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Microchip Liquid Chromatography and Capillary Electrophoresis Separations in Multilayer MicrodevicesFuentes, Hernan Vicente 21 November 2007 (has links) (PDF)
In this dissertation, several microfabricated devices are introduced to develop new applications in the area of chemical analysis. Electrochemical micropumps, chip-based liquid chromatography systems and multilayer capillary electrophoresis microdevices with crossover channels were fabricated using various substrates such as poly(dimethylsiloxane) (PDMS), glass, and poly(methyl methacrylate) (PMMA). I have demonstrated pressure-driven pumping of liquids in microfabricated channels using electrochemical actuation. PDMS-based micropumps were integrated easily with channel-containing PMMA substrates. Flow rates on the order of ~10 µL/min were achieved using low voltages (10 V). The potential of electrolysis-based pumping in microchannels was further evaluated for pressure driven microchip liquid chromatography (LC). Two micropumps were connected with reservoirs for sample and mobile phase, situated at the ends of microchannels for sample injection and separation, respectively. Columns micromachined in glass were coated covalently with an organic stationary phase to provide a separation medium. A pressure-balanced sample injection method was developed and allowed the injection of picoliter sample volumes into the separation channel. Fast (<40 s) separation of three fluorescently tagged amino acids was performed in a 2.5-cm-long microchip column with an efficiency of 3300 theoretical plates. Improved electrode designs that eliminate the stochastic formation of bubbles on the electrode surface will enhance pumping reproducibility. Multilayer polymeric microdevices having fluidically and electrically independent crossover channels were made using phase-changing sacrificial layers (PCSLs). High-performance electrophoretic separations of fluorescently labeled amino acids were carried out in multilayer PMMA microchips. Neither pressure nor voltage applied in a crossover channel resulted in negative effects on the separation quality in the main fluidic path. A fifty-fold reduction in crossover volumes was achieved in next-generation multilayered microchips. The ability to make minimal dead volume crossover channels facilitated the design and operation of multichannel array microdevices with a minimum number of electrical and fluidic inputs. Replicate electrophoretic separation of two peptides was performed in parallel for three independent microchannels connected to a single sample reservoir. My work demonstrates the value of PCSLs in making complex microfluidic structures that should expand the application of micro-total analysis systems.
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Development of Monolithic Stationary phases for Cation-Exchange Capillary Liquid Chromatography of Peptides and ProteinsChen, Xin 22 February 2011 (has links) (PDF)
This dissertation focuses on the preparation of polymeric monolithic capillaries for ion exchange chromatography of peptides and proteins, since polymeric monoliths have shown promise for providing improved protein separations. Characteristics of monolithic columns include low back pressure, simplicity of fabrication and biocompatibility. Preparation of strong and weak cation-exchange monolithic stationary phases in 75 μm I.D. capillaries by direct in situ copolymerization was achieved using various functional monomers including sulfopropyl methacrylate, phosphoric acid 2-hydroxyethyl methacrylate, bis[2-(methacryloyloxy)ethyl] phosphate and 2-carboxyethyl acrylate with polyethylene glycol diacrylate and other PEG materials. The resulting monoliths provided excellent ion exchange capillary LC of peptides and proteins with good run-to-run [relative standard deviation (RSD) < 1.99%] and column-to-column (RSD < 5.64%) reproducibilities. Narrow peaks were obtained and peak capacities of over 20 were achieved. Dynamic binding capacities of over 30 mg/mL of column volume for lysozyme were measured. A single monomer was used to synthesize a phosphoric acid containing monolith to improve its stability and reproducibility. The monolith was synthesized from only BMEP in 75 μm I.D. UV transparent fused-silica capillaries by photo-initiated polymerization. A dynamic binding capacity (lysozyme) of approximately 70 mg/mL of column volume was measured. Efficiencies of 52,900 plates/m for peptides and 71,000 plates/m for proteins were obtained under isocratic conditions. Good reproducibilities were achieved. Zwitterionic monolithic columns based on photo-initiated copolymerization of N,N-dimethyl-N-methacryloxyethyl-N-(3-sulfopropyl)ammonium betain and poly(ethylene glycol) diacrylate were prepared in 75 μm I.D. fused silica capillaries for hydrophilic interaction chromatography. Inverse size exclusion chromatography was used to characterize the pore structure of the resulting monolith. A typical hydrophilic interaction chromatography mechanism was observed when the organic content in the mobile phase was higher than 60%. Good separations of amides, phenols, and benzoic acids were achieved. The effects of mobile phase pH, salt concentration, and organic modifier content on retention were investigated.
