| Capillary electrophoresis(CE),benefited from exceptional advantages such as high separation efficiency,short analysis time,versatile operation modes,low sample and reagent consumption,has developed into an effective separation technology which is widely applied in the fields of biomedicine,food,environment,and so on.Limited to small inner diameter(typically 25-100 ?m)of capillary,the injection volume is often in nanoliters,and the effective optical path-length for detection is usually in micrometers,thus leading to a low sensitivity for CE analysis by using traditional absorbance detection.Laser-induced fluorescence detection(LIF)is one of the highest sensitive detection techniques for CE at present.Capillary electrophoresis-laserinduced fluorescence detection(CE-LIF)combined the advantages of high separation efficiency and excellent sensitivity is very suitable for the analysis of trace substances in complex samples.However,the available wavelengths of commercial lasers for LIF detector are still insufficient,and many analytes of interest show no native fluorescence,which greatly limit the development and application of LIF detectors.Recently,the advances of laser technology have provided more possibilities for LIF detection.Diode lasers have received increasing attention and developed as the main excitation source in(miniaturized)LIF detector because of their advantages of rich wavelength varieties,low-cost,long-life,and small-size.However,the application of diode laser-induced native fluorescence is relatively rare at present,and there is a lack of fluorescence labeling reagents and derivatization strategies matching these wavelengths,which to some extent limits the promotion and application of such diode laser-induced fluorescence detectors.The purpose of this paper is to construct the confocal blue / violet light-excited LIF detector with 445 nm / 405 nm diode laser as excitation source and then establish a complete CE-LIF system coupled with LIF detection.And to expand the application of blue and violet laser-induced fluorescence detector,the application of 445 nm LINF was established with curcuminoids as the target analytes;and the fluorescence labeling reagents and corresponding fluorescence derivatization methods suitable for the 405-nm LIF were developed using coumarin fluorophore as the parent.This dissertation can be divided into four chapters:Chapter one: This chapter briefly introduces the capillary electrophoresis-laser induced-fluorescence detection(CE-LIF)technique,including the separation modes and detectors for CE,and the principle,structure and related progress of LIF detector.This chapter focused on the available laser sources for LIF detector,and systematically summarized the applications of capillary electrophoresis-laser induced native fluorescence(CE-LINF).In addition,the fluorescence labeling reagents for LIF detectors were also summarized briefly.Chapter two: A commercial blue laser diode(445 nm)was used as an excitation source to construct a confocal laser-induced fluorescence detector and then a complete capillary electrophoresis system coupled with LIF detection was established.With three major curcuminoids,curcumin,demethoxy curcumin(DMC)and bisdemethoxy curcumin(BDMC)as target analytes,a micellar electrokinetic chromatography(MEKC)method was proposed using mixed micelles consisting of Triton X-100 and SDS to sensitize the native fluorescence of curcuminoids and enhance their separation efficiency.Fluorescence spectra revealed that the mixed micelles induced fluorescence synergism could enhance the signals of three curcuminoids by 77-,57-,and 47-fold for curcumin,DMC,and BDMC.Under the optimal conditions,a baseline separation of three curcuminoids was achieved within 10 min and the detection limits were found to be 4.1,2.6,and 0.4 ng/mL for curcumin,DMC,and BDMC,respectively.Furthermore,the developed MEKC-LINF method was validated in terms of precision,linearity,accuracy and successfully applied for the determination of three curcuminoids in turmeric,medicinal turmeric liniment,curry seasoning,and human urine samples.Chapter three: Based on the previous chapter,a set of violet light-excited CELIF system was constructed using 405 nm diode laser as the excitation source.And then,a commercial fluorescent dye,7-(diethylamino)coumarin-3-carboxylic acid(DEAC-C)was introduced as derivatization reagent and corresponding derivatization strategy for DEAC-C labeling sulfonamides was developed for 405 nm LIF detection.After systematic optimization,three sulfonamides including hydrochlorothiazide(HCTZ),chlorothiazide(CTZ)and chlortalidone(CTD)could be efficiently labeled by DEAC-C in the presence of cyanuric chloride and triethylamine(1.5%)in acetonitrile at 50 oC for 180 min.Based on the laboratory-built CE-LIF system,a robust method was then proposed for the separation of DEAC-C labeled sulfonamides by micellar electrokinetic chromatography(MEKC).Under the optimized conditions,a baseline separation of three sulfonamides was achieved within 15 min,and the detection limits were determined to be 0.24,0.29,and 0.23 nM for HCTZ,CTZ,and CTD,respectively.Furthermore,the developed MEKC-LIF method was successfully applied for the analysis of three sulfonamides in complex pharmaceutical and physiological samples.Chapter four: Based on the previous chapter,7-(Diethylamino)coumarin-3-carbohydrazide(DEAC-H)was synthesized as derivatization reagent for 405 nm LIF detection.And corresponding derivatization strategy was proposed for aldehydes using glyoxal(Gly)and methylglyoxal(Mgly)as target analytes.After optimization,aldehydes could be efficiently labeled by DEAC-H in the presence of 30 ?M HCl in acetonitrile at 50 oC for 60 min.Compared with the reaction of carboxylic acid with sulfonamide,the reaction of hydrazide with aldehyde is more efficient.Therefore,combined with the high sensitivity of LIF detection,the developed method is expected to be applied to the determination of such compounds in atmospheric fine particulate matter. |
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