Development And Application Of New Capillary Eletrophoresis-Chemiluminescence System

In narrow terms, capillary electrophoresis (CE) usually means the capillary zone electrophoresis (CZE). which was performed in a narrow-bore open quartz tube filled with separation electrolyte. The separation efficiency of this technology is relied on the differential migration of ionic species in an electric field by their charge, frictional forces and hydrodynamic radius. In simple terms, it was designed to separate species based on their size to charge ratio. Introduced in1981, CE has been extensively investigated and applied for its high performance, rapid analysis and environmental friendliness; now, it has become an important liquid separation technology besides the traditional HPLC. For the micro volume of injection sample, CE confronts challenges for sensitive detection of the electrophoresis-separated analytes. To solve this problem, various technologies, such as fluorescence detection, UV-Vis absorbance detection and electrochemical detection were adopted to improve this micro separation technology. Being highly sensitive with simple equipment, chemiluminescence (CL) was expected to be an efficient detection method for CE. In practice, the CL reaction system was complicated and hard to be controlled. It made the explorations of CE-CL detection get in various difficulties.Using the CL reactions of luminol and TCPO (bis(2,4,6-trichlorophenyl)oxalate), this dissertation focused on finding new CE-CL detection methods to expand the application of this technology. Indirect CE-CL determination of saccharides and phenols were realized; ultra-fast CL reactions were discovered, making static reaction cell perform well as a compact CE-CL interface; the mechanism of TCPO CL reaction and the catalytic properties of hemin were further understood. Six parts were included in this dissertation, as following:Chapter one was the introduction. First, the development and theories of CE technology were overviewed involving the corresponding detection methods. The CL detection was introduced with its theories and characters. These CL reaction systems, which were frequently used in CE-CL, were especially displayed with various CE-CL interfaces, investigated status and developing tendency. The goals and significances of this dissertation were also introduced here.In chapter two, we designed a compact CE-CL system with a static detection cell, using an ultra-fast CL reaction of TCPO-H2O2system by the catalysis of imidazole in THF (tetrahydrofuran). There are three parts in this investigation. First, we recorded an ultra-fast CL duration of TCPO-H2O2reaction system, which was catalyzed by imidazole in the solvent of THF. A strong CL emission appeared with the reaction starting, going and finishing in0.6s. Based on this observation, a compact CE-CL system was realized using a static reaction cell for CL detection. With this simplest interface, the peak-spreading and-tailing caused by a redundant CL duration were avoided in CE-CL electropherograms. Then, relied on the CL system of imidazole-TCPO-H2O2-R6G (rhodamine6G), a NACE (nonaqueous capillary electrophoresis) method was applied to monitor the hydrolysis process of R6G in alkaline solutions with this compact CE-CL system. The relative rate constants and activation energy were gained. Adequate separation efficiency, stability and reproducibility with LOD (limit of detection) of6×10-8mol/L,7×10-8mol/L for R6G and R6G-COOH were gained in experiment. The CL duration of TCPO system was quite slow in normal conditions, and its mechanism did not be pointed out very clearly. So we further observed the discovered unusual fast CL duration (the fastest one in reports of TCPO) and investigated the effects of catalyst (imidazole), oxidant (H2O2) and solvents on the CL intensity and duration. With these data, a new intermediate was suggested in the CL reaction process of TCPO-H2O2catalyzed by imidazole, followed with a possible mechanism. Chapter three, a compact CE-CL system was developed for carbohydrate analysis withluminol-KIO4-K3Fe(CN)6reaction system. In this investigation, an ultra-fast CL reaction was realized with frequently-used CL reagent of luminol. In alkaline solution, a strong CL emission could be accomplished in0.65s by luminol with proper concentration of K3Fe(CN)6as catalyst and KIO4as oxidant. It made the CE-CL detection system performed efficiently with a static reaction cell. Using this system, we observed negative signals with carbohydrates in the CE-CL baseline of luminol and realized