Study Of On-Column Preconcentration And Two Dimensional Capillary Electrophoresis Technology And Application

In this paper, a novel dual concentration method, sweeping with electrokinetic injection and analyte focusing by micelle collapse, has been developed. Then the new dual concentration method was applied to the two-dimensional (2D) capillary electrophoresis (CE) for analysis of complex samples for the simultaneous increasing of sensitivity, resolution and resolving power. The integrated concentration and 2D-CE method was successfully applied to the concentration and separation of flavoids in Herba Leonuri sample. Moreover, a new method based on gold nanoparticle amplification of capillary electrophoresis was developed, and applied to the enrichment and seperation of Escherichia coli (E.coli) in scallop extract. The thesis concludes five chapters:The first chapter is the preface overview of capillary electrophoresis. Firstly the basic principles of capillary electrophoresis and progress was introduced, citing the common mode of capillary electrophoresis separation methods and sample introductions.Then the capillary electrophoresis detection method - optical detection and electrochemical detection was introduced. Subsequently, on-line samples preconcentration methods which can improve the detection sensitivity of CE were described in detail, inclouding isotachophoresis, stacking and sweeping techniques. Finally, the two-dimensional capillary electrophoresis separation techniques were described.In chapter two, capillary zone electrophoresis (CZE) was applied to determine eight flavonoid compounds in Herba Leonuri, named apigenin, kaempferol, quercetin, phlorizin, hyperoside, quercitrin, rutin and hesperetin. The effect of several parameters was investigated, including voltage detection, separation voltage, the running buffer pH, running buffer concentration and so on. Sodium phosphate buffer was selected for its best resolving power. Under the optimum conditions, the 8 analytes were well separated within 20 min at a separation voltage of 15 kV and a detection potential of 0.85 V in a 40 mM Sodium phosphate buffer (pH 8.5). the detection limit (S/N = 3) was 1.14×10^-7-7.85×10^-7 g/mL.In chapter three, micellar electrokinetic capillary chromatography (MEKC) was employed to determine mentioned eight flavonoid compounds in Herba Leonuri. The important factors, such as pH, running buffer concentration, injection time, were optimized. We chose 90 mM sodium phosphate solution (pH 7.0) containing 5 mM sodium dodecyl sulfate(SDS) as buffer solution. At the separation voltage 15 kV and the detection potential of 0.85 V, the eight substances in real samples with other impurities peaks were well separated.In chapter four, a novel integrated concentration/separation approach involving online combination of sweeping with electrokinetic injection and analyte focusing by micelle collapse (AFMC) with heart-cutting twodimensional (2D) capillary electrophoresis (CE) in a single capillary was developed for analysis of Herba Leonuri. First, a new sweeping way with an electrokinetic injection preconcentration method was developed to inject a large volume sample solution and significantly enhanced detection sensitivity. Then, the preconcentration scheme was integrated to the 2D-CE to provide significant analyte concentration and extremely enhance resolving power. The sample was preconcentrated by sweeping with electrokinetic injection and separated in first dimension micellar electrokinetic chromatography (MEKC). Then, only a desirable fraction of the first dimension separation was transferred into the second dimension of the capillary by pressure and further analyzed by capillary zone electrophoresis (CZE) acting as the second dimension. As the key to successful integration of MEKC and CZE, an AFMC step was integrated between the two dimensions to release analytes from the micelle interior to a liquid zone and to overcome the sample zone diffusion caused by mobilization pressure. The injected sample plug length for flavonoids under 15 kV for 60 min was experimentally estimated as 546 cm. The dual concentration methods resulted in the increased detection factors of 6000-fold relative to the traditional pressure injection method. The relative standard deviation (RSD) values of peak height, peak area, and migration time were 2.7-4.5 %, 1.9-4.3 %, and 4.7-6.8 % (n = 10), respectively. The limits of detection (S/N = 3) were in the range of 7.3-36.4 ng/L, and the theoretical plate numbers (N) were in the range of 1.7-4.3×10⁴ plates/m. This method has been successfully applied to determine flavonoids in Herba Leonuri. The pharmacokinetic study also demonstrated that the proposed concentration/separation method was convenient and sensitive and would become an attractively alternative method for online sample concentration and separation in complex samples.In chapter five, we use enrichment of nano-gold amplification with capillary zone electrophoresis (CZE) electrochemical (EC) detection to detect Escherichia coli (E.coli). We added a diameter of 10 nm of nano-gold to the solution of E. coli to achieve enrichment, and found that the followed detection signal of E. coli could be increased significantly. The CZE was performed in a running buffer of a TBE buffer (4.5 mmol/L Tris-4.5 mmol/L H₃BO₃-0.1 mmol/L EDTA, pH 8.4), and other experimental conditions were as follows: a detection potential of -0.7 V, the separation voltage 12 kV, electrokinetic injection 10 s and a injection voltage of 15 kV. Under optimal conditions, the E. coli in scallop extract was successfully detected.