Physical Sciences and Mathematics Commons^™

Capillary Electrophoresis: Method Development And Application In Biomolecule Analysis, Lindsay Marie Veltri

Graduate Theses, Dissertations, and Problem Reports

Abstract

Capillary Electrophoresis: Method Development and Application in Biomolecule Analysis

Lindsay Veltri

Biomolecule analysis is essential in disease diagnostics and the development of therapeutic agents. There is a vast array of methods available for biomolecule analysis, however, capillary electrophoresis is well-suited due to minimal sample consumption (~nanoliters of solution) and fast analysis times. In this report, capillary electrophoresis was used to analyze glycosylation of therapeutic proteins. Additionally, a capillary electrophoresis-mass spectrometry method is reported for the analysis of proteins.

Post-translation protein modifications like glycosylation, play an important in protein structure and function. Modifications of protein glycosylation can lead to improved …

Development And Implementation Of A Novel Voltage-Free Interface For Capillary Electrophoresis-Mass Spectrometry Separations Of Proteins, Courtney J. Kristoff

Graduate Theses, Dissertations, and Problem Reports

Capillary electrophoresis-mass spectrometry is a powerful technique for high-throughput and high efficiency separations combined with structural identification. Electrospray ionization is the primary interface used to couple capillary electrophoresis to mass analyzers; however, improved designs continue to be reported. A new interfacing method based on vibrating sharp-edge spray ionization is presented in this work to overcome the challenges of decoupling applied voltages and to enhance the compatibility with separations performed at near-neutral pH. The versatility and ease of use of this ionization source is demonstrated using β-blockers, peptides, and proteins. The cationic β-blocker pindolol was injected electrokinetically and detected at concentrations …

Improving Coupled Enzyme Assays With Capillary Electrophoresis, Thu Hoai Nguyen

LSU Doctoral Dissertations

The goal of this project is to address limitations of coupled enzyme assays using capillary electrophoresis (CE). In some enzyme assays, it is ineffective to directly detect products and substrates spectroscopically (absorption or fluorescence). Coupling the enzyme-catalyzed reaction of interest with another enzyme-catalyzed reaction allows for indirect detection of product formation or substrate depletion for the target enzyme. While conventional coupled enzyme assays are facile for kinetic measurements, they can be problematic for inhibition screening due to spectral and inhibitor interferences. To address these limitations of coupled assays, CE has been used to develop enzyme assays for acetyl-coenzyme A carboxylase …

Separation Of On-Column Labeled Model Proteins With Packed Capillary Electrophoresis, Yingxu Hao

Open Access Theses

Protein drugs are increasingly developed in the pharmaceutical company. Under the regulation of FDA, high purity of therapeutic proteins needs to be maintained. Before putting those drugs in the market, fast and efficient method is in need to achieve homogeneity. Traditionally, polyacrylamide gel electrophoresis (PAGE), capillary electrophoresis (CE), and size-exclusion chromatography (SEC) are used for the purification process. These methods have the disadvantages of low time and cost efficiency, and this quality assurance process has become the bottleneck of production. In our group, sub-micron silica colloidal particles with polyacrylamide layer on the surface are packed inside capillaries to increase the …

Silica Colloidal Crystals As Emerging Materials For The High-Throughput Sizing Of Proteins By Packed Capillary Electrophoresis, Nadine K. Njoya

Open Access Dissertations

Protein heterogeneity is currently one of the leading problems in formulation of therapeutic proteins. The effectiveness of therapeutic proteins is diminished when they exhibit heterogeneity due to aggregates and degradation by-products. Furthermore, guidelines set forth by the FDA require therapeutic proteins to have high stability as well as high purity. With development time on the order of years and costs incurred in the millions, pharmaceutical companies are investing more time in finding fast and efficient ways to screen for protein heterogeneity. Current methods such as native polyacrylamide gel electrophoresis (PAGE), size-exclusion chromatography (SEC) and capillary electrophoresis (CE) are time consuming, …

On The Capillary Electrophoresis Of Monohydroxy Metabolites Of Polycyclic Aromatic Hydrocarbons And Its Application To The Analysis Of Biological Matrices, Gaston Knobel

