Study On The Application Of Indirect Capillary Electrophoresis With Amperometric Detection

Capillary electrophoresis (CE) is a kind of liquid phase separation technology which is also called as high performance capillary electrophoresis (HPCE). It has the characteristics of high efficiency, high speed, minimal consumption of solvent, etc. CE has been used to separate inorganic ions, organic acids, DNA, proteins, biological macromolecules and enantiomers, etc. It has great potential to be applied in analytical chemistry, biological chemistry, food chemistry, environmental chemistry, clinical chemistry and so on. In this part, the history of CE development has been retrieved, and the principle, the separation modes, the instrumental system, the technological characteristics of CE, and especially the method of CE with amperometric detection (CE-AD) have been introduced. According to the research area of this thesis, the application of CE in food and clinical analysis has been reviewed.In this thesis, indirect CE-AD has been used for the determination of preservatives in foods, the mark compounds of phenylketonuria (PKU) in urine samples of infants, as well as lactic acid content in body fluids of lactating postpartum women.A novel capillary electrophoresis and amperometric detection method was achieved by adding an electroactive additive (3,4-dihydroxybenzylamine,3,4-DHBA) to the running buffer, so that both electroactive and non-electroactive food preservatives were simultaneously determined. The end-capillary 300μm diameter carbon disc electrode offers favorable signal-to-noise characteristics at potential of +1100 mV (vs. SCE). Under the selected optimum conditions, four electroactive preservatives (methylparaben, ethylparaben, propylparaben and butylparaben) and two non-electroactive preservatives (potassium sorbate and sodium lactate) were well separated in a 75 cm length of 25μm i.d. and 360μm o.d. fused silica capillary at a separation voltage of 16 kV in a 40 mmol/L borate running buffer (pH 8.2) containing 12 mmol/L SDS and 0.018 mmol/L 3,4-dihydroxybenzylamine (DHBA). The detection limits (S/N=3) rang from 1.06×10-8 to 2.73×10-6 g/mL. This method has been successfully employed for the determination of both electroactive and non-electroactive preservatives in several food commodities.An indirect capillary electrophoresis with amperometric detection (CE-AD) has been developed for the simultaneous determination of the mark compounds of phenylketonuria (PKU), including electroactive phenylpyruvic acid and non-elecroactive phenylacetic acid, in infant urine samples. Several important parameters such as running buffer additive, sodium dodecyl sulfate (SDS) concentration and working electrode potential were investigated. Under the optimum conditions, phenylpyruvic acid and phenylacetic acid could be well separated in 60 mmol/L borate running buffer (pH 7.8) containing 20 mmol/L SDS and 0.018 mmol/L 3,4-dihydroxybenzylamine (DHBA). The detection limits (S/N=3) were 0.50 and 0.92 mg/L, respectively. The concentrations and peak heights of phenylpyruvic acid and phenylacetic acid were in good linearities in the range of 3.0-1.0×10-3 mg/L and 2.0-5.0×10-3 mg/L, respectively. The RSD of the peak heights and migration times for two marked compounds were 4.4%,3.2%(phenylpyruvic acid) and 2.2%,4.3%(phenylacetic acid), respectively; and this method is interference free from the coexisting compound uric acid in urine samples. The proposed method has been successfully applied for the simultaneous determination of two PKU mark compounds in infant urine samples after simple preparation, which could provided an alternative for the early noninvasive diagnosis of PKU infants.An indirect high-performance capillary electrophoresis with amperometric detection (CE-AD) method has been developed for the determination of lactic acid (LA) in body fluids of lactating postpartum women. Several important factors such as the running buffer additive and concentration, the working electrode potential, the pH value and concentration of running buffer, the separation voltage and injection time were investigated. Under the optimum conditions, LA could be well separated with co-existing interferences including uric acid (UA) in real samples in a 90 cm length capillary at a separation voltage of 12 kV in a 4.0×10-6 g mL-1 3,4-dihydroxybenzylamine (DHBA)/40 mmol/L H3BO3-Na2B4O7 buffer (pH 7.8). The linearity between peak current and concentration of LA was over three orders of magnitude with detection limit of 5.0×10-7g mL-1 (S/N=3). This proposed method has been successfully used for the study on the effects of moderate exercise on LA content in the breast milk and urine samples of lactating postpartum women, and the assay result showed that the LA content in breast milk and urine can return to preexercise level through 60 min and 120 min rest, respectively, although the LA level does increase (usually 4-6 times) both in breast milk and urine samples at 10 min postexercise.