Study Of The Methods For Microscale Separations And Analysis

Miniaturizing of the analysis system has been an important task for the development of analytical chemistry. In the miniaturized system, high detection sensitivity and high sample throughput can be obtained, the analysis cost and reagent consumption can be greatly reduced, and therefore integrated device for automated analysis may be possible. In recent years, microfluidic-based technique has attracted extensive research interest. Application of the microchips in various analytical fields and development of the function-integrated microchips have been of great desire. The goal of this research is to establish the new separation method which is based on microfluidic electrophoresis chips, and to develop the microfluidic separation, detection and sample pretreatment methods for the future analysis on integrated microchips. The research work in this thesis is divided into five chapters.In Chpter 1, micro analysis techniques are summarized. Capillary-based separation methods, including capillary liquid chromatography, capillary electrophoresis and microfluidic multi-dimensional separation methods are introduced, and corresponding detection techniques for sensitive detection are included. Sample pretreatment methods in microchannels, such as sample purification, preconcentration and derivatization methods are reviewed. The research background is demonstrated.In Chapter 2, a novel comprehensive two-dimensional separation system that couples capillary high performance liquid chromatography (cHPLC) with microchip electrophoresis (chip CE) is first devised and demonstrated. Reversed phase cHPLC is used as the first dimension, and chip CE acts as the second dimension to perform fast sample transfers and separations. A valve-free gating interface is devised simply by inserting the outlet-end of LC column into the cross-channel on a specially designed chip. A home-made Confocal laser induced fluorescence detector is used to perform on-chip high sensitive detection. The cHPLC effluents are continuously delivered to the chip and pinched-injections of the effluents every 20 seconds are employed for chip CE separation. Gradient elution of cHPLC is carried out to obtain the high efficiency separation. Free zone electrophoresis is performed with triethylamine buffer to achieve high-speed separation and prevent sample adsorption. Such a simple-made comprehensive system is proved to be effective. Peptides of the FITC-labeled tryptic digests of bovine serum albumin are fairly resolved and detected with thiscomprehensive two-dimensional system.In Chpter 3, capillary electrophoresis (CE) separation and laser induced fluorescence (LIF) detection methods are developed for the analysis of doping reagents. In the great sports games, abuse of doping reagent has become an outstanding problem and it is very necessary to develop the fast, sensitive and automated methods for high throughput doping analysis. Integrated microfluidc operations on a chip can provide a new way to realize such an aim. In order to fulfill the miniaturizings and integrations, it is necessary to develop first the analysis methods in capillaries. In this chapter, LIF detection methods, either in direct mode or in indirect mode, are developed for the detection of doping reagent in the capillaries, which provides the possibility for the future detection in microchips. Capillary zone electrophoresis (CZE) separation with direct LIF detection for the determination of fluorescein isothiocyanate (FITC) derivatized stimulants (ephedrine, phenylpropanolamine and heptaminol) is demonstrated. Stimulants in human urine are extracted by organic solvents and then derivatized with FITC. Low concentration stimulants of 22 ng/mL in human urine can be detected within 10 minutes. Operation conditions for derivatization and separation are discussed. Micellar electrokinetic chromatography (MEKC) separations for FITC-derivatized amphetamines are also investigated. Meanwhile, indirect LIF detection methods are developed for the detection of four acidic diuretics separated by CZE. With an optimized electrophoretic buffer system, fast separation of four diuretics (ethacrynic acid, chlorthalidone, bendroflumethiazide and bumetanide) can be performed within 3 minutes with the detection limits of 0.2-2 μg/mL. The impacts of buffer components including the concentrations of the electrolytes, fluorescence probe and the organic additives are demonstrated.In Chpter 4, sample pretreatment methods employing capillary solid phase extraction and solid phase derivatization are developed for the CZE-LIF detection of ephedrine in human urine. The novelty of this research includes: 1), with UV-initiated polymerization and slurry packing methods, solid supports with certain bed length for sample pretreatment can be immobilized in the capillaries at any desired location, suggesting that the solid supports may be preparared in microchannels without any complex micromachining process. 2), dual phase solid supports for sample pretreatment are demonstrated for the first time. By coupling porous polymer monolith with chromatographic packing materials, high efficiencies for solid phase extraction and derivatization are obtained. 3), solid phase derivatiozation of ephedrinewith FITC are realized for the first time. Compared with solution derivatization, higher derivatization efficiencies can be obtained in the solid supports and the extraction and derivatization can be operated in sequence with the same solid supports. 4), microwave assisted solid phase derivatiozation technique is presented for the first time. High efficiency of solid phase derivatization and high speed of microwave assisted reaction are coupled. The derivatization time is shortened from several hours to several minutes. In this chapter, three kinds of capillary pretreatment columns are prepared and tested for the pretreatment of ephedrine in both water solutions and urines. It is proved that precolumns with porous polymer/GDX-502 dual solid supports are most suitable. With the optimized operation process, ephedrine with the absolute mass of 10 ng in urine can be purified, preconcentrated and detected.In Chapter 5, an initial research for the determination of amino acid sequence in proteins is reported. Edman degradation reaction is modified and miniaturized in the capillaries packed with solid supports. By using of novel coupling reagent FITC, the degradation products can be analyzed by fast CZE and sensitive LIF detection. The miniaturizing process is explored and the detection sensitivity is demonstrated. This research can provide the possibility of realizing the integrated Edman degradation and sequencing methods in the microchannels.To sum up, this thesis focuses on the development of novel methods for microfluidic analysis. Novel 2-d separation system coupling capillary HPLC and microchip CE is established and the integrated analysis methods of sample pretreatment, separation and detection for doping reagents are developed. Miniaturized solid phase Edman degradation for the determination of protein sequence is explored.