Development And Optimization Of High-precision Quantitative Capillary Electrophoresis And Its Application On Illegal Additives

Capillary electrophoresis(CE) is a modern scientific analytical technique,which has the characteristics of high column efficiency, fast separation, less solvent consumption and can be used in many separation modes. It developed very quickly at the beginning when it was commercialized. CE has been predicted to take the place of HPLC and become the mainstream analytical instrument in micro separation field. However, after three decades of development, CE is not popularized as expected, due to some drawbacks in the instrumentation, especially the defect of the sample introduction. Conventional sample introduction in CE mainly includes electrokinetic injection and hydrodynamic injection, both have some drawbacks. The former is prone to sample discrimination and the latter is not suitable for high viscosity of the samples and buffer. Moreover, both the above mentioned methods need the capillary inlet dip into the sample vial, then move back to the buffer vial. The movement of capillary-inlet between the sample and buffer not only causes cross contamination, but also disturbs the equilibrium of electrochemistry in the separation capillary and, therefore, sacrifices the accuracy and reproducibility in quantitative analysis.To change the status quo, we constructed a high-precision quantitative capillary electrophoresis system with the introduction of nano-injector, and optimized the experimental parameters to stable and reliable operation. Besides, we combined the system with microflow evaporative light scattering detector(μ-ELSD) system successfully, and obtained good results on the analysis of illegal adulterants in weight loss products.The full text is divided into five chapters:In chapter 1, we briefly introduced the background and significance of the project. Then described the sample introduction of traditional CE, and summarized the problems and difficulties in this injection methods. Then, we reviewed the reported nano-injector technology in CE in detail.In chapter 2, we constructed and optimized the high-precision quantitative capillary electrophoresis system. In the process of optimized the system, we improved the structure of electric decoupler, and found the optimal ratio of pressurized flow to EOF. In addition, we also investigated the column efficiency of qCE, and compared it with traditional CE.In chapter 3, we combined the qCE system with μELSD system. We briefly introduced the μELSD, and discussed the application prospect of the combination with q CE system. The most important factor is the development of the connection mode of the interface. We finished five ways of the connection mode, and evaluated each of mode respectively to get an optimal connection scheme. At last, we tested properties through the separation of glucose and nucleoside.In chapter 4, we applied this system in determining the illegal adulterants in weight loss products, mainly including fenfluramine, sibutramine, pseudoephedrine, amfebutamone, clenbuterol and norepinephrine. The experimenal conditions including the type of buffer, buffer concentration and buffer p H value were studied, we also studied the optimal ratio of pressurized flow to EOF, and found an optimum temperature condition. The optimal condition comprises an electrolyte containing 20 mM NaH2PO4/H3PO4 at pH 2.0 and with the capillary temperature at 15℃ and voltage of 15 kV, the detection wavelength of 195 nm. The capillary have an inner diameter of 50 μm, with effective length of 40 cm(total length of 60 cm). The syringe pump flowrate was set at 0.6 μL/min with split ratio of 10:1(in the absence of the electric field). At last, a series of pretreatment and quantitative analysis was made in real samples including tablets, teabags and capsules, the results were satisfactory.In chapter 5, we summarized the main innovation of this project, and its development direction and application prospects were also discussed.This dissertation constructed an improved version of high-precision, quantitative capillary electrophoresis on the basis of our previous research, and optimized the major modules of this system. The column efficiency of 254,000 plates/m was achieved with the repeatability in terms of relative standard deviation(RSD) lower than 2%. Experimental results demonstrated that the system meets the requirements of a commercial instrument and may be used in quality control(QC) and quality assurance(QA) in pharmaceutical industry.