Electric field gradient focusing (EFGF) utilizes a hydrodynamic flow and an electric field gradient to focus and concentrate charged analytes and order them in a separation channel according to electrophoretic mobility. Elution can be achieved by decreasing the applied voltage or increasing the hydrodynamic flow. EFGF has the advantages of concentrating a large volume (100 micro-L) of target proteins without significant band broadening and simultaneously removing unwanted components from the sample. Two types of EFGF devices have been investigated to concentrate and separate proteins: a fiber-based EFGF device and a hydrogel-based EFGF device. Using fiber-based EFGF with UV detection, a concentration factor as high as 15,000 and a concentration limit of detection as low as 30 pM were achieved using bovine serum albumin as a model protein. I also demonstrated the potential of using fiber-based EFGF for quantitative protein analysis. Simultaneous desalting and protein concentration as well as online concentration of ferritin and simultaneous removal of albumin from a sample matrix were also performed using this fiber-based EFGF system. In the approach of utilizing hydrogel-based EFGF, online concentration of amyloglucosidase indicated a concentration limit of detection of approximately 20 ng/mL (200 pM) from a sample volume of 100 micro-L. Both voltage-controlled and flow-controlled elution methods were demonstrated using a 3-component protein mixture. Concentration of human α1-acid glycoprotein with simultaneous removal of human serum albumin was also described. A tandem EFGF system, which integrates fiber-based and hydrogel-based EFGF sections, was also investigated to selectively concentrate and separate proteins in a mixture. By carefully controlling the voltages applied to both sections, charged analytes with high mobilities were trapped in the fiber-based section, analytes with intermediate mobilities in the hydrogel-based section, and analytes with low mobilities not at all. A 3-way switching valve was incorporated in the system to purge the analytes with high mobilities periodically. Selective concentration and separation of myoglobin from a mixture were performed using the tandem EFGF system. Based on the experimental results described in this dissertation, EFGF shows potential for selective isolation, concentration, and quantitation of trace analytes such as proteins in biomedical samples.

Polymeric materials have seen increasing use as microfluidic device substrates due to their low cost and the simplicity of templated fabrication procedures. I showed that poly(methyl methacrylate) (PMMA) microdevices could be enclosed in a boiling water bath, which allowed the seal to form more quickly than in conventional approaches, and enabled microchannels to remain hydrated throughout the bonding process. Microchip capillary electrophoresis (µ-CE) devices were fabricated using water-based enclosure, and a mixture of fluorescently labeled amino acids was separated in 30 s in these microchips. To create more robust capillary electrophoresis (CE) microdevices with improved separation performance, phase-changing sacrificial materials were developed for solvent bonding of polymer microchips. Devices were fabricated by filling channels in embossed PMMA with a heated liquid that formed a solid sacrificial layer at room temperature. The sacrificial material prevented the bonding solvent and softened PMMA from filling the channels. Once the sealing step was finished, the sacrificial layer was melted and removed, leaving enclosed microchannels. These solvent-welded devices withstood internal pressures >2,200 psi, and 300 CE runs were performed on a single microchip without any loss of separation performance. Furthermore, CE separations of peptides and amino acids were completed in ~10 s, with peak efficiencies of 43,000 theoretical plates. Electric field gradient focusing (EFGF), which uses a combination of pressure-driven flow and an electric field gradient to separate charged species according to their electrophoretic mobilities, was explored for protein analysis. Capillary-based EFGF devices were characterized; mixtures of four proteins were resolved, band focusing dynamics were studied, and analytes were enriched 10,000-fold. EFGF was miniaturized further to a microfluidic platform. Phase-changing sacrificial layers were employed to interface an electric field gradient enabling semi-permeable copolymer with microchannels. Because of decreased channel dimensions, EFGF microchips produced narrower bands and yielded threefold higher resolution compared with capillary-based devices. Beyond providing improved performance for polymer-based µ-CE and EFGF, the advances in microchip fabrication technology presented here should be applicable broadly in interfacing microfluidics with hydrogel structures, for example in sample pretreatment.

Ultrahigh pressure liquid chromatography (UHPLC) makes it possible to use very small particles (< 2 µm) as packing materials to provide high column efficiencies. Results from a careful comparison of small porous and nonporous particles show that when the particle size is small enough (< 2 µm), both porous and nonporous particles give excellent performance, and the differences in column efficiencies between porous and nonporous particles become insignificant. Columns packed with bare diamond particles could separate small molecules, especially polar molecules, however, severe tailing occurred for less polar compounds. The polybutadiene coated diamond particles gave greater retention and better separation of small molecules compared to bare particles, although no improvement in column efficiency was observed. Changes in surface bonding of thermally hydrogenated diamond particles was achieved by chemical modification using various organic peroxides with or without reagents containing long carbon chain functional groups. It appears that the alkyl groups were attached onto the diamond surface with limited coverage. LC experiments did not demonstrate good separation; however, changes in LC behavior were observed. A repetitive solvent programming approach was successfully applied to the analysis of a continuous sample stream in microbore LC. Each analysis cycle consisted of three steps: pseudo-injection, elution and rinse. In the pseudo-injection step, elution with a non- or poor-eluting solvent produced a concentrated sample plug due to on-column focusing. Factors influencing peak symmetry, resolution and analysis cycle length were investigated. Quantitative analysis of a continuous sample stream is possible under certain operating conditions. Electric field gradient focusing (EFGF) devices with distributed resistor substrates could focus proteins in the separation channel, however, the focused bands were not stable, and the repeatability was poor due to the formation of bubbles and pH gradient in the separation channel. Both fiber-based and porous glass capillary-based planar EFGF devices with changing cross-sectional area (CCSA) channels were constructed and evaluated with the aid of a home-made scanning laser-induced fluorescence detection system. The fiber-based CCSA EFGF devices gave poorer performance compared with glass capillary based devices. Porous glass capillary-based EFGF devices could focus single proteins and separate mixtures of two to three proteins.