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Polymeric Monolithic Stationary Phases for Capillary Reversed-phase Liquid Chromatography of Small MoleculesLiu, Kun 29 January 2014 (has links) (PDF)
Highly cross-linked monoliths prepared from single cross-linking monomers were found to increase surface area and stability. Therefore, seven cross-linking monomers, i.e., 1,3-butanediol dimethacrylate (1,3-BDDMA), 1,4-butanediol dimethacrylate (1,4-BDDMA), neopentyl glycol dimethacrylate (NPGDMA), 1,5-pentanediol dimethacrylate (1,5-PDDMA), 1,6-hexanediol dimethacrylate (1,6-HDDMA), 1,10-decanediol dimethacrylate (1,10-DDDMA), and 1,12-dodecanediol dimethacrylate (1,12-DoDDMA), were used to synthesize highly cross-linked monolithic columns in 75-µm i.d. capillaries by one-step UV-initiated polymerization using dodecanol and methanol as porogens for reversed-phase liquid chromatography (RPLC) of small molecules. Selection of porogen type and concentration was investigated in detail. Isocratic elution of alkylbenzenes at a flow rate of 300 nL/min was conducted for all of the monoliths. Gradient elution of alkylbenzenes and alkylparabens provided high resolution separations. Several of the monoliths demonstrated column efficiencies in excess of 50,000 plates/m. Monoliths with longer alkyl-bridging chains showed very little shrinking or swelling in solvents of different polarities. In addition, highly cross-linked monolithic capillary columns poly(1,6-HDDMA), poly(cyclohexanediol dimethacrylate) [poly(CHDDMA)] and poly(1,4-phenylene diacrylate) [poly(PHDA)], were synthesized and compared for RPLC of small molecules. Isocratic elution of alkylbenzenes was performed using 1,6-HDDMA and CHDDMA monolithic columns. Gradient elution of alkylbenzenes using all three monolithic columns showed good separations. Monolithic columns formed from 1,6-HDDMA, which had a linear alkyl-bridging chain structure, exhibited the highest column efficiencies (86,000 plates/m). Optimized columns showed high permeability and high run-to-run and column-to-column reproducibilities. Monoliths prepared from controlled/living polymerization was demonstrated exhibiting narrower molecular weight distribution and more homogeneous cross-linked structures due to the reversible character of this polymerization method. Thus, monolithic columns were developed from three cross-linking monomers, i.e., 1, 12-DoDDMA, trimethylolpropane trimethacrylate (TMPTMA) and pentaerythritol tetraacrylate (PETA) using organotellurium-mediated living radical polymerization (TERP) in 150-µm i.d. capillaries for RPLC of small molecules. Selection of the polymerization conditions for the 1,12-DoDDMA monolirh was investigated in detail. Isocratic elution of alkylbenzenes was achieved with good efficiency (47,700 to 64,200 plates/m for uracil) using all monolithic columns prepared using TERP.
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Integrating glycomics, proteomics and glycoproteomics to understand the structural basis for influenza a virus evolution and glycan mediated immune interactionsKhatri, Kshitij 10 July 2017 (has links)
Glycosylation modulates the range and specificity of interactions among glycoproteins and their binding partners. This is important in influenza A virus (IAV) biology because binding of host immune molecules depends on glycosylation of viral surface proteins such as hemagglutinin (HA). Circulating viruses mutate rapidly in response to pressure from the host immune system. As proteins mutate, the virus glycosylation patterns change. The consequence is that viruses evolve to evade host immune responses, which renders vaccines ineffective. Glycan biosynthesis is a non-template driven process, governed by stoichiometric and steric relationships between the enzymatic machinery for glycosylation and the protein being glycosylated. Consequently, protein glycosylation is heterogeneous, thereby making structural analysis and elucidation of precise biological functions extremely challenging. The lack of structural information has been a limiting factor in understanding the exact mechanisms of glycan-mediated interactions of the IAV with host immune-lectins. Genetic sequencing methods allow prediction of glycosylation sites along the protein backbone but are unable to provide exact phenotypic information regarding site occupancy. Crystallography methods are also unable to determine the glycan structures beyond the core residues due to the flexible nature of carbohydrates. This dissertation centers on the development of chromatography and mass spectrometry methods for characterization of site-specific glycosylation in complex glycoproteins and application of these methods to IAV glycomics and glycoproteomics. We combined the site-specific glycosylation information generated using mass spectrometry with information from biochemical assays and structural modeling studies to identify key glycosylation sites mediating interactions of HA with immune lectin surfactant protein-D (SP-D). We also identified the structural features that control glycan processing at these sites, particularly those involving glycan maturation from high-mannose to complex-type, which, in turn, regulate interactions with SP-D. The work presented in this dissertation contributes significantly to the improvement of analytical and bioinformatics methods in glycan and glycoprotein analysis using mass spectrometry and greatly advances the understanding of the structural features regulating glycan microheterogeneity on HA and its interactions with host immune lectins.
