Analytical Chemistry Commons^™

Investigation Of Warfarin–Human Serum Albumin Binding By Ultrafast Affinity Extraction Using Single- And Dual-Column Affinity Microcolumn Systems, Samiul Alim

Department of Chemistry: Dissertations, Theses, and Student Research

This dissertation examined the interaction of warfarin with human serum albumin (HSA) by using affinity chromatography, with particular emphasis on ultrafast affinity extraction (UAE) and affinity microcolumns. HSA affinity supports were prepared by immobilizing HSA onto diol-bonded silica via Schiff base chemistry and used to prepare affinity microcolumns for chromatographic studies under physiological-like conditions at pH 7.4 and 37.0 °C. UAE studies were performed to investigate the effects of residence time, flow rate, and column configuration on the measured apparent free fraction of warfarin and on the calculated apparent binding parameters. The results showed that the measured apparent free fraction …

Synthesis And Study Of High-Spin Stable Organic Radicals For Electrical Conductors And Mannosamine Nitroxide For Mri Contrast Agents, Shuyang Zhang

Department of Chemistry: Dissertations, Theses, and Student Research

In the first project, we describe the synthesis of an ambient stable high spin organic diradical 4 based on the Blatter moiety. The high-spin (S = 1) organic diradical 4, which consists of two Blatter radical moieties in a conjugated structure, exhibits a nearly exclusive population (88%) on triplet ground state at room temperature as a consequence of a large single-triplet energy gap (ΔEST = 0.5 kcal/mol). The target diradical molecule is synthesized over five steps with structural confirmation by single-crystal X-ray diffraction. The thermogravimetric analysis (TGA) shows the onset of decomposition at ~264 oC, indicating the diradical molecule has …

Oxygen Binding Thermodynamics Of Human Hemoglobin In The Red Blood Cell, Kyle K. Hill

Department of Chemistry: Dissertations, Theses, and Student Research

We report for the first time the binding constants and Hill numbers for oxygen in the red blood cell under physiological conditions. When compared to our results for hemoglobin in solution, our results show conclusively that hemoglobin binds oxygen more tightly and with lower co-operativity when packed in the red blood cell. At 18°C, these differences are striking: the respective half-saturation values are 15.57 µM (in red blood cells) and 18.83 µM (in solution), with corresponding Hill numbers of 2.475 (in red blood cells) and 2.949 (in solution). The optical complications that arise from high turbidity of red blood cell …

The Application And Development Of Metabolomics Methodologies For The Profiling Of Food And Cellular Toxicity, Jade Woods

Department of Chemistry: Dissertations, Theses, and Student Research

Metabolomics is a rapidly growing field of study. Its growth reflects advancements in technology and an improved understanding of the impact of the environment on metabolism. As a result, metabolomics is now commonly employed to investigate and characterize human and plant metabolism. The first chapter of this thesis provides an introduction to metabolomics and an overview of the protocols for sample preparation, data collection and statistical analysis. The second thesis chapter describes in explicit detail the step-by-step process of extracting and analyzing metabolites collected from mammalian cells, specifically brain tissue with a focus on Parkinson’s disease. The chapter highlights important …

Evaluation Of The Overall Binding Of Acetohexamide And Tolbutamide With Methyl Glyoxal-Modified Hsa By High-Performance Affinity Chromatography, Ashley G. Woolfork, David S. Hage

Department of Chemistry: Dissertations, Theses, and Student Research

High performance affinity chromatography (HPAC) was used in a zonal competition format to investigate the overall binding of two sulfonylurea drugs with modified human serum albumin (HSA). The in vitro modifications of this protein that were examined included glycation, as occurs in the presence of elevated glucose levels during diabetes, and the formation of advanced glycation end-products (AGEs) due to the reaction of HSA with methylglyoxal (MGo). The sulfonylurea drugs acetohexamide and tolbutamide, which are used to treat type II diabetes, were each injected onto 10 mm x 2.1 mm i.d. affinity microcolumns that contained normal HSA and HSA modified …

Analysis Of Hydroxychloroquine Interaction With Serum Proteins By High Performance Affinity Chromatography, Kyungah Suh, Sadia Sharmeen, David S. Hage

Department of Chemistry: Dissertations, Theses, and Student Research

Personalized medicine or precision medicine has been an emerging market for better treatments of patients under varying physiological conditions.To improve the development of personalized medicine, a higher understanding of drug-protein binding between serum transport proteins and the unbound form of pharmaceuticals is needed. Human Serum Albumin (HSA) and Alpha1-acidglycoprotein (AGP) play a crucial role in pharmacokinetics as they are two most abundant transport serum proteins in body circulatory system. Inter-individual variabilities of serum proteins, such as the physiological condition of patients, have been believed to alter the drug-serum protein binding. Fig. 1 shows an example of the cause of inter-individual …

Electropolymerization And Characterization Of Thin Film Dielectrics, Christopher White Ii

Department of Chemistry: Dissertations, Theses, and Student Research

Graphene is a two-dimensional allotrope of carbon, with several superlative properties, including high electron and hole conductivities, high tensile strength, and chemical robustness. Its chemical and physical properties make it attractive for in use in a myriad of applications, including transistors. However, graphene’s chemical inertness and its physical and electrical properties’ dependency on its conjugated structure make building composite materials with graphene difficult. In 2015, Lipatov, et. al. demonstrated that a film derived from electropolymerized phenol could be used as a dielectric layer on transistor devices fabricated from exfoliated graphene. This thesis extends that research, detailing experiments of phenol and …

Analysis Of Drug–Protein Interactions During Diabetes By High-Performance Affinity Chromatography, Pingyang Tao

Department of Chemistry: Dissertations, Theses, and Student Research

High-performance affinity chromatography (HPAC) is a type of liquid chromatography in which solutes are separated based on their binding to a stationary phase that is a biologically-related agent. Sulfonylurea drugs have significant binding to proteins in blood, with most of this binding is believed to occur with human serum albumin (HSA). HSA is known to be modified as a result of the high serum levels of glucose that is present during diabetes, which may also alter the function of HSA as a binding agent for many drugs in circulation. Some modifications of HSA that can occur during diabetes involves early …

Development Of Nanomaterial Supports For The Study Of Affinity-Based Analytes Using Ultra-Thin Layer Chromatography, Allegra Pