Study On Capillary Electrophoresis With Laser-induced Fluorescence And Its Application On Environmental Contaminants

Capillary electrophoresis (CE) is an important and powerful technique in the area of liquid-phase separations. The separation principle is based on the phenomena that ionized molecules of different mass or/and charge will travel with different velocities through a fused silica capillary under the force of a high-voltage electric field. Due to the different velocities, the analytes can be separated and then detected by the detector. Thanks to its high separation efficiency, short analysis time, low cost, environmental friendliness and small sample consumption, CE has been widely flourishing in many areas such as food analysis, pharmaceutical analysis, biochemical analysis, and environmental analysis etc. The prominent advantage of laser-induced fluorescence is its high sensitivity. CE-LIF combins their merits so that fits to the trace analysis of biological samples and environmental samples.Our living environment became worse and worse with the development of economy. Brominated flame retardants (BFRs) are used in a number of industries in the manufacture of polymers and synthetic resins to reduce the risk of fires. Recently, for BFRs especially TBBPA, great concern has been aroused because of their wide spread, stable structures and uncertain risk to human health. But research on neurotoxicity of BFRs is still less. Pesticide residue is also an important environment problem which caused contamination to water, soil, air and so on. Food security derived from pesticide residue is worrying.In this dissertation, we laid our emphasis on the research of environment pollution and its effects and established methods to determine glutathione (GSH and GSSG) and thyroid hormone (T4 and T3) after rats'exposure to TBBPA, and carbamate pesticides by capillary electrophoresis (CE) method coupled with laser-induced fluorescence (LIF) detection. The dissertation includes four chapters:Chapter 1. IntroductionIn this chapter, development, the principle and applications of capillary electrophoresis were outlined. The characteristics of BFRs and pesticide residue were introduced, and the applications of capillary electrophoresis in environmental analysis were highlighted, as well as the meanings of this paper.Chapter 2. Determination of GSH and GSSG by Capillary Electrophoresis with Laser-Induced Fluorescence Detection and its application on the Rats after Exposure to TBBPAGSH and GSSG play a great role in some biological process, and GSH/GSSG is a biomarker of oxidative stress. We study the neurotoxicity of TBBPA through determination GSH and GSSG in rats'brain and serum. A capillary electrophoresis (CE) method coupled with laser-induced fluorescence (LIF) detection was developed for the separation and detection of GSH and GSSG after derivatization by fluorescein isothiocyanate (FITC). The separation and detection conditions were optimized in terms of buffer concentrations, buffer pH, separation voltage and injection time. Under the optimum experiment conditions, GSH and GSSG were derivatized with FITC in 50 mmol/L pH 9.9 of borate buffer and separated in 50 mmol/L pH 9.7 of borate buffer. Linear relationships between the peak area and concentrations of derivatized analytes were obtained (3-1000 nmol/L). The detection limits for GSH and GSSG were 1.0×10-9mol/L and 3.0×10-9mol/L(S/N=3), respectively. The method was applied to analysis of GSH and GSSG in hippocampus of rats after exposure of TBBPA.Chapter 3. Determination of T4 and T3 by Capillary Electrophoresis with Laser-Induced Fluorescence Detection and its application on the Rats after Exposure to TBBPAThe amino acid type thyroid hormones, thyroxine (T4) and triiodothyronine (T3), secreted by the pituitary gland are compounds of critical importance in regulating a number of biological processes. In this paper, a method based on capillary electrophoresis with laser-induced fluorescence has been developed for the simultaneous separation and determination of T4 and T3 after derivatization with FITC. The derivatization conditions were optimized in terms of buffer systems, ratios of derivatization, buffer concentrations and buffer pH. And the effects of several important factors about separation were also investigated, such as buffer concentrations, buffer pH, separation voltages and injection time. Under the optimum conditions, T4 and T3 were derivatized in phosphate buffer (pH 9.2,25 mmol/L), and then separated within 10 min in pH 8.9,40 mmol/L borate buffer containing 15% acetonitrile (v/v). The detection limits were as low as 3.0×10-9 mol/L and 1.0×10-9 mol/L (S/N=3) for T4 and T3, respectively. The method has been applied to analyze T4 and T3 in rats'serum and pituitary to study the toxicity of BFRs.Chapter 4. Dtermination of the rate constants and activation energy of carbamate pesticides hydrolysis by capillary electrophoresis with laser-induced fluorescence detectionCarbamates are an important pollutants found in various environmental matrices. A capillary electrophoresis (CE) with laser-induced fluorescence (LIF) method was used for determination of hydrolysates of (3,5-dimethylphenyl)N-methylcarbamate(XMC), 2,3,5-trimethacarb and mercaptodimethur. The method was based on simultaneous hydrolysis and derivatization reaction between the produced methylamine and 4-chloro-7-nitrobenzo-2-oxa-1,3-diazol (NBD-Cl). Effects of several key factors, such as time and temperature for hydrolysis and derivatization, pH values and concentrations of both running buffer and derivatization buffer, voltage applied and injection time were all investigated to obtain the optimum conditions for determination. The three analytes were respectively mixed with NBD-Cl in 20 mmol/L pH 9.8 borate buffer and the derivatives got good peaks in the running buffer of 50 mmol/L pH 8.5 borate buffer. The detection limits (LOD) obtained by the method were 3.0×10-9,1.0×10-9 and 3×10-9 mol/L, respectively. Relative standard deviations for the studied compounds varied from 2.22% to 3.37%(n=7).