| Capillary electrophoresis (CE) is a powerful liquid phase separation technique. It has been increasingly recognized as an important analytical separation technique due to its easy operation, ultra-small sample volume, high separation efficiency and environmental friendliness. The theory and application research of CE has been flourishing in wide areas such as biochemical analysis, food analysis, pharmaceutical synthesis, environmental monitoring and medical evaluation etc. However, its detection sensitivity is seriously limited due to the small volume sample injection, short optical path length as well as detector restrictions, which can not meet the needs for the analysis of trace components in real-world samples.So far, the main research subject in CE is to improve the detection sensitivity. Amperometric detection (AD) coupled with CE has many advantages over other detection methodologies in that it provides good selectivity and high sensitivity, and easy manipulated procedure and so on. Recently, many researchers have been focusing on the online sample pre-concentration techniques to enhance CE sensitivity. Therefore, it would be a promising strategy for the separation chemistry, if an online hyphenated sample stacking approach coupled with CE-AD could be established.In this paper, we mainly focus on the development of CE-AD system to analyze biological samples (serum or urine). After surmounting some difficulties in electrochemical detection, we do some further researches via hyphenation of online pre-concentration techniques coupled with CE-AD to improve detection sensitivities. The detailed profile in this dissertation is shown as follows:1. Brief introduction of CE.In this part, we summarized the development and theory background of CE technology, involving its separation models, various detection methodologies, as well as many online pre-concentration techniques utilized in CE. 2. Simultaneous determination of neuroactive amino acids in serum by capillary zone electrophoresis with amperometric detectionA quantitative determination of six neuroactive amino acids (NAAs) was performed by capillary zone electrophoresis with amperometric detection (CZE-AD). This CZE-AD method utilized two electrolytes:the borate solution flowing in a capillary has the NAAs-separation effects, and the sodium hydroxide (NaOH) solution filled in the detection reservoir for the amperometric analysis of NAAs. The following experimental parameters were optimized:the working’electrode potential, the pH value, the component, and the concentration of running buffer, the separation voltage, and the injection time on CZE-AD. Then, under the optimum conditions, the six NAAs could be completely separated in30min and had well-shaped AD responses at0.75V (versus SCE) on a copper electrode. The linear calibration range of NAAs was from5×10-4to5×10-6mol L-1with the limits of detection (LODs) ranging from10"6to10-7mol L-1(S/N=3), and the relative standard deviations (RSDs) of the migration time and peak area were0.45～0.55and3.8-6.3%, respectively. Moreover, this method has succeeded in human serum analysis, and the determined contents of the six NAAs in human serum were in an average recovery range of85.3～117.9%, which confirmed the validity and practicability of this method.3. Sensitive measurement of polyols in urine by capillary zone electrophoresis with amperometric detection using on-column complexation with borateLittle is known about human polyol metabolism, but recent studies indicate that abnormal polyol concentrations in body fluids are related to several diseases. In this study, a rapid and sensitive method for the determination of seven major polyols in urine including two groups of polyol isomers, C5-polyols (Rib+Arb+Xyl) and C6-polyols (Sor+Gal+Man), was developed using capillary zone electrophoresis with amperometric detection (CZE-AD). The effects of the working electrode potential, pH, running buffer components and concentrations, separation voltage and injection times were investigated. Under the optimised conditions, seven types of polyols could be perfectly separated via the formation of anionic polyol-borate complexes in a borate buffer solution. Highly linear current responses to the polyol concentrations were obtained with good correlation (0.9984<R2<0.9997), and the LODs ranged from1.33×10-6to5.8×10-7mol L-1(S/N=3). The proposed method has been successfully used to detect polyols in urinary samples from healthy subjects and diabetes patients, demonstrating accurate and reliable results. This method has potential applications in the recognition of inborn errors affecting polyol metabolism.4. Sensitive analysis of aminoglycoside antibiotics via hyphenation of transient moving substitution boundary with field-enhanced sample injection in capillary electrophoresisA novel field-enhanced sample injection (FESI) coupled with a transient moving substitution boundary (MSB) method in capillary electrophoresis (CE) was developed for aminoglycoside antibiotics (AGs) analysis using18-crown-6-tetracarboxylic acid (18C6H4) as a pseudostationary phase. Results indicated that the stacking mechanism of MSB relied on the substitution reaction between18C6H4-bonded AGs complexes and Na+at the substitution boundary. The stacking mechanism as well as important parameters governing pre-concentration and separation have been investigated in order to get maximum resolution and sensitivity. Under optimised conditions, using a sample prepared in a low-conductivity matrix, the limits of detection (LODs) for streptomycin, neomycin, and kanamycin were0.62,5.9and8.6nmol L-1(S/N=3), respectively, and the detection sensitivities were improved940,692, and415folds, respectively. The method also gave accurate and reliable results in the analysis of aminoglycoside antibiotics in river water samples. 5. Combination of field-amplified sample stacking and transient trapping for amino acids stacking using capillary electrophoresis-amperometric detectionWe reported a novel field-enhanced sample injection (FESI) integrated transient trapping (Tr-trapping) for the sensitive analysis of amino acids using capillary electrophoresis (CE) coupled with amperometric detection (AD). Analytes was prepared in low-conductivity phosphate buffer (pH=2.62), which allowed for the introduction of a large amount of sample into the capillary. Tr-trapping occurred by the partial-filling (PF) of the pseudostationary phase (PSP) plug before the sample injection. The enrichment mechanism and important parameters governing both stacking and separation were all investigated. With the linear calibration ranging from2.5×10-4to1.25×10-6mol L-1, this novel FESI-Tr-trapping method allows for the limits of detection (LODs) of amino acids at very low concentrations (7.0～67.4nmol L-1) and yields10-to44-fold inprovement in detection sensitivity.6. On-line sample preconcentration technique for analysis of biogenic amines in urine based on dynamic pH junction in capillary electrophoresis-amperometric detectionWe report an on-line sample preconcentration method for simultaneous analysis of six biogenic amines (dopamine, epinephrine, norepinephrine, tyramine, tryptamine and serotonin) based on a dynamic pH junction coupled with capillary electrophoresis-amperometric detection (CE-AD). The technique is predicated on a sharp pH junction generated at the boundary between acidic sample and basic background electrolyte (BGE) zone. Under optimised conditions, all analytes could be successfully focused and well separated within20min, with high efficiency and sensitivity (LODs at S/N=3ranging from5.34to68.3nmol L-1). This method was first applied to urinary sample analysis and obtained satisfactory recoveries, without off-line preconcentration or derivatization process. The results suggest the potential of this approach for the analysis of real biological samples. |
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