an indirect determination of rhamnose, D-fructose, sucrose and β-cyclodextrin with adequate sensitivities, linear ranges and reproducibilities. As a novel analysis method for carbohydrates, it performed with simple equipment to achieve rapid determination of mono-, di-and oligo-saccharides with adequate sensitivities (10-5mol/L). This job expanded the application area of CE-CL detection and provided a new approach to realize simplified CE-CL system. In chapter four. the indirect CE-CL carbohydrate analysis was further investigated, α-,β- and y-cyclodextrins (CDs) were used as a mixed sample to find out the efficiency of this indirect CE-CL method for the determination of oligo-saccharides; further, it was applied to detect the cyclodextrin additives in a real sample. Here, the alkaline and dimethylsulfoxide (DMSO) contents in separation electrolyte were studied to find out their effects on the saccharide separation and detection. A higher content of DMSO would lead more stable electrophoresis and better separation, while the baseline became weaker with longer operation time. So the separation voltage was increased to24kV to determine those CDs in24min with a separation electrolyte of40%DMSO. The limits of detection for α-, β-and y-CD were6.0×10-5,6.5×10-5,5.5×10-5mol/L correspondingly. In this job, electroosmotic flow (EOF) was regulated by DMSO to improve the resolution of analytes in CE. With the determination of cyclodextrin additives (β-,γ-CD) in wax gourd tee drink, it’s confirmed to be useful in practice.In chapter five, an improved sheath flow CE-CL system was utilized to perform accurate determination of vitamin B12(VB12) in drugs based on the catalytic ability of VB12(Co(Ⅱ)). which was reduced from VB12(Co(Ⅲ)), in CL reaction of luminol-H2O2.There are two sections in this chapter. In the first one, a previously lab-constructed sheath flow CE-CL system was improved. An equipment of "gravity driving-pressure control" was set up to control the flow velocity of CL reagents in the outer channel. By this way, the time window for the CL detection of analytes could be calculated and modified easily. The light resistant container was enhanced. To remove the interference of inner-appearing bubbles on CE, we shifted the cathode of separation voltage closer to the outlet of capillary. In the second section, sodium hydrosulfite (Na2S2O4) was found to an competent reagent to pre-reduce VB12(Co(Ⅲ)) into VB12(Co(Ⅱ)) gently. In this process, some interfering CL noise was also introduced and CE was used to remove it. In20min, an accurate CL detection of VB12could be accomplished with limit of detection (LOD)2×10-1mol/L (S/N=3) by CE-CL method. It’s applied to determine VB12in tablets, gained satisfactory results.Chapter six was focused on the catalytic characters of hemin as an efficient CL catalyst and its potential application in CL detection. With this exploration, we realized an indirect CE-CL detection for phenols analysis in hernin-luminol-H2O2system. There are two sections in this chapter. Section one performed static injection tests to observe catalytic characters of hemin in CL reaction of luminol-H2O2. It’s observed that hemin did best in a neutral solution compared with acidic or alkaline medium. Though halide ions, such as Br, F-could further enhance the CL signal catalyzed by hemin, it’s found to be difficult while we tried to couple this CL system with CE, caused by the self-polymerization of hemin to hinder CE from going smoothly. In section two. quite high concentration of NH3·H2O was added in the DMSO aqueous solution of hemin-luminol to gain a stable CE-CL baseline (the electrophoresis of hemin could perform smoothly). With experiments, it’s confirmed that the achievement of this CE-CL process was mostly due to the coordination between NH3and Fe(Ⅲ) of hemin to eliminate the interference of self-polymerization. The significance of this result is obvious for the application of hemin in CE-CL technologies. As a further job, we explored to detect phenols by indirect CE-CL method:five phenols could be separated and determined in15min with corresponding LODs of4.8×10-6mol/L (o-sec-butylphenol),4.9×10-6mol/L (o-cresol),5.4×10-6mol/L (m-cresol),5.3×10-6mol/L (dichlorophenol) and7.1×10-6mol/L (phenol). In the following static injection tests, four kinds of phenols were found to be inhibitive for the CL emission of hemin-luminol-H2O2, while dichlorophenol did enhance the emission. So, we tend to attribute this novel indirect CE-CL determination coming from ions displacement of luminol by phenols in CE process.