Browsing by Subject "Capillary electrophoresis"
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Item Applications of multiphoton-excited photochemistry to microsecond capillary electrophoresis, photolithography, and the development of smart materials(2010-08) Ritschdorff, Eric Thomas; Shear, Jason B.; Holcombe, James A.; Stevenson, Keith J.; Vanden Bout, David A.; Schmidt, ChristineLaser-based techniques have become essential tools for probing biological molecules in systems that demand high spatial and temporal control. This dissertation presents the development of micro-analytical techniques based on multiphoton excitation (MPE) to promote highly localized, three-dimensional (3D) photochemistry of biologically relevant molecules on submicron dimensions. Strategies based on capillary electrophoresis (CE) have been developed for the rapid separation and spectroscopic analysis of short-lived photochemical reaction products. High-speed separation and analysis are achieved through a combination of very high electric fields and a laser-based optical system that uses MPE for both the generation and detection of hydroxyindole photoproducts on the time scale of microseconds. MPE was also used for the development of photolithographic techniques for the creation of microstructured protein-based materials with highly defined three-dimensional (3D) topographies. Specifically, a multiphoton lithographic (MPL) technique was developed that used a low-cost microchip laser for the rapid prototyping of 3D microarchitectures when combined with dynamic optical masking. Furthermore, MPL was used to create novel “smart” biomaterials that reproducibly respond with tunable actuation to changes in the local chemical and thermal environment. The utility of these materials for creating biocompatible cellular microenvironments was demonstrated and presents a novel approach for studying small populations of microorganisms. Finally, through the development of a multifocal approach that used multiple laser beams to promote the photocrosslinking of biological molecules, the speed and versatility of MPL was extended to allow both the parallel fabrication of 3D microstructures and the rapid creation of large-scale biomaterials with highly defined spatial features.Item Capillary electrophoresis with multiphoton-exited fluorescence : native fluorescence, enzymatic assays, and ultra-fast separations(2001-08) Okerberg, Eric Steven; Shear, Jason B.Item Capillary-based microanalytical systems(Texas Tech University, 1998-08) Boring, Charles BradleyMiniaturization is a trend common to science and technology. Miniaturization into the capillary domain has been the most prevalent approach for improving chromatographic techniques. The advantages of moving from conventional size columns into the capillary domain include higher efficiencies, better mass limits of detection, low reagent consumption, and a small sample requirement. Optical absorption detection is the most common detection mode in capillary-based separation systems such as capillary liquid chromatography (LC) or capillary electrophoresis (CE). A simple, versatile, and inexpensive design for an on-column optical absorbance detector has been investigated. Detector performance is comparable to commercial detectors in both the ultraviolet and visible regions. The component cost for the detector, including data processing electronics, is only a fraction of the cost of commercial instruments. Miniaturization of both separation columns and ancillary components for capillary-based systems has made field portable liquid chromatographs feasible. A fully computer controlled, field portable capillary scale suppressed conductometric ion chromatograph (IC) that fits In a standard size briefcase has been fabricated. The system uses a capillary scale electrodialytic sodium hydroxide generator on the high-pressure side of a syringe pump to provide isocratic or gradient operation with excellent eluent purity. The system provides detection limits in the sub- to low parts per billion range, with mass limits of detection >100 times better than standard IC systems. Further, this capillary ion chromatograph has been coupled to a wet effluent parallel plate diffusion denuder for the determination of soluble lonogenic atmospheric trace gases. This resulting gas phase analyzer requires low reagent volumes for operation while providing limits of detection in the low parts per trillion range for sampled gases.Item Characterization of biological chromophores using fast electrophoretic analyses and multiphoton-exited fluorescence(2001-08) Gordon, Mary Jane Sia; Shear, Jason B.A fast and highly sensitive technique has been developed for characterizing mixtures of biological molecules that should be useful for various applications, including investigations of enzymatic reactions with fast chemical kinetics, analysis of transient photochemical reactions, and probing neurotransmitter secretion. In this dissertation work, multiphoton excitation (MPE), a high sensitivity detection approach, and capillary electrophoresis (CE), a fast separation strategy, have been coupled to rapidly characterize primary amine neurotransmitters, to perform fast electrophoretic analyses of photochemical reactions, and to probe changing biochemical microenvironments. Nonfluorescent neurotransmitter molecules have been separated and detected by virtue of fluorogenic-labeling strategies with mass detection limits (< 50,000 molecules) that should prove useful for various single-cell studies. Also, this technique has been extended to rapidly probe spectroscopically indistinct components in solution. Extremely short capillary lengths and large applied electric fields are used to extend the speed of CE to sub-second time regimes. Previously, others have demonstrated that fast CE can be accomplished using optical gating, a technique in which sample injection is controlled by modulation of photobleaching at the separation channel inlet. In these studies, this strategy has been adapted to an "inverse" optical photogating mode with CE, where fluorescent packets of photogenerated molecules are created and detected by MPE. Two instrumental configurations, defined by the alignment of the laser light with the capillary ("end on" and "side on" geometries) and the separation distance, are constructed to achieve fast separations. Using these approaches, this work demonstrates the capacity to analyze dynamic biochemical microenvironments and track transient reaction products on a several-hundredmicrosecond to several-hundred-millisecond time scale. These fast analysis strategies have provided additional information on transient species on millisecond