| Capillary electrophoresis (CE) and high performance liquid chromatography (HPLC) are two kinds of liquid phase analysis techniques. They both are powerful separation and accurate qualification tools in drug analysis, food analysis, environment chemistry and biochemistry.Compared to the HPLC, CE administrates the merits of higher efficiency and lower assay cost. However, There are some defectives for the existing CE techniques, CE combined with ultra-violet (UV) has the poor sensitivity because the tiny sample injection volume cause the short detection light-path; CE combined with laser-induced fluorescence detector (CE-LIF) can only determine a few analytes because the limited derivatization reagent can be used, besides, the derivatization procedure also introduce the reagents interferes. CE combined with mass spectrophy (CE-MS) is an expensive technique, and now it is on trial in lab for its elevated background noise. Since the electrochemical detection (ED), especially amperometric detection (AD) can produce the direct electrochemical response to the most compounds existing in nature, including saccharides, amino acids, peptides, etc.. CE-AD technique is fast developed and used in many fields. CE-AD based on the end-column detection configuration is characterized by high sensitivity, large linear range, simple instrumental structure and lower assay cost. Now, the researches on the new-type chemical-modified electrodes with high sensitivity, the composite electrode for the multi-indexes detection, new buffer system for the CE-AD, the conjugation interface are growing interesting.As a proven qualification technique, the reversal pharse liquid chromatography combined with the ultra-violet detection is widely used for the biochemistry research. However, because the UV detector is non-selective and the biosamples are generally very complicated, the applicable pretreatment technique for the HPLC-UV should be focused on investigation to achieve the good assay result.This paper is composed of six chapters,Chapter one gave a brief introduction on CE principle and applications.In chapter two, a type of composite electrode was fabricated with Cu2O powder, carboxyl functionalised multi-wall carbon nanometer tube (MWCNT-COOH), using paraffin oil as binder. Its electrocatalysis effect to polyphenols was demonstrated by the comparative study of the carboxylic graphite powder composites. In order to optimize the composition of the composite, scanning electron microscopy (SEM) was employed for surface morphology characterization while cyclic voltammetry was used for evaluation the amperometric responses to analytes. The experiments demonstrated that the composite electrode with the proportion of Cu2O and MWCNT-COOH at 3:1 (w/w) simultaneously showed best signal enhancement effects on polyphenols and sugars in alkenine background electrolytes. As amperometric detector in CZE-AD, the selected composite electrode displayed the most adequate performance in 100 mmol·L-1 NaOH, maintaining good electrocatalytic properties to polyphenols and sugars simultaneously; lowering LODs of the polyphenols to 10-8 mol·L-1 magnitudes (S/N=3); keeping good linear in three order of magnitudes and good resistance to passivation. Finally, two tobacco samples were analysed by proposed method.Chapter three used different buffers in detecting and separating sections of CZE-AD for simultaneous determination of sugars, amino acids, epigallocatechin gallate (EGCg) and ascorbic acid in oolong tea infusion. Operated in a wall-jet configuration,100 mmol·L-1 NaOH was used in detecting cell to lead the electrocatalysis oxidation behaviors of the analytes on a 300μm diameter copper-disc electrode (working electrode); while in separating capillary, a mild alkaline running buffer consisting in a mixture of 30 mmol·L-1 borate and 40 mmol·L-1 phosphates charged and carried analytes to detecting end. The methodology research was performed for system stability and suitability. Under the optimal CE conditions, analytes could be separated within moderate time period. Good linearity between peak area and concentration existed over three orders of magnitudes; lower RSD and LOD were achieved. The oolong tea infusion was assayed and result was satisfactory.Chapter four reported on component analysis of Asparagus Officinalis Linn polysaccharide by determination of its hydrolyzed monosaccharides using CZE-AD. A copper disk electrode was used as working electrode and its diameter was investigated for matching the untreated fused silica capillary. The buffer concentration, separation voltage, injection volume had also been studied to get the optimal conditions of CZE-AD. Experimental results showed that seven monosaccharides were baseline separated by employing 120 mmol·L-1 NaOH as running buffer with excellent linear responses and satisfactory repeatability. It was found that Asparagus polysaccharide was composed of galactose, glucose, rhamnose, arabinose and xylose at a mole ratio of 17.8:5.5:1.0:14.2:17.2. Compared with other methods, the determination of polysaccharide composition by CZE-AD has the merits of rapidness, accuracy, and lower-volume sampling. The reasons of the analysis difference by different methods were also discussed. Chapter five presented simultaneous determination of four active ingredients in Naoxueshuan Tablets, including ferulic acid, protocatechuic aldehyde, caffeic acid and protocatechuic acid. The effects of working electrode potential, pH and concentration of running buffer, separation voltage and injection time on CZE-AD were investigated. Under the optimum conditions, the four analytes could be perfectly separated within 18 min. A 300μm diameter carbon-disc electrode had a good response at+0.95 V (versus SCE) for the four analytes. The response was linear over three orders of magnitude with detection limits (S/N=3) as low as 10-8 or 10-9 g/mL for the analytes. The assay results were satisfactory with recoveries in the range of 85.2-93.0% and RSD less than 3.3%.Chapter six described a isocratic reversal phase HPLC method for the quantification of adenosine nucleotides (ATP, ADP, AMP) and NAD(H) in mouse pancreaticβ-cells. The pretreatments before HPLC were focused on study by comparing the recoveries of analytes for the frequently-used extraction techniques. Perchloric acid (PCA) route is optimal extraction technique. The extract was measured by an isocratic separation on a Agela Venusil AQ C-18 reversed phase column (250mm×4.6mm,5μm) with a mixture of 90% phosphate buffer (50 mmol·L-1, pH 6.5) and 10% methanol as the eluent. UV absorbance at 265 nm was used. The limits of detection of ATP-2Na, ADP-2Na, AMP-2Na were 0.0684,0.1208,0.1024μg/mL (S/N=3) respectively; NAD and NADH were 0.095 and 0.044μg/mL (S/N=3) respectively. All analytes were linear within the concentration range of 0.04 to 1μg/mL. The overall recoveries of analytes were over 90% and RSD for peak areas were below 15%. |