Electronic Theses and Dissertations

Polycyclic aromatic hydrocarbons (PAH) are a class of environmental pollutants consisting of a minimum of two fused aromatics rings originating from the incomplete combustion of organic matter and/or anthropogenic sources. Numerous possible anthropogenic and natural sources make the presence of PAH ubiquitous in the environment. The carcinogenic nature of some PAH and their ubiquitous presence makes their chemical analysis a topic of environmental and toxicological importance. Although environmental monitoring of PAH is an important step to prevent exposure to contaminated sites, it provides little information on the actual uptake and subsequent risks. Parent PAH are relatively inert and need metabolic …

Analysis Of Protein Aggregates And Synthetic Nanoparticles Using Capillary Electrophoresis With Light Scattering And Fluorescence Detection, Suresh Chandra Regmi

LSU Doctoral Dissertations

The goal of the research presented in this dissertation is to develop capillary electrophoresis (CE) based analysis techniques for the characterization of individual particles including amyloid beta (Aâ) peptide aggregates and to apply these methods to study the aggregation of Aâ peptides. Protein self-assembly into ordered aggregates is a common and important process in normal biological function, but this process is also involved in several diseases. Amyloid beta peptide aggregation is linked to Alzheimer’s disease. Thioflavin T (ThT) has been used as a fluorescent probe for the detection of Aâ aggregation. Based on previous observations of false positive fluorescent signals …

Laser Ablation Sample Transfer For Mass Spectrometry, Sung-Gun Park

LSU Doctoral Dissertations

In this research, a new ambient sampling technique for mass spectrometry was developed that uses an infrared laser to ablate materials under ambient conditions that are captured in a solvent or on a surface. An infrared optical parametric oscillator (OPO) laser system at 3 μm wavelength was focused onto samples for ablation at atmospheric pressure. The ablated materials were transferred to a solvent or surface. For off-line electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI) analysis, the ablated material was captured in a static solvent droplet that was deposited on a MALDI target or flow-injected into a nanoelectrospray source. The …

Microfluidic Devices With Integrated Sample Preparation For Improved Analysis Of Protein Biomarkers, Pamela Nsang Nge

Theses and Dissertations

Biomarkers present a non-invasive means of detecting cancer because they can be obtained from body fluids. They can also be used for prognosis and assessing response to treatment. To limit interferences it is essential to pretreat biological samples before analysis. Sample preparation methods include extraction of analyte from an unsuitable matrix, purification, concentration or dilution and labeling. The many advantages offered by microfluidics include portability, speed, automation and integration. Because of the difficulties encountered in integrating this step in microfluidic devices most sample preparation methods are often carried out off-chip. In the fabrication of micro-total analysis systems it is important …

In-Capillary Reduction And Digestion Of Proteins At Nano-Liter Volume Using Capillary Electrophoresis, Thasneen Naina Nazeema

Electronic Thesis and Dissertation Repository

To analyze the proteins present in biological samples several major steps are involved. One of the crucial steps in proteomics has been sample preparation. Prior to mass spectral analysis proteins containing disulfide bonds must be reduced which is followed by digestion. In this work, a novel way of performing protein reduction and digestion at nano-liter volume using capillary electrophoresis is presented. In this work, zone passing CE technique was utilized to analyze the reduction products in CE. After the successful in-capillary reduction, digestion of protein was also performed. A pH mediated stacking was used to bring the reduced protein and …

Analysis Of Adp-Ribosylation And Protein Sample Preparation By Capillary Electrophoresis, Ming Huang

Electronic Thesis and Dissertation Repository

ADP-ribosylation is one of many biologically important protein post-translational modifications (PTMs) that received considerable research interest in the past decade. The first part of this work presents the analysis of ADP-ribosylation, with special focus on the catalytic domain of an enzyme ARTD-10. The application of capillary electrophoresis (CE) is proposed in this work to separate the in vitro enzymatic mono-ADP-ribosylated ARTD-10 catalytic domain and its unmodified form. Another exciting aspect of CE application with growing interest is the sample preparation. The effectiveness and efficiency for in-vial reaction, in-capillary mixed reaction and on-capillary-tip reaction by performing methyl esterification on phophopeptides were …

Migration Behaviour Of Discontinuous Buffers In Capillary Electrophoresis During Protein Enrichment, Ting Li

Electronic Thesis and Dissertation Repository

To enrich protein at sub-microliter sample volumes, our group has developed a discontinuous buffer system which consists of an acid and a base inside a capillary. Upon voltage application, the protein moves towards and stacks at the pH junction. Prior to this work, we only focused on the protein enrichment and have not investigated the migration behaviour of various ions in this system. However, it is important to understand the mechanism of the migration of these ions, because the pH junction formation and further the protein enrichment are based on it. In this work, the ion migration patterns at the …

