Investigation On Chemiluminescence Assay And Capillary Electrophoresis Coupled With Chemiluminescence

Chemiluminescence (CL) is defined as the emission of light from an electronically excited state species which is produced during the course of a chemical reaction, and is observed when the electronically excited product or intermediate formed decays to the ground state with a photon emitted. There are many merits on CL such as simple cheap optical systems requiring no light sources which avoiding the effects of stray light and the instability of light source, and thus providing low background with excellent sensitivity, wide linear range, rapidity, simplicity and so on. But its weakness is thought to be its poor selectivity. Up to now, the CL analysis has been widely applied in clinical, biological and environmental fields and is becoming one of the most powerful techniques for use in analytical chemistry. The predominant development of CL analysis nowadays is focused on the development of new CL system, the combination with other techniques such as flow injection (FI), capillary electrophoresis (CE), high performance liquid chromatography (HPLC) and so on while the uses of established CL reaction systems still attract much interest.Capillary electrophoresis emerged in the early 1980's as a highly efficient means of separation, which is performed in capillary tube and driven by electric field. It brings a profound revolution following the separations of GC and HPLC and develops the analysis technology from a level of microlitre to nanolitre. And CE-CL has made it possible to analyze a single cell and single molecule. The most interesting achievements focus on the separation of protein, saccharide, chiral compounds and the DNA sequencing, as well as single cell and single molecule detection. Yet CE has potential applications in many research areas, such as molecular kinetics and early diseases diagnosis. Much attention has been given to the analysis of metal ions and small organic species since 1990's. The relatively short separation times, simple instrumentation and low operational costs make CE an attractive alternative. Nevertheless, the small volumes manipulated in capillary dimensions pose a real challenge for detectors in achieving sensitivity. The problem has created a demand for a new form of detection, which would be relatively simple, widely applicable, good sensitive, and providing quantitation.Combining CE with CL is a new approach with the high sensitivity and highselectivity for analyzing the small organic molecules in complex Chinese herbal medicines and has become the most powerful technique in analytical chemistry.In the thesis, the sensitivity and the selectivity of old CL system was improved and the new CL system was discovered, and the corresponding compounds were determined with flow injection CL method. Also, due to CL's simple optical system and low background nature, CL has become an ideal detection method for CE. CE-CL technique has received significant attention over the past several years. In this work, the new approaches for detection of some polyphenols in Chinese herbs were exploited. Because the capillary can be easily regenerated, CE is ideal for analyzing "dirty" samples. The research results obtained in the thesis are listed as follows:1. Investigation new CL system and improvement old sensitivity of CL system combined with flow injection techniqueFlow injection analysis (FIA) is now well established as a powerful sample handling method for analysis in laboratory, which is compatible with a wide range of detection systems. In its most basic form, it is a convenient means of delivering samples to detector in a controlled and reproducible manner. The combination of FIA with CL detection is becoming increasingly important in various fields because of high sensitivity, wide linear dynamic range, reproducibility, simplicity and rapidity.1.1. CTMAB enhanced CL determination of uric acid using luminol-K3Fe(CN)6-K4Fe(CN)?i systemA CL method using FI was developed for the determination of uric acid based on the enhancement CL intensity of luminol-K3Fe(CN)6-K4Fe(CN)6 in the presence of CTMAB and the uric acid species. The linear range was 7.0×10-10 -9.0×10-7mol/L with a detection limit (3σ) of 2.58×10-10mol/L, which was about two orders of magnitude lower than that reported in literatures. The proposed method was used for the determination of uric acid in real samples. And the possible mechanism is proposed.1.2. Investigation of the photodecomposition of hemin and trace amount determination of hemin with FI-CL methodUsing H2O2-Na2CO3-NaOH system, the study on the photodecomposition behavior of hemin under ultraviolet light and solar light were carried out and thedetermination of hemin was developed coupled with simple flow injection technique. The results showed that the decomposition reaction of hemin in different light irradiations corresponded with a first-order reaction. And then the determination of hemin was completed by the CL emission from the reaction of hemin with H2O2 in aqueous carbonate. The linear range was 2.20×10-10 to 6.88×10-7mol/L and the detection limit was 2.2×10-11 mol/L (3a). The relative standard deviation (RSD) was 2.82% for ten independent detections of 1.72×10-8 mol/L hemin. The proposed method was successfully applied for the analysis of hemin in pharmaceutical formulations and animal blood with a recovery of 96-108%. A possible CL mechanism of the present system was discussed, and free radicals were suggested to be involved in this reaction.1.3. Photolytic kinetics study and determination of hemin by FI-CLA FI-CL method has been developed and applied to the study of the photolytic kinetics of hemin and the determination of hemin. The proposed