| HPLC is the fastest developing and the most widely used fields in analytical chemistry, based on its separation characteristic, the enthusiastic adoption of HPLC method has had a great impact on clinical chemistry environmental chemistry, biochemistry, toxicology, and many other fields. However, not all compounds are accessible to direct analysis by chromatography due to the limitations of properties of the compounds themselves and the detect means. Therefore, developing new highly sensitive fluorescent reagents using chemistry derivatization reaction to alter the chemical physics properties of target compounds to make them easy to be analyzed is the purpose of this thesis. The principles and applications of fluorescence detection and fluorescence reagents and methods are introduced. We designed and synthesized several novel fluorescence reagents with the different chromophores. The optimal derivatization conditions, separation behavior and sensitivity are also investigated.Currently many kinds of fluorescent reagents have been developed, However, there are still disadvantages in their applications. In order to overcome some drawbacks. We synthesized four fluorescent reagents based on matrix compounds of acridone and carbazole by means of chemically modifying. They are 2-(9-carbazole)-ethyl chloroformate (CEOC), 9-(2-hydroxylethyl)-acridone (HEA), 9-(2- hydroxylethyl)-carbazole (HEC), and 2-(9-acridone)-ethyl chloroformate (AEOC).A systematic research on the optimal derivatization and separation conditions with HPLC method using 2-(9-carbazole)-ethyl chloroformate (CEOC) and 9-(2-hydroxylethyl)-acridone (HEA) as derivatizing reagents for the determination of amino acids, amines and carboxylic acids were investigated for the first time. At the same time, The composition of theoptimal mobile phase and the influences of many other factors were also investigated. In addition, the applications of these fluorescent reagents A>r the separation of more complicated samples such as the amino acids from fodder and liquid state white egg hydrolysates, multi-peptide, the amine compounds from sea water were carried out with satisfactory results. The detection limits are at the level of/wo/ with relatively wide linear rangesThe MEKC (micellar electrokinetic chromatography) separation behavior of nine kinds of derivatized amino acid using 2-(9-carbazole)ethyl chloroformat(CEOC) as derivatizing reagents has been studied under 254nm ultraviolet detection. The effect factors of migration time and separation selectivity have been investigated by changing the concentration of SDS, buffer, organic solvent and pH value of buffer. The optimal conditions are obtained as follows: the separation voltage is 18Kv; The concentration of borate buffer, SDS, and acetonitrile are 20 mmol/L, 30 mmol/L, and 3% (v/v) respectively. The chromatography exhibits a good resolution for the separation of amino acid derivatives with ultraviolet detection.Preparation of several novel fluorescent derivatization reagents /.Preparation of 9-(2-hydroxyethyl)-carbazole(HEC): Carbazole-9-yl-acetic acid was conveniently prepared by a modifying method. A mixture of ethylbromoacetate in methyl-sulfoxide (DMSO) was added by dropwising to a solution containing carbazole, potassium hydroxide and methyl-sulfoxide (DMSO) in a round-bottom flask, the reaction was kept at 95 癈 for 1 h. The resulting solution was poured into a mixture of water and potassium hydroxide. After hydrolying, the solution was filtered-off and the pH of solution was adjusted to 2.0~3.0 with HC1 solution, the precipitation was removed and washed with water. The rough product was recrystallied from toluene to afford a white crystal. 9-(-2-hydroxyethyl)-carbazole (HEC) was synthesized by the reacton ofcarbazole-9-yl-acetic acid with reductant LiAlH4 by refluxing in ether. The mixture was extracted with ether. The solvent was removed by evaporation under reduced pressure. The residue was extracted three times with heated hexane. The white product was filtered and recrystallied fro... |
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