| High performance capillary electrophoresis (HPCE) has received a great amount of attention as an attractive separation technique in recent years due to the following characteristics: simplicity, high efficiency, rapid, clear, low cost, easy automation and short analysis time. At present, HPCE has been applied in many fields, such as life sciences, biomedicine and environment detection. CE separations coupled with highly sensitive detector had become more and more necessary. So, the research of detector was very important for developing the CE methodology. So far, some employed CE detectors were following: ultraviolet visible, laser induced fluorescence, chemiluminescence, mass spectrum, nuclear magnetic resonance and electrochemistry detector. The laser induced fluorescence was the most sensitive detector among the above detectors for high lighteness, straight and small wavelength range. But, the conventional laser induced fluorescence detector suffered from the light reflecting and scattering on capillary surface when the laser resource pass though the capillary wall and reach the detection window in the capillary, for which that the excitation light excited analytes from outside of the capillary at the detection window, which could result in high background noise and low detection sensitivity. At the same time, fluorescence intensity would be weaken when detect the fluorescence from outside of the capillary at the detection window. To avoid above problems, and develop some new capillary electrophoresis detection system and establish highly sensitive method, based on research of our group, two parts researches as follow were performed.1. A simple, rapid and sensitive method was developed for routine analysis of riboflavin in beverage, green tea and urine by capillary electrophoresis with in-column optical fiber laser-induced fluorescence detection (LIF). The difference between the novel detector we used and others was the laser resource guided into the capillary right at the detection window by an optical fiber. The laser resource of maximum wavelength 474 nm excited directly the analytes in the buffering solutions of detection window. The detector avoided the light reflecting scattering and refracting on capillary surface and improved detection sensitivity and decreased background noise. Riboflavin (RF), commonly called vitamin B2, is a water-soluble vitamin. Benefiting from intrinsic fluorescent nature, RF can be directly detected at very low amounts using CE-LIF.The electrophoresis was performed in an uncoated fused-silica capillary (50cm×100μm I.D.) with a running buffer of 10.0 mmol·L- 1 borate at pH 9.6 and an applied voltage of 18 kV. All HPCE procedures were conducted at room temperature. A 474nm diode-pumped solid state blue laser was used as the excitation light source. All solutions and samples detected by the home-building in-column optical fiber laser induced fluorescence detector.Under the conditions described above, the regression equation of standard curve was y = 71.44±1.72 x + 41.25±0.24 with r2= 0.998 for 95% confidence interval of the regression equation, where y is the peak height (mV) of the fluorescence of RF and x is the concentration (μmol·L- 1) of RF. The limit of detection (LOD) of the method was 3.0 nmol·L- 1 obtained by with 99% level of confidence according to regression analysis and s.d. of blank. The relative standard deviations were all less than 5.0%. The linear ranges were from 0.05-20.0μmol·L- 1. The recoveries of the method for determination of riboflavin were 95.3±2.9, 105.5±3.9 and 94.3±1.7 percent for the beverage, green tea and urine samples, respectively. The reproducibility of the migration time and the peak height were 5.48±0.07min. (RSD=1.30%, n=5) and 142.7±3.7 (RSD=2.98%, n=5), respectively. At the same time, the typical electropherogrames of RF 8.0nmol·L- 1 was illustrated. In order to verify the accuracy of the proposed method, the content of RF in beverage and green tea samples were analyzed and list by the fluorometric method of American Organization of Analytical Chemists (AOAC). To investigate the metabolism of RF in healthy body, all electropherogrames of urine samples and corresponding concentrations of riboflavin in the period of 13 h after the ingestion of vitamin B2 tablets were illustrated.2. The conventional laser induced fluorescence detector must make use of laser as the excitation source, such as argon ion and helium-cadmium laser. These lasers suffered from limit of alternative excitation wavelength, expensive cost, bulky volume, short lifetime and high power consumption, which limited the application of capillary electrophoresis laser induced fluorescence detection methodology. A LED induced fluorescence (LIF) detection system of CE has been constructed. The novel detection window was design with in-column optical fiber and sliver film gathering light by reflection. In this reach, the 452 nm wavelength of LED was choice. Coating a uniformity and smooth sliver film onto the half of detection window, the sliver film was formed a concave mirror for the half capillary was concave. Befitting the concave mirror, the light resource and the fluorescence of the analytes could be gathered and detected. As a result, the sensitivity of the detector was increased. Determination of riboflavin by capillary electrophoresis with in-column optical fiber and sliver film gathering light LED induced fluorescence detection. Comparing with the detector without sliver film, the concentration of the riboflavin decrease five times when S/N=3.
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