The Detection Of Carbonyl Compounds By Pre-column Derivatives With High Performance Liquid Chromatography Fluorescence Detector

Carbonyl compounds are extensively existed in biologyical resources.Although often present in low concentrations,they are of vital importance in various biologycal activites.Therefore,the development of effective and sensitive analytical methods for carbonyl compounds has become a hot research topic.However,direct analysis of carbonyl compounds is often hindered by two major factors:1.The low abundance of carbonyl compounds in complex biological samples;2.Most carbonyl compounds have no chromophores or fluorophores;To overcome those disadvantigies,proper derivatization methods are often required.High Performance Liquid Chromatography?HPLC?has been widely used in many fields of industry and scientific research,due to its powerful separation and analysis capacities.HPLC often combines with various detectors,in which fluorescence detector,compared with other detectors,is irreplaceable due to its high sensitivity,selectivity and wide linear range.High Performance Liquid Chromatography combined with fluorescence detector?HPLC-FD?has become an ideal candidate for the determination of carbonyl compounds in analytical chemistry.In the study,we aimed at developing a novel derivatization method to facilitate the sensitive analysis of carbonyl compounds by HPLC-FD.The major contents of this thesis were described as below:In the first part,the fluorescent properties,including?_ex and?_em values of three novel coumarin compounds:4-??aminooxy?methyl?-7-hydroxy-coumarin,4-??aminooxy?methyl?-6-chloro-7-hydroxy-coumarin and 4-??aminooxy?methyl?-7-met-hoxy-coumarin have been analyzed.The quantum yeilds of each coumarin co-mpounds were calculated.The result shows?_ex and?_emm of 4-??aminooxy?methyl?-7-hydroxy-coumarin are 325 and 470 nm in acetic acid-sodium acetate buffer,respectively and its fluorescence quantum yeild is measured as 0.98 in 10%ACN solution,reflecting its high fluorescence properties.In the second part,4-??aminooxy?methyl?-7-hydroxy-coumarin has been successfully used as a fluorescent label to tag a standard oligosaccharid Maltoheptaose for the first time.The chromatographs obtained from HPLC-FD detection shows three derivative peaks,indicating the products have three isomers.This hypothesis has been confirmed by the following mass spectrometric experiments.The fluorescent derivatization conditions have been optimized,including the reaction temperature,reaction time,pH,concentration of sodium acetate buffer,reactants ratio,the amount of catalyst and so on.In order to improve chromatographic analysis and increase detection sensitivity,the oxime derivative was therefore hydrogenated to a single,more stable product,using sodium cyanoborohydride as a soft reducing agent.In the third part,both NBD-AO and 4-??aminooxy?methyl?-7-hydroxy-coumarin were selected as fluorescent labeling reagents to derivatize with jasmonic acid?JA?,HPLC-FD analysis shows that 4-??aminooxy?methyl?-7-hydroxy-coumarin can readily react with JA,while NBD-AO shows no obvious reactivity.It has been hypothesized that NBD-AO can not derivatize with JA,due to steric hindrance and fluorescent quenching effect.To enchance the separation of derivative products of4-??aminooxy?methyl?-7-hydroxy-coumarin and JA,the mobile phase were adjusted by adding 0.1%trifluoroacetic acid in case of peak tailing and baseline drifting.