| Nonaqueous capillary electrophoresis(NACE) is a rapidly developing analytical platform, and it has experienced rapid growth in recent years. It has been widely used in areas such as pharmaceutical analysis, environmental analysis, biological analysis, and so on. NACE presents nuberous superiorities over aqueous capillary zone electrophoresis(CZE). The most significant ones are: flexible change of selectivity, higher separation efficiencicy, weak adsorption of analytes on capillary wall, good compatibility with different detectors, etc; Expecially, NACE demonstrates distinct and particular merits in analytical applications of all kinds of substances, such as poor water-soluble analytes, water-instability analytes, neutral or uncharged analytes and chiral substances.Laser induced fluorescence(LIF) is one of the most sensitive detection methods of capillary electrophoresis(CE), also firstly and still commonly used now in microfluidic capillary electrophoresis(μCE), due to benefits associated with high sensitivity, strong specificity and fast response speed.In this paper, a Chip-NACE(for seaparation)-LIF(for detection) method to analyse some bioactive components in traditional herbs was established by integrating NACE into a glass chip of a few square centimeters. The main four-part contents are summarized as follows: The first chapter review critically gives an overview on the theoretical and practical advances in NACE, including basic theoretical aspects, its advantages compared with aqueous CZE, selection of organic solvents and background electrolytes, detection methods, sample preconcentration, application in real samples(Traditional Chinese Medicine, TCM) and the future research trends.In the second chapter, we introduced an effective, uncomplicated and sensitive method of Chip-NACE-LIF to determine the content of sanguinarine(SA) and chelerythrine(CHE) in the seeds of Macleaya cordata(Willd.) R.Br. 40 m M ammonium acetate-acetic acid-water buffer(apparent p H*=2.90) containing 50 %(v/v) formamide is electrophoresis buffer solution. The separation voltage is 1800 V, injection time is 60 s and the detection length is 4 cm. Baseline separation of SA and CHE was achieved within only 120 s. Good linearity can be obtained in the range of 0.3-600 μg·m L-1 with a correlation coefficient of 0.9998 for SA and in the range of 0.15-550 μg·m L-1 with a correlation coefficient of 0.9993 for CHE, and lower limit of detection(LOD) was achieved thanks to the high sensitivity of LIF detection(2.0 ng·m L-1 and 5.0 ng·m L-1 for SA and CHE, respectively). The method established on microchip is susceptivity, rapid and low sample consumption, which can be used in the assay of alkaloids with very similar structures in Macleaya cordata and Chelidonium majus L.In the third chapter, an unprecedente Chip-NACE-LIF method has been developed for the separation and simultaneous determination of free anthraquinones in Rhubarb. Some key parameters affecting the separation, such as buffer concentration, apparent p H value, the applied voltage, proportion of water, were discussed systematacially. Optimum complete separation of physcion and rhein was accomplished in less than 150 s using a non-aqueous buffer system of 20 m M ammonium acetate, 60 % dimethylsulfoxide, 20 % formamide and 20 % water at 1.8 k V. Satisfactory LOD values were achieved for the studied compounds considering their appearance in natural extracts. Low limits of detection were 0.085 μg·m L-1 for physcion and 0.077 μg·m L-1 for rhein. The regression curves were used for the quantification of physcion and rhein, which showed a linear range of 1.44-28.43, 0.98-28.42 μg·m L-1, respectively, with correlation coefficients of 0.9995 and 0.9998. The method is sample, yare, accurate and well reproducible, which also successfully used to simultaneous analysis of the two related alkaloids with the recoveries of 101.4 % and 97.8 %(for physcion and rhein, respectively), hopefully, this method could be used practically for the quality control of herbs and their complicated medicinal prearations containing the two bioactive components.In the fourth chapter, we made an elementary exploration of chip preparative electrophoresis, for it is our aim to obtain monocomponent substance after NACE separation. Accordingly, this chapter addressed channel design of the chip, width of separation channel, injection time and test of injection methods with a variety of configurations, including a simple cross, a double-T and a triple-T configuration. The resulting designs were optimized and confirmed experimentally by investigating the effect of different configurations on the separation speed and resolution of two uncomplicated and stable fluorescent dyes(Rh123 and Flu), then applied for the analysis of Rh123 and SA. It turned out that the optimum five-channel microchip could allow the formation of volumetrically defined sample plugs in shorter time, then, analyzed electrophoretically. |
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