method was based on the luminescent properties of H2O2-RhB-NaOH system and the addition of SDS as photostability-stabilizer and emission-sensitizer. The results showed that the decomposition reaction of hemin in the presence and absence of SDS under ultraviolet light corresponded with a first-order reaction. However, the presence of SDS in the system decreased the photolytic rate of hemin 1.8 times than that of in the absence of SDS. And then the hemin was determined in real samples. A possible CL mechanism of the present system was discussed, and free radicals were suggested to be involved in this reaction.1.4. Determination of tetracycline, chlortetracycline and oxytetracycline by flow-injection with inhibitory CL detection using copper (II) as a probe ionThis work reported an indirect FI method for the determination of the tetracyclines(TCs) drugs tetracycline(TC), chlortetracycline(CTC) and oxytetracycline(OTC) using Cu(II) as a probe ion. The CL reaction was induced on-line and injection of the sample produced negative peaks as a result of the Cu(II) complexation by the analytes, causing the decreased CL intensity of the Cu(II)-catalysed reaction between luminol and H2O2. The heights of the peaks were proportional to the drug concentrations. The linear range was 3.6×10-8-1.0×10-5, 1.1×10-7-1.0×10-5 and 1.9×10-7-1.0×10-5 mol/L for TC, CTC and OTC, respectively. The method was applied to. the determination of TCs inpharmaceutical formulations and human urine with recoveries in the range 95-105%. A simple detection mechanism was proposed. 2. Sensitive and selective on-line CL detection in CE for the polyphenols in Chinese herbs2.1. On-line CL determination protocatechuic aldehyde and protocatechuic acid in pharmaceutical preparations by capillary electrophoresisCE with on-line direct CL detection was first used in detecting protocatechuic aldehyde (PAH) and protocatechuic acid (PA) in their pharmaceutical preparations. It was found that the weak CL produced from the reaction of luminol with ferricyanide in an alkaline solution was strongly increased by PAH and PA which was separated by CE. Parameters affecting separation process and CL detection have been examined in detail. Under the optimum conditions, the baseline separation of PAH and PA was obtained within 6 min. The RSD for the analysis of PAH and PA was less than 1.1 % for the migration time and 1.6 % for the peak height. The detection limits of PAH and PA were 7.0×10-8 mol/L and 5.0×10-8 mol/L, respectively. The proposed method has been satisfactorily applied to the determination of PAH and PA in Salvia Miltiorrhiza pharmaceutical preparations. The CL reaction mechanism was discussed.2.2. On-line enhanced CL detection of rutin and chlorogenic acid by capillary zone electrophoresisA CE with enhanced CL method has been developed for the simultaneous determination of rutin and chlorogenic acid. The electrophoretic buffer(pH 8.5) containing 8.0 mmol/L borate and 0.28 mmol/L luminol was used in separating the analytes and the post-column CL reagent was 0.125 mol/L K3Fe(CN)6 in 0.05 mol/L NaOH. The effects of other factors such as the acidity of running buffer, the applied voltage and the injection time were investigated to find the optimum conditions. The baseline separation was completed within 5 min. The detection limits (3a) of rutin and chlorogenic acid were 0.33 μmol/L (50.6 fmol) and 0.15 μmol/L (12.6 fmol), respectively, which was 2 orders of magnitude lower than that of CE-inhibited CL method. The method was applied to the analysis of rutin and chlorogenic acid in cigarettes and pharmaceutical preparations and the matrix in the sample solution have not the influence on the CL detection.2.3. CE separation and on-line CL detection of pharmacologically activecomponents with SDS as an additiveIn this work a new method for determination of five active components including rutin (RT), protocatechuic aldehyde (PAH), chlorogenic acid (CA), luteolin (LT) and protocatechuic acid (PA) has been developed based on capillary electrophoresis with CL method. In order to improve resolution, we examined the effect of SDS, methanol and acetonitrile in a running buffer solution on the electropherogram. The results showed that only the addition of SDS improved the resolution, and the addition of methanol or acetonitrile worsen the resolution and the sensitivity. The baseline separation of the analytes was obtained within 10 min. The concentration detection limits (S/N=3) were 5.5×10-8, 3.0×10-8, 4.5×10-8, 2.0×10-8 and 1.2×10-8 mol/L for RT, PAH, CA, LT and PA, respectively.2.4. On-line inhibited CL detection of glucoside and baicalin separated by CE A capillary electrophoresis with inhibited CL method was employed for thedetermination of stilbene (2,3,5,4' -tetrahydroxystilbene-2-O-β-D-glucoside) and baicalin. After being separated by CE, the analytes were determined by inhibited CL of luminol-K3Fe(CN)6. Under the optimum conditions, the baseline separation of stilbene and baicalin was obtained within 4 min. The RSD for the analysis of stilbene and baicalin was less than 0.9% for the migration time and 2.5% for the peak height. The detection limits of stilbene and baicalin were 3.0×10-6 mol/L (mass detection limit 60 fmol) and 1.2×10-6 mol/L (mass detection limit 24 fmol), respectively. This work provided a useful method for the analysis of traditional Chinese medicines.2.5. Chemiluminescence detection of molecule level polyphenol in capillary electrophoresisSingle-molecule detection (SMD) is becoming more and more popular in the scientific community and is on the threshold to become a technique for laboratory use. We report the methodology of molecule-level polyphenol based on CE-CL. The detection limit is 5.21×10-16mol/L(6 molecules) for gossypol.