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Advancing Single-Cell Proteomics Through Innovations in Liquid Chromatography and Mass SpectrometryWebber, Kei Grant Isaac 02 April 2024 (has links) (PDF)
Traditional proteomics studies can measure many protein biomarkers simultaneously from a single patient-derived sample, promising the possibility of syndromic diagnoses of multiple diseases sharing common symptoms. However, precious cellular-level information is lost in conventional bulk-scale studies that measure tissues comprising many types of cells. As single cells are the building blocks of organisms and are easier to biopsy than traditional bulk samples, performing proteomics on a single-cell level would benefit clinicians and patients. Single-cell proteomics, combined with mass spectrometry imaging, can be used to analyze cells in their microenvironment, preserving spatial information. We have previously used laser-capture microdissection to isolate single motor neurons from tissue and analyze them in our single-cell proteomics platform. However, our sampled population of cells was necessarily limited by the low throughput of the measurement platform, and by the sensitivity of our liquid chromatography-mass spectrometry system to debris introduced in the laser-capture microdissection isolation workflow. In the work described in this dissertation, we dramatically improved the throughput of single-cell proteomics, created a method for removing insoluble debris that clogged our liquid chromatography-mass spectrometry system, and developed a high-performance, low-cost method for nanoflow gradient formation. Together, these methodologies will increase the depth of information and the number of biological replicates that can measured in single-cell proteomics. We hope that these technologies will be applied to future liquid chromatography systems to enable large scale single-cell proteomics studies of tissues. This will reveal the cellular origins of disease on a multimolecular level, while keeping important spatial information. Thus, we expect the technologies and ideas developed here to play a key role in understanding the cellular proteomics in biomedical and clinical settings.
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The chromatography and detection of various metabolites along the tryptophan-kynurenine-nicotinic acid pathway with application to plasma and homogenized rat kidney and liver /Markus, George Eugene. January 1982 (has links)
No description available.
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High pressure liquid chromatographic methods for the separations of derivatized alpha-keto acids and thiamine phosphate estersHemming, Bruce Clark 01 August 1977 (has links)
Improved methods have been developed for separation of a-keto acid derivatives and for thiamine and its phosphate esters. Linear gradient reverse phase chromatography using ion-pair formation provides baseline resolution for a seven-component homologous series of a-keto acid dinitrophenylhydrazones having increasing carbon chain lengths. Branched-chain keto acids can also be separated. Aldehyde derivatives were examined as possible interfering compounds. Peak identification in biological material was confirmed for pyruvate by an enzymic peak shift technique. Monitoring near 365 nm permits low nanogram detection. Preliminary studies of keto acids in biological material illustrate the method's applicability. The same system with slight changes separates the thiamine phosphate esters and is compared with anion exchange chromatography. The thiamine method involves effluent oxidation to form the respective fluorescent thiochrome esters for selective fluorometric detection.
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The Application of Metabolomics to the Evaluation of the Celllular ToxicityWang, Yu 09 June 2014 (has links)
No description available.
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Functionalized Octatetrayne as Novel Carbon Media for Capillary Liquid ChromatographyLiu, Jiayi 22 May 2015 (has links)
No description available.
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Predictive Value of Steroidal Hormones to Type 2 Diabetes and Metabolic SyndromeBunch, Dustin R. 12 May 2016 (has links)
No description available.
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