| Pressurized capillary electrochromatography (pCEC) based on the theory and technique of capillary electrochromatography (CEC) is emerging as an environmental friendly micro-separation technology in recent years. Compared with capillary zone electrophoresis (CE), where only charged compounds can be separated,bothcharged and neutral analytes can be separated simultaneously in pCEC. Moreover, pCEC can provide higher column efficiency than high performance chromatography (HPLC). Even more importantly, compared with traditional CEC, gradient elution mode can be achieved and some problem associated withJoule heating effect, like bubble formation and dryout of the column can bealsoovercome in pCEC due to the introduction of pump pressure. The appearance of pCEC followed the trend of high performance and miniaturization in the development of modern analytical instrumention. Thus, pCEC have been applied to the analysis of more complicated samples in life science, traditional Chinese medicine research, drug analysis, environment monitoring and food safety, as well as other popular research fields.Evaporative light-scattering detector (ELSD) has been widely used as a universal detector for HPLC since the1990s. The application of ELSD doesn’t depend on the molecular structure and optical properties of analytes. ELSD only requests the evaporitivity of analytes to be lower than that of the mobil phase. Recently, ELSD has displayed the huge advantage for the detection of natural products, Chinese herbal medicines, lipids, surfactants and carbohydrates.Therefore, the aim of this dissertation focuses on the development of a platform based on pCEC coupledto micro-ELSD (μELSD)and its applications. Some key parts such as evaporation assembly, nebulization assembly, and micro light scattering module were designed. Subsequently, the interface technique of pCEC with μELSD was investigated through optimizing the operation conditions such as evaporation, nebulizer and light scattering parts. The performance of pCEC with μELSD was valuated in detail, and then a method based on pCEC-μELSD was applied successfully for the analysis of synthetic and natural sweeteners in food samples. pCEC-μELSD as an efficient and economical technology hasgreat potential for the analysis of complicated samples in various research fields。The content of this dissertation is as follows:Chapter1:The research background of pCEC and ELSD including their advances history, basic principle and application is systematically summarized. According to their characteristics, the feasibility of the coupling of two techniques was proposed. The purpose and significanceof this dissertation wereput forward。Chapter2:In order to investigate the miniaturization of the evaporative light-scattering detector and understand the mechanism of μELSD, the structure of evaporation assembly, nebulizer assembly and light scattering cell were designed in detail in this chapter. The finalized structure of micro-fluid nebulizer was composed of nebulization nozzle and nebulization capillary. The finalized mirco-evaporator was based on cylindrical conical tube which was connected to heating resistor and covered with insulation sleeve. Sheath liquid assembly equipped with supplementary carrier gas was used to connect the evaporative annular tube to light scattering detector cell. The laser and photomultiplier were used, respectively, as the light source and scattering light collector of μELSD. Light trap was also designed in the structure of μELSDin order to avoid the reflected light. The electronic circuit system that designed for μELSD can be used to adjust and monitorvarious parameters of μELSDin real time such as evaporation temperature, flow rate of carrier gas and system carrier gas pressure. Chapter3:The parameters of evaporation assembly, nebulizer assembly and light scattering cell were optimized in this chapter. The influence of nebulization nozzle and matching nebulization capillary on atomizing effect was investigated. The finalized size of nebulization nozzle and matching nebulization capillary were410μm and20μm I.D.×360μm O.D,respectively. The influenceof the position of capillary on the signal to noise ratio and repeatability was also investigated. It is indicated that the best signal to noise ratio can be abtained under the condition of10cm length,7.5mm inlet diameter and1.0outlet diameter of cylindrical conical tube. Moreover, three different light scattering detector cells were made to evaluate the influence of detector cell size on the signal to noise ratio. Finally, the largestone(65mm i.d.x60mm high)was choosen to increase the signal to noise ratio. Therefore, the detection limit of μELSD was caculated as0.1ng (S/N=3, direct injection with glucose sample),the noise of baseline was0.8μV,the baseline drift was0.182mV/h, the reproducibility of peek aera (RSD6) was0.4%, and the reproducibility of peek height(RSD6) was0.3%. The platform of pCEC coupling toμELSD was built successfully.Chapter4:Optimization and validation of pCEC with μELSD detector were processed. A method base on pCEC-μELSD was developed for the determination of a mixture of fructose, glucose and sucrose in food. TSK Amide-80capillary column (150mm x200μ m,5μm) was selected. The analytes can be separated within13min under-3kV of operation voltage. The mobile phase was chosen as80%acetonitrile in DI water. The linear range of the method was from0.01to1.0μg for each analytes. The detection limit was4ng. Recoveries within88.1%-103.9%and RSD within0.9%-3.3%(n=6) indicated that adaptability and stability of pCEC with μELSD are comparable with commercial HPLC-ELSD.Chapter5:Based on the technique of the pCEC-μELSD, a method was established for analysis of a mixture of erythritol, xylitol, sorbitol, maltose and lactitol in the sugar-free food. Some operation conditions of ELSD such as evaporated temperature and flow rate of carrier gas and the chromatographic condition were optimized. The influence of pH value on the signal to noise ratio was also investigated. The linear range of each analyetes was from0.01to2.5μg (correlation coefficient greater than0.998).The average recovery was between82.4%-107.2%under three different level by using standard addition method and the relative standard deviation (RSD) was less than3.9%(n=6). The detection limit (LOD, S/N=3)was between4to11ng. Contrast to conventional method based on HPLC with ELSD, analysis speed was improved obviously. This method then was applied for analyses of two real samples. The proposed method provides a fast and effective method for the quantitative detection of sugar alcohol in sugar-free foods.Chapter6:Based on the technique of the pCEC-μELSD, ananalytical method was established to detect simultaneously nine common sweetener. The operation conditions of ELSD and the separation condition were both optimized. The linear range of each sweetener was from7ng to4.2μg (correlation coefficient greater than0.998).The average recovery was between85.4%-110.4%under three different level by using standard addition method and the RSDwas less than3.6%(n=6). The detection limit (LOD, S/N=3)was between7ng to42ng. This method was applied for three reall xylitol samples and the targeted analytes were separated and detected accurately. The proposed method provides a sensitive and stable means for the determination of two kinds of sweetener simultaneously. |
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