and faster time scales, and may prove valuable in analyzing cellular processes in real-time.Item Development and applications of capillary-based analysis systems(Texas Tech University, 1995-08) Liu, ShaorongNot availableItem Fluorescent Reagents to Improve the Analytical Infrastructure of Capillary Electrophoretic Separations(2012-07-16) Li, Ming-ChienTwo types of fluorescent molecules had been designed and synthesized to improve the analytical infrastructure of capillary electrophoretic separations. First, a hydrophilic version of the permanently cationic acridine-based fluorophore, HEG2Me2-DAA was synthesized. HEG2Me2-DAA has a lambda^ex max of 490 nm which matches the 488 nm line of the commonly used argon ion laser. The emission spectra of HEG2Me2-DAA are pH-independent. HEG2Me2-DAA was used in capillary electrophoresis with an aqueous background electrolyte and was found to be free of the detrimental peak tailing of the acridine orange-based fluorophore that was caused by adsorption on the inner wall of the fused silica capillary. Bovine serum albumin was labeled with excess of the designed amine reactive reagent and the lowest concentration at which the tagged bovine serum albumin was tested was 15 nM. Chicken ovalbumin was also labeled with FL-CA-PFP and analyzed by capillary isoelectric focusing (cIEF) with LIF detection. The pI values of the tagged proteins shifted in the alkaline direction by about 0.02 compared to the pI values of the non-tagged proteins. A tri-functional probe intended to enable selective enrichment and selective detection of a variety of molecules (e.g., natural products, pharmaceuticals, inhibitors, etc.) was also designed and synthetized by combining FL-CA with biotin and an azide group in a "proof-of-principle" level experiment. In cIEF, the profile of the pH gradient can only be determined with the help of pI markers. A large set of pyrene-based fluorescent pI markers was rationally designed to cover the pI range 3 to 10. To prove the feasibility of the proposed synthetic approach, the subgroup of the pI markers having the greatest structural complexity was synthesized and characterized. The classical zone electrophoretic pI determination methods failed due to severe chromatographic retention of the APTS based pI markers on the capillary wall. Exploratory work was done to design a new pI value determination method that combines the advantages of the immobilized pH gradient technology of the OFFGEL instrument and the carrier-ampholyte-based IEF technology. The method aspects of cIEF have also been improved in this work. The new segmented loading method yielded a more linear pH gradient than the previously known cIEF methods. To exploit a unique property of the newly developed fluorescent pI markers, we used them as pyrene-based ampholytic carbohydrate derivatizing reagents. The pI4 carbohydrate derivatization reagent proved advantageous over 8-aminopyrene-1,3,6-trisulfonic acid (APTS): the pI4 conjugates have higher molar absorbance at 488 nm than the APTS conjugates and become detectable in positive ion mode of MS affording better detection sensitivity.Item Investigations in capillary liquid chromatography(Texas Tech University, 1997-08) Mo, YouwenNot availableItem Reverse direction capillary electrophoresis: theory and application(Texas Tech University, 1995-08) Dunn, Connie DeeThe history of Capillary Electtophoresis can be ttaced back over a century, as noted by Li (1). In 1800, Nicholson and Carlisle discovered electtolysis which is the decomposition of a compound into its ions by the passage of an electrical current through a solution of the compound (2). Soon thereafter, in 1808, Reuss (2) reported an interesting phenomenon involving electricity and its mobilizing effect on liquid matter. He used a U-shaped glass tube apparatus to demonsttate electtoosmosis, the movement of solvent in a field of applied potential. Two platinum wires were fused at the bottom of the tube. Powdered quartz was placed in the bottom between the wires. The tube was filled with water and a circuit was established. Soon after, hydrogen and oxygen gases were produced at the positive pole. Simultaneously, the water level rose at the negative pole and fell at the positive pole. Once the connection was broken, the water returned to its original position. This phenomenon was reproducible. The movement was not appreciable in systems that did not have the quartz powder in the bottom. Similar experiments led Reuss to propose a hypothesis that particles were ttansported from one pole to the other by galvanic current (2). This educed other experiments involving electricity as a cause of motion in fluids. In 1833, Faraday stated that the mass of a substance liberated from an electtolyte is proportional to the total quantity of electricity applied (2).Item Tracking neuronal content using capillary electrophoresis with multiphoton excitation of fluorescence(2005) Wise, Dana Diane; Shear, Jason B.Capillary electrophoresis with multiphoton-excited fluorescence detection (CEMPE) allows low-background analysis of many spectrally distinct biological fluorophores using a single long-wavelength laser. This work demonstrates the methodical transformation of CE-MPE from a proof-of-concept instrument to a reliable and powerful workhorse for complex cellular samples. Preparation of cell extracts and their long-term storage prior to CE-MPE analysis have also been exhaustively characterized (Chapter 4). The process is suitable for extractions at 2 to 3 hour intervals over a day or more, or as frequently as every hour for shorter durations. With these methods, answers were obtained for hypothesis-driven research—answers not readily available from other techniques. For example, evidence suggested intracellular levels of vitamin B3 (nicotinamide) derivatives might exhibit a circadian rhythm in suprachiasmatic nuclei neurons. Therefore, Chapter 4 presents the tracking of these cofactors over 24 – 48 h periods in extracts prepared from an immortalized biological clock cell line. Chapter 5 extends this single-fluorophore work to investigate hypothesized intracellular changes in both indole and nicotinamide derivatives during depolarization-induced upregulation of serotonergic phenotype, using cells immortalized from the raphe nuclei of the brain. Chapter 5 also demonstrates detection of riboflavin (vitamin B2) derivatives in cell extracts, and proposes several relevant continuation experiments. Finally, Chapter 6 broadens the capabilities of CE-MPE to neutral analytes, such as melatonin, for the circadian investigation of multiple analytes in cells immortalized from the pineal gland, another clock-like area of the brain.