11h Benzo [a] Carbazole As The Mother Of New Fluorescent Reagent Preparation And Applied Research 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 scientific research and application, such as chemistry, biochemistry, clinical sciences, pharmacology, neurology, environmental science and quality control. However, not all compounds are suitable to be directly analyzed by HPLC because of the limitation of the compound properties and the very low contents of the corresponding compounds. Currently, fluorescence detection is still recognized to be one of the most sensitive detection technologies. Therefore, derivatization of analytes, such as fatty acids, aliphatic amines, amino acids,with fluorescence labeling reagents has been widely adopted. It can alter the chemical and physical properties of target compounds and make them easier to analyze. In this paper, we designed and synthesized three novel fluorescence labeling reagents based on 11H-benzo[a]carbazol . 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: fluorescence theory and fluorescence labeling technology were introduced systematically.The history and basic theory about HPLC were simply introduced.The The applications of fluorescence reagents by HPLC were summarized.Chapter two: The syntheses of three fluorescence labeling reagents 2-(11H-benzo[a]carbazol-11-yl) ethyl 4-methylbenzenesulfonate(BCETS)2-(11H-benzo[a]carbazol-11-yl) ethyl carbonochloridate (BCEC-C1) 2-(11H-benzo[a]carbazol-11-yl) isopropyl chloroformate (BCPC-C1) were described, and followed by their structural characterization and spectral properties. Chapter three: Pre-column derivatization methods for the sensitive determination of fatty acids, using 2-(11H-benzo[a]carbazol-11-yl) ethyl 4—methylbenzenesulfonateas labeling reagents followed by HPLC-MS analysis have been developed. The optimal derivatization and chromatographic separation conditions using BCETS as labeling reagents were elucidated. The contents of free fatty acids from real samples such as soil, bryophyte were determined. Fluorescence spectra of representative BCETS-C₈ derivative in the presence of halide ions.Chapter four: Pre-column derivatization methods for the sensitive determination of aliphatic amines, respectively, using 2-(11H-benzo[a]carbazol-11-yl) ethyl carbonochloridate (BCEC-Cl) 2-(11H-benzo[a]carbazol-11-yl) isopropyl chloroformate (BCPC-Cl) as labeling reagents followed by HPLC-MS analysis have been developed. The optimal derivatization and chromatographic separation conditions using BCEC-Cl and BCPC-Cl as labeling reagents were evaluated, respectively. The contents of aliphatic amines from real samples such as shrimp catsup were determined. Studies on derivatization conditions indicated that aliphatic amines could rapidly and smoothly react with BCEC-Cl and BCPC-Cl reagents under very mild conditions with maximal derivatization yields close to 100% with excellent linearity, good repeatability and higher sensitivity.Chapter five: Pre-column derivatization method for the sensitive determination of amino acids using the labeling reagents 2-(11H-benzo[a]carbazol-11-yl) ethyl carbonochloridate (BCEC-C1) followed by HPLC-MS analysis has been developed. Additionally, the contents of twenty amino acids from hydrolyzed bovine serum albumin and rape bee pollen were determined using BCEC—Cl as labeling reagent.