Study And Application Of Coating Technique And Indirect Laser-induced Fluorescence Detection For Capillary Electrophoresis

Capillary electrophoresis(CE)has emerged as a separation technique.Due to its high resolution,efficiency,short analysis time,simplicity and low sample and solvent consumption,CE has proved to be a highly efficient separation technique.In recent years, much attention has been devoted to the improvement the separation selectivity of CE.CE is a powerful tool especially for the determination of big biological molecules.However, the adsorption of proteins on the capillary is a problem that we concerned.It is necessary to manipulate the surface charge of the capillary to prevent wall adsorption of proteins. Coating technique is broadly used in CE to prevent the adsorption of proteins.In addition, laser induced fluorescence(LIF)detection has high sensitivity and can be adapted to different separation modes of CE.So it has become an important detection technique in the field of drug analysis and life science.In this dissertation,new separation and analysis systems were brought forward based on CE-LIF detection and laser light scattering research.Based on the previous literatures,the following major innovation woks were carried out in this dissertation:(1)The long-chained,silanizing agent chloro(dodecyl)dimethylsilane(CDDS)was investigated as a semipermanent coating in micellar electrokinetic capillary chromatography(MEKC).AFM image obviously showed that CDDS coating can covalently bond on the surface of the capillary by rinsing the capillary with CDDS solution and forms a monolayer flat.CDDS semipermanent coating had great stability on the surface of fused-silica capillary due to the formation of covalent bonding. (2)A rapid,sensitive and reproducible chloro(dodecyl)dimethylsilane(CDDS) semipermanent coating-micellar electrokinetic capillary chromatography(MEKC) method to analyze ephedrine(E)and pseudoephedrine(PE)after derivatizated with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazol(NBD-Cl).(3)We developed a sensitive method using micellar electrokinetic chromatography with laser-induced native fluorescence detection for the simultaneous separation and determination of epirubicin,daunorubicin and idarubicin in human serum sample.(4)We developed a rapid and simple method using indirect laser-induced fluorescence detection(ILIFD)for the simultaneous determination of matrine(MT),sophoridine (SRI)and sophocarpine(SC).The method was operated simply without derivatization and would redound to the application range of LIF detection.(5)A new method using indirect laser-induced fluorescence detection(ILIFD)was developed for the simply and rapidly determination of alpinetin and cardamonin by micellar electrokinetic chromatography.This dissertation consists of six chapters.Chapter 1:This chapter reviewed the progress of coating technique for capillary electrophoresis,especially for the trends and applications of permanent,coating, semi-permanent coating and dynamic coating.Chapter 2:The long-chained,silanizing agent chloro(dodecyl)dimethylsilane (CDDS)was investigated as a semipermanent coating in micellar electrokinetic capillary chromatography(MEKC).CDDS coating had great stability due to the formation of covalent bonding with the silanol groups on the surface of fused-silica capillary and remained stable for over 100 minutes after removal of the rinse step of CDDS solution. Migration time reproducibility was less than 0.97%RSD from run to run and less than 1.56%RSD from day to day.Chapter 3:This chapter presents a rapid,sensitive and reproducible chloro(dodecyl)dimethylsilane(CDDS)semipermanent coating-MEKC method to analyze ephedrine(E)and pseudoephedrine(PE)after derivatizated with 4-chloro-7- nitrobenzo-2-oxa-1,3-diazol(NBD-Cl).The baseline separation was achieved within 4 min with running buffer composed of 20 mM borate,8 mM SDS(adjusted pH 9.8).The new method was successfully applied to the analysis of pharmaceutical preparations with recoveries in the range of 96.3-102.4%.Chapter 4:This chapter describes a sensitive method using micellar electrokinetic chromatography with laser-induced native fluorescence detection for the simultaneous separation and determination of epirubicin,daunorubicin and idarubicin in human serum sample.The method was successfully applied to the analysis of spiked serum samples and two patient serum samples with satisfactory results.Chapter 5:We developed a rapid and simple method using indirect laser-induced fluorescence detection(ILIFD)for the simultaneous determination of matrine(MT), sophoridine(SRI)and sophocarpine(SC).This developed method was applied to the analysis of these three bio-alkaloids in herb plants with recoveries in the range of 93.1-101.5%.In this work,although the detection sensitivity was lower than that of direct LIF,the method was operated simply without derivatization and would redound to the application range of LIF detection.Chapter 6:This chapter developed a new method using indirect laser-induced fluorescence detection(ILIFD)for simply and rapidly determining alpinetin and cardamonin by micellar electrokinetic chromatography.With an optimized electrophoretic buffer system,alpinetin and cardamonin can be separated and determined with 7.5 min with the detection limits of 1.4 and 1.1μg/mL based on equation,1.9 and 1.5μg/mL based on an S/N ratio of 3,respectively.This new method was applied to the analysis of these two compounds in pharmaceutical preparations with recoveries in the range of 93.2-108.2%.