Miniaturized Fractionation And Purification Of Analytes Of Interest In Complex Mixtures: Protein Enrichment And Purification With Capillary Electrophoresis And Identification Of Pesticide Chemicals In Bio-Oils, Christina J. Booker

Electronic Thesis and Dissertation Repository

The ability to isolate and detect an analyte of interest is vital for many different facets of life today, from disease detection to environmental safety. The goal of the method development discussed within this thesis is to isolate and identify unique analytes from complex matrices and to do so in a scaled down approach. Removal of an analyte from a diverse background of molecules is often required for successful detection and determination of analyte properties. As well, many samples are only available at low volumes and at trace levels so miniaturized techniques that only require small sample volumes are designed …

Integrated Affinity Column Capillary Electrophoresis Microdevices For Biomarker Analysis, Weichun Yang

Theses and Dissertations

In this dissertation, microfluidic systems that integrate antibody-based sample preparation methods with electrophoretic separation are developed to analyze multiple biomarkers in a point-of-care setting. To form an affinity column, both monolith materials and wall-coated channels were explored. I successfully demonstrated that monolith columns can be prepared in microfluidic devices via photopolymerization. The selectivity of monolith columns was improved by immobilizing antibodies on the surface. These affinity columns can selectively enrich target analytes and reduce the signal of contaminant proteins up to 25,000 fold after immunoaffinity extraction. These results clearly demonstrate that microchip affinity monoliths can selectively concentrate and purify target …

Capillary Electrophoretic Separations Of Enzyme Inhibitors With Activity-Based Detection, Xiaoyan Yan

LSU Master's Theses

A technique for separating and detecting enzyme inhibitors was developed using capillary electrophoresis with an enzyme microreactor. The on-column enzyme microreactor was constructed using one or two NdFeB magnets in two configurations to immobilize alkaline phosphatase-coated superparamagnetic beads with diameters of 2.8 µm inside a capillary before the detection window. Enzyme inhibition assays were performed by injecting a plug of inhibitor into a capillary filled with an alkaline phosphatase substrate, AttoPhos. Product generated in the enzyme microreactor was detected by laser-induced fluorescence. Inhibitor zones electrophoresed through the capillary, passed through the enzyme microreactor, and were observed as negative peaks due …

On-Line Laser Desorption/Ionization Mass Spectrometry, Damien A. Narcisse

LSU Doctoral Dissertations

Interfaces for on-line laser desorption/ionization mass spectrometry are presented in this dissertation. An on line laser desorption interface allows samples to be introduced directly into the mass spectrometer for high-throughput applications. For this research, a linear time of flight mass spectrometer was constructed with an ionization source designed to accept various laser desorption interfaces. A ball inlet interface was used for continuous analyte deposition at atmospheric pressure and vacuum ionization. A solvent-based cleaning system and a separate capillary for MALDI matrix delivery was utilized for continuous on line sampling with the ball inlet interface. Microfluidic devices were brought into contact …

Capillary Electrophoresis Used As An Alternative To Hplc For Pharmaceutical Analysis Of Antifungal Agents, Christopher N. Lechnou

Full-Text Theses & Dissertations

HPLC is a commonly used analytical tool in the pharmaceutical industry for the characterization of drug potency and purity. However, HPLC analysis can be very time consuming, use large quantities of organics, and thus costly and not environmentally friendly. In this study, we describe an alternative to HPLC that can be used for pharmaceutical analysis. Capillary electrophoresis is being utilized increasingly for biochemistry and analytical chemistry applications. With advances in auto samplers and improvements in injection precision, the potential for this instrumentation to be used in conjunction or as an alternative to HPLC is currently being evaluated in analytical laboratories. …

I. Characterization Of Sulfonated Phthalocyanines By Mass Spectrometry. Ii. Characterization Of Siaa, A Streptococcal Heme-Binding Protein Associated With A Heme Abc Transport System, Brian R. Sook

Chemistry Dissertations

Sulfonated phthalocyanines were characterized using capillary electrophoresis and mass spectrometry. Derivatives investigated included the copper, cobalt, zinc and metal-free sulfonated phthalocyanines. The electropherograms of commercially available copper phthalocyanine-3,4',4'',4'''-tetrasulfonic acid and 4,4',4'',4'''-tetrasulfonic acid were very different, consistent with the latter compound having a structure that is not fully sulfonated. Matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) were used to characterize the sulfonated phthalocyanines. Mass spectral evidence was obtained for a pentasulfonated species of both the metal-free phthalocyanine and zinc phthalocyanine when these species were made by sulfonation of the metal-free phthalocyanine (followed by zinc insertion in the latter case). Many …

