| Capillary electrophoresis (CE) is a powerful separation technique with fast separation speed, high separation efficiency and low sample consumption. For its short optical path and small sample volume, the detection sensitivity of ultraviolet-visible (UV-Vis) spectrophotometry is not satisfied. Laser-induced fluorescence (LIF) is one of the most sensitive methods to be used in CE detection. It can both enhance the detection sensitivity and improve the selectivity. So, it extends the application of CE, especially for biological and medical samples. Pre-column derivatization needs additional manipulations and stable derivatives. Morover, in pre-column derivatization of LIF, an analyte molecule with more than one labeled site may result in multiple separation peaks. However, the problem of multiple peaks can be avoided with post-column derivatization method, because the separation is based on the native analytes. The post-column LIF method is also suitable for high speed on-line derivatization detection. On the other hand, the fluorescence intensity suffers from the short optical detection path, and the S/N ratio decreases with the light scattering and background emission from the wall of a conventional on-column optical detection cell. A windowless fluorescence cell can resolve the problems of scattering and background emission from the conventional cell walls, and expand the optical path length.The auther reviews the sensitive detection methods of CE, fluorescence detection principle, instrumental structure, derivatization detection methods and post-column derivatization reactors of CE - LIF. The main tasks of the thesis are to assemble a simple CE - LIF system, to develop a simple and convenient post-column reactor for CE - LIF, which can be applied to analyze the kanamycin residues in foodstuff, such as milk and animal tissue. In addition, a windowless fluorescence detection method is also proposed. The research contents and results of the thesis are as follows:1. A simple CE - LIF system was assembled. The detection system was based on the collinear or confocal optic configuration arrangement. The experiments were optimized by the effects of diaphragm and PMT valtage on detection performance. The discussion was made about the method of improve the detection sensitivity. The concentration detection limit (CLOD) and mass detection limit (MLOD) of the riboflavin and was 9.0×10-9 mol/L (3.4×10-6 g/L) and 18 amol, respectively. The linear calibration range was 3.0×10-8-1.0×10-5 mol/L. The relative standard deviiatios (RSD) of the method was 2.4% (n=5).2. A post-column reactor with coaxial-gap mode was developed for LIF in CE. The reactor could be assembled simply and conveniently without any micromanipulation, in which a thin polyimide sleeve of 10-mm length obtained from the capillary coating was used to align and connect the separation and reaction capillary with a 10 - 20μm gap. Naphthalene-2,3-dicarboxaldehyde (NDA) and 2-mercaptoethanol (2-ME) were used as derivatization reagents and delivered into the reaction capillary through the annulus between the separation capillary and polyimide sleeve and the gap of two capillaries by gravity. A reaction distance from the gap to detection point could be shortened to 5 mm. For the post-column reactor of CE-LIF, several configuration parameters were optimized, including liquid level difference between the derivatization solution and outlet buffer, annular dimension between the etched separation capillary and polyimide sleeve, and reaction distance etc. for amino acids, the detection limits ranged from 8.0×10-8 to 1.0×10-6 mol/L and linear calibration ranges were more than two orders of magnitude, e.g. 5×10-7-1×10-4 mol/L for glycine. The separation efficiency ranged from 1.35×105 to 1.67×105 theoretical plates. The method was applied to the analysis of amino acids in vinegar samples successfully.3. An analytical method of capillary zone electrophoresis (CZE), post-column derivatization and LIF detection with the homemade coaxial-gap reactor was proposed for the determination of kanamycin A, amikacin and tobramycin, kanmycin components of aminoglycoside antibiotics (AGs). The CZE separation was performed with 50 mmol/L sodium acetate buffer (pH 5.0) containing 0.5 mmol/L cetyltrimethyl ammonium bromide to reverse the electroosmotic flow in separation capillary. The derivatization reagent solution contained 1.0 mmol/L NDA, 8.0 mmol/L 2-ME and 35 mrnol/L sodium tetraborate buffer (pH 10.0) in 30% (v/v) methanol. The detection limits ranged from 3.6×10-5 to 5.2×10-5 g/L and the linear calibration concentrations were in the range of 1.1×10-4-5.0×10-2 g/L. The proposed method was verified by measuring the spiked aminoglycosides in milk samples and animal issues samples after a simple sample pretreatment with trichloroacetic acid. The recovery of the amiinoglycosides ranged from 81.6% to 95.3% (n=4).4.A windowless detection cell combined with capillary electrophoresis for laser-induced fluorescence detection was proposed. The detection cell was simply and conveniently assembled using polyimide coating sleeve, in which the thin polyimide sleeve obtained from the capillary was used to align two capillaries with a 140μm gap. The effect of gap distance and running voltage on the contour of the windowless detection cell was investigated by imaging the windowless cell under a microscope. The experimental results validated the feasibility of the theory of the windowless detection method. Under optimized conditions, the windowless detection method presented lower LODs by compare with the on-column detection method. The detection sensitivity was improved about 15-fold for riboflavin. The linear calibration concentrations more than two orders of magnitude were obtained in the range of 5.0×10-9-1.0×10-6 mol/L for flavins. The separation efficiency of windowless detection method ranged from 1.0×105-2.4×105 theoretical plates. The RSDs were in the range of 3.7%-5.8% (n=5). The method was applied to the analysis of flavins in spinach and lettuce leaves. Since the windowless detection cell was simply assembled and eliminated the adsorption of matrix impurities, it could be employed in optical detection modes in CE.
|
PDF Full Text Request