of the most powerful methods to separate complex mixures,just as fluorescence detection is thought to be one of the most sensitive detection means.Then using their advantage could satisfy the request of the ultra trace analysis.Owing that not all of the samples could emit fluorescence,it is necessary to use the fluorescence derivatization.Therefore,derivatization of analyses,such as fatty acids, aliphatic amines,with fluorescence labeling reagents has been adopted in this paper.It can alter the chemical and physical properties of target compounds and make them easier to analyze. Three kinds novel fluorescence labeling reagents based on benzo[b]acridin-12(5H)-one and 2-(pyren-1-yl)-1H-benzo[d]imidazole were designed and synthesized,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 fluorescence theory and fluorescence labeling technology were introduced systematically.The history and basic theory about HPLC were simply introduced. The applications of fluorescence reagents for labeling of carboxyl and amine compounds in HPLC were summarized.Chapter two:Three kinds fluorescence labeling reagents,2-(12-oxobenzo [b]acridin-5(12H)-yl)ethyl-4-methylbenzenesulfonate(BAETS),2-(2-(pyren-1-yl) -1H-benzo[d]imidazol-1-yl)ethyl-4-methylbenzenesulfonate(PBITS) and 2-(12-oxobenzo [b]acridin-5(12H)-yl)acetic acid(BAAA),were synthesized,described and followed by their structural characterization and spectral properties.Chapter three:Pre-column derivatization methods for the sensitive determination of fatty acids,using BAETS and PBITS labeling reagents followed by HPLC-MS analysis have been developed.The optimal derivatization and chromatographic separation conditions using BAETS and PBITS as labeling reagents were elucidated.The contents of free fatty acids from real samples such as auricularia auricula,stellera chamaejasme were determined.Studies on derivatization conditions indicated that fatty acids could rapidly and smoothly react with the labeling reagents under very mild conditions,with maximal derivatization yields close to 100%, with excellent linearity,good repeatability and higher sensitiviy.Chapter four:Pre-column derivatization method for the sensitive determination of bile acids using the labeling reagent BAETS followed by HPLC - MS analysis has been developed. We set up the method of bile acids pre-column derivatization high-performance liquid chromatography with fluorescence detection,by optimizing derivatization and chromatographic separation conditions.Additionally,we use the method in the determination of serum samples, and give highly sensitive bile acids detection.Chapter five:Pre-column derivatization methods for the sensitive determination of aliphatic amines,using BAAA as labeling reagent followed by HPLC-MS analysis have been developed.The optimal derivatization and chromatographic separation conditions using BAAA as labeling reagent were evaluated.The contents of aliphatic amines from real samples such as water was determined.