Bioanalytical Methods For Studies Of Homocysteine And Novel Cardiovascular Disease Indicators, Arther T. Gates

LSU Doctoral Dissertations

This dissertation explores the development of analytical methods for studies of CVD biomarkers and related biomolecular indicators. Initially, spectroscopic studies were conducted to investigate the chemical reactivity of homocysteine (Hcy), an independent CVD risk factor and serological biomarker. Consequently, we proposed an alternate theory for in vivo Hcy clearance based on spontaneous pyridoxal tetrahydrothiazine (PT) formation from Hcy and pyridoxal. The validity of PT-assisted Hcy clearance was further evaluated by use of capillary electrophoretic methods, which allowed rapid monitoring of protein oligomerization in PT-protein reaction mixtures. The results of these studies suggest that PT formation is a plausible mechanism for …

Microchip Liquid Chromatography And Capillary Electrophoresis Separations In Multilayer Microdevices, Hernan Vicente Fuentes

Theses and Dissertations

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). …

Capillary Electrophoresis Of Proteins With Selective On-Line Affinity Monoliths, Jenny Marcela Armenta Blanco

Theses and Dissertations

The analysis of proteins in biological fluids by capillary electrophoresis (CE) is of interest in clinical chemistry. However, due to low analyte concentrations and poor concentration limits of detection (CLOD), protein analysis by this technique is frequently challenging. Coupling preconcentration techniques with CE greatly improves the CLOD. An on-line preconcentration-CE method that can selectively preconcentrate any protein for which an antibody is available would be very useful for the analysis of low abundance proteins and would establish CE as a major tool in biomarker discovery. To accomplish this, an on-line protein G monolithic preconcentrator CE system for enrichment and separation …

Membrane-Based Protein Preconcentration Microfluidic Devices, Yi Li

Theses and Dissertations

Interest in microchip capillary electrophoresis (CE) is growing due to the rapid analysis times provided and small sample input requirements. However, higher-concentration samples are typically needed because of the small (~pL) detection volumes in these devices. I have made membrane-based protein preconcentration systems in capillary and microchip designs to increase the detectability of low-concentration biological samples. A photopolymerized ion-permeable membrane interfaced with a microchannel in poly(methyl methacrylate) (PMMA) formed the preconcentrator. When a voltage was applied between the sample reservoir and the ionically conductive membrane in a capillary-based system, R-phycoerythrin was concentrated more than 1,000 fold, as determined by laser-induced …

Development Of Analytical Techniques For The Analysis Of Submicron Particles And Protein Aggregates, Yohannes H. Rezenom

LSU Doctoral Dissertations

The goal of the research presented in this dissertation is to develop analytical techniques for the analysis of heterogeneous mixtures of submicron particles and protein aggregates. In Chapter 2, a simple and reproducible technique for constructing perfectly aligned gaps in fused-silica capillaries has been developed for postcolumn reagent addition with capillary electrophoresis (CE) to take advantage of laser-induced fluorescence (LIF) detection. This technique uses laser ablation with a Nd:YAG laser to create gaps of 14.0 ¡Ó 2.2 ƒÝm. These structures have been used for reagent addition for postcolumn derivatization with LIF detection and have been tested for the separation of …

The Determination Of Probes For The Indirect Detection Of Anions In Capillary Zone Electrophoresis Using Light Emitting Diode Detection, Peter Ashley Balding

Theses : Honours

This study investigated the potential of a number of compounds to act as probes for the indirect detection of inorganic and organic anions separated by capillary zone electrophoresis and using light emitting diodes (LED) as the light source. Desirable characteristics of the probe anion included: high molar absorptivity, moderate to high electrophoretic mobility, and having a wavelength of maximum absorbance near the maximum emission wavelength of a LED. A preliminary study led to the evaluation of eleven possible compounds. Two of these, p-nitrophenol and 4-hydroxy-3,5-dinitrobenzoic acid were selected for further study as they fulfilled the criteria previously mentioned. The optimised …

An Evaluation Of The Enantiomeric Recognition Of Amino Acid Based Polymeric Surfactants And Cyclodextrins Using Spectroscopic And Chromatographic Methods, Bertha Cedillo Valle

