| Capillary electrophoresis(CE)in conjunction with laser-induced fluorescence(LIF) detection,with its utmost separation efficiency and detection sensitivity,has documented a powerful tool for the determination of a variety of compounds,especially for pharmaceutical and bio-analytical applications.However,there are not many analytes of interest applicable by CE that naturally possess a high fluorescence intensity.Also, analysis of native fluorescence compounds is limited by their differing excitation wavelengths,and their compatibility with available laser sources.To overcome this drawback,derivatization of the analytes with a fluorophore that fluoresces with an excitation wavelength near that of the laser is an important technique for improving sensitivity in CE.Therefore,derivatization reaction plays an important role in CE-LIF method.Generally,derivatization procedures can be performed before(pre-capillary), during(in-capillary)or after(post-capillary)the electrophoretic separation.The most suitable approach depends on the reason why a derivatization procedure is introduced, the physicochemical properties of the analyte and reagent,ect.However,the derivatization reaction in many cases is tedious and time consuming,which is the bottleneck for analysis time and sample throughput.On the basis of previous literatures,this dissertation focused on the development of rapid and automatic derivatization reactions,and some original studies were carried out as follows:1.A selective and sensitive method was developed for simultaneous determination of catecholamines and amino acids by micellar electrokinetic chromatography(MEKC) with laser-induced fluorescence detection(LIF)using NBD-Cl as derivatization reagent; 2.A comparative study was performed to investigate the influence of different derivatization and separation behaviors in aqueous and nonaqueous media on the fluorescence intensity using NBD-labeled catecholamines as test compounds by CE with LIF detection.The mechanisms of derivatization and separation in nonaqueous media were elucidated in detail;3.The potential of NBD-F as in-capillary derivatization reagent is described,and two rapid and sensitive MEKC-LIF methods with in-capillary derivatization were developed for the fully automated of organophosphorus pesticides,ephedrine and pseudoephedrine,respectively;4.The possibility of microwave-assisted accelerated derivatization for CE-LIF was investigated using FITC as derivatization reagent,and then the developed method was used for the rapid and sensitive determination of histidine,1-methylhistidine and 3-methylhistidine in human urine;5.An approach combined optical spectroscopic techniques and modeling studies was depicted to probe the differences in human serum albumin(HSA)binding affinity for pyrrolo[2,3-d]pyrimidine antifolates enantiomers.This dissertation consists of seven chapters:Chapter 1:This chapter reviewed the progress of derivatization reaction for capillary electrophoresis,especially for the trends and applications of on-line and in-capillary derivatization.Chapter 2:A selective and sensitive method was developed for separation and simultaneous determination of catecholamines and amino acids by MEKC-LIF using NBD-Cl as derivatization reagent.And the detailed derivatization mechanism and sensitization effect of SDS were discussed.Under optimum conditions,two catecholamines and eleven amino acids were separated in a short 13 min analysis time and the relative standard deviations for migration time and peak area were less than 0.60%and 6.50%,respectively.The method was successfully applied for the quantification of catecholamines and amino acids in Portulaca oleracea L.,human urine sample and mixed injection sample.Chapter 3:Based on the Chapter 1,a novel method based on separation by nonaqueous capillary electrophoresis(NACE)combined with LIF detection was developed and compared with classic aqueous modes of electrophoresis in terms of resolution of solutes of interest and sensitivity of the fluorescence detection. Catecholamines derivatized with NBD-Cl were chosen as test analytes for their subtle fluorescence properties.In aqueous systems,capillary zone electrophoresis(CZE)was not suitable for the analysis of test analytes due to complete fluorescence quenching of NBD-labeled catecholamines in neat aqueous buffer.The addition of micelles or microemulsion droplets into aqueous running buffer can dramatically improve the fluorescence response,and the enhancement seems to be comparable for micellar electrokinetic chromatography(MEKC)and microemulsion electrokinetic chromatography(MEEKC).As another alternative,NACE-LIF procedure was proved to be superior,providing high sensitivity and short migration time.Under respective optimum conditions,the NACE procedure offered the best fluorescence response with 5-24 folds enhancement for catecholamines compared to aqueous procedures.In addition, the mechanisms of derivatization and separation in nonaqueous media were elucidated in detail.These discussions would significantly extend the application area of NACE-LIF method.Chapter 4:This chapter developed a rapid and sensitive method using in-capillary derivatization in conjunction with MEKC-LIF for the fully automated analysis of organophosphorus pesticides.The potentiality of NBD-F as in-capillary derivatization reagent was described.The unique feature of this MEKC method comes from that the capillary is used as a small reaction chamber.In in-capillary derivatization,the sample and reagent solutions were injected directly into the capillary by tandem mode,followed by an electrokinetic step(5 kV,30 s)to enhance the mixing efficiency of analytes and reagent plugs in accordance with their different electrophoretic mobilities.Standing a specified time(7.5 min)for reaction,the derivatives were then immediately separated and determined.Careful optimization of the derivatization and separation conditions allowed the determination of glufosinate,AMPA and glyphosate with detection limits of 2.8,3.6 and 32.2 ng/mL,respectively.The method was successfully applied to the analysis of spiked river water sample with satisfactory results.Chapter 5:Based on the Chapter 4,this chapter describes an automatic rapid approach for in-capillary derivatization of ephedrine(E)and pseudoephedrine(PE)and subsequent sensitive determination of the derivatives by MEKC-LIF using NBD-F as fluorescent reagent.Several parameters for in-capillary derivatization and subsequent MEKC separation were systematically investigated.Under these optimized conditions,a baseline separation of two analytes was achieved within 10 min and the derivatization concentration limits of detection were found to be 4.8 ng mL-1for E and 1.6 ng mL-1for PE,respectively.The method was validated in terms of precision,linearity,accuracy and successfully applied for the determination of the two alkaloids in ephedra herb and its preparations.Chapter 6:A novel microwave-assisted accelerated derivatization method was developed for CE-LIF using FITC as derivatization reagent.Compared with traditional water-bath derivatization(50℃,4 h),the derivatization reaction can been complete within 2.5 min at 700 W.The developed method was successfully applied for the rapid and sensitive determination of histidine,1-methylhistidine and 3-methylhistidine in human urine.Chapter 7:An approach combined fluorescence,Fourier transform infrared spectroscopy(FT-IR),circular dichroism(CD)and molecular modeling study was applied to probe the differences in HSA binding affinity for pyrrolo[2,3-d]pyrimidine antifolates enantiomers(L/D-PPGA).The results indicated that the two enantiomers can bind to HSA via two types of sites(subdomainⅡA andⅢA)by hydrophobic interaction and hydrogen bond,which can be supported by competition binding and molecular modeling.Compared with L-PPGA,a stronger interaction between D-PPGA and HSA was observed.
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