LSU Doctoral Dissertations

The scope of this dissertation is to explore the enantiomeric recognition properties of amino acid based polymeric surfactants using a combination of analytical techniques including chromatographic and spectroscopic methods. Chapter 1 includes an introduction to chirality and chiral separations using capillary electrophoresis. Using cyclodextrin modified micellar electrokinetic chromatography (CD-MEKC), the enantioseparation of three binaphthyl derivatives using native alpha- and beta-cyclodextrins in combination with various diastereomers of chiral polymeric surfactants (PS) was examined. A reversal of enantiomeric order was observed with the enantiomers of (1,1 S-binaphthyl-2,2 S-diamine (BNA) and (1,1 S-binaphthol (BOH) due to a competition between the two chiral selectors …

Analysis Of Clinically Important Compounds Using Electrophoretic Separation Techniques Coupled To Time-Of-Flight Mass Spectrometry, Zlatuse Durda Peterson

Theses and Dissertations

Capillary electrophoretic (CE) separations were successfully coupled to time-of-flight mass spectrometric (TOFMS) detection for the analysis of three families of biological compounds that act as mediators and/or indicators of disease, namely, catecholamines (dopamine, epinephrine, norepinephrine) and their O-methoxylated metabolites (3-methoxytyramine, norepinephrine, and normetanephrine), indolamines (serotonin, tryptophan, and 5-hydroxytryptophan), and angiotensin peptides. While electrophoretic separation techniques provided high separation efficiency, mass spectrometric detection afforded specificity unsurpassed by other types of detectors.

Both catecholamines and indolamines are present in body fluids at concentrations that make it possible for them to be determined by capillary zone electrophoresis coupled to TOFMS without employing any …

Solid-Phase Dna Sequencing Reactions Performed In Micro-Capillary Reactors And Solid-Phase Reversible Immobilization In Microfluidic Chips For Purification Of Dye-Labeled Dna Sequencing Fragments, Yichuan Xu

LSU Doctoral Dissertations

The research presented in this dissertation involves micro-capillary reactors for solid phase DNA sequencing, the identification of dye terminator sequencing fragments with time-resolved methods, and purification of dye-labeled DNA fragments using solid- phase reversible immobilization in microfluidic chips. Solid-phase micro-reactors have been prepared for DNA sequencing applications using slab gel electrophoresis. A PCR product was immobilized to the interior wall of a fused-silica capillary tube via a biotin-streptavidin linkage. Solid-phase sequencing was carried out in micro-capillary reactors using a four-lane, single color dye primer chemistry strategy. The read length was found to be 589 bases. The complementary DNA fragments generated …

Capillary And Microdevice Electrophoretic Tools For Genetic Analyses: Heteroduplex Analysis For Tuberculosis Drug Susceptibility And Ligase Detection Reaction For Colorectal Cancer Detection, Gloria Thomas

LSU Doctoral Dissertations

The traditional format of electrophoresis, the slab gel, is quickly being replaced by capillary and microdevice platforms, which offer improvements in cost, resolution, speed, quantitation and automation in genetic analyses. These techniques also employ a variety of separation matrices while the slab gel is limited to agarose and polyacrylamide. The research presented here explores the use of these electrophoretic formats for the detection of single nucleotide mutations using two genetic models associated with human disease. Slab gel based heteroduplex analysis (HDA), a popular mutation scanning method, uses a specially designed universal heteroduplex generator (UHG) containing controlled variation to enhance the …

Synthesis, Characterization, And Applications Of Novel Pseudostationary Phases In Micellar Capillary Electrophoresis For Separation Of Chiral And Archiral Compounds H[Electronic Resource], Cevdet Akbay

LSU Doctoral Dissertations

The research presented in this dissertation involves the synthesis, characterization, and the use of novel surfactants, including both micelles and vesicles, as pseudostationary phases in micellar capillary electrophoresis (MCE) for the separation of achiral and chiral compounds. Separation of environmental pollutants such as 2 to 6-ring polycyclic aromatic hydrocarbons (PAHs) was achieved using poly(sodium undecylenic sulfate). A baseline separation of all 16 PAHs was possible for the first time in MCE by a single-surfactant system. In addition, a surfactant with a phosphated head group, i.e., di(2-ethylhexyl)phosphate (DEHP), was also introduced as a novel pseudostationary phase for separation of 21 weakly …

Dendritic Polymers As Bonded Stationary Phases In Capillary Electrochromatography, Chiayu Helen Chao

Seton Hall University Dissertations and Theses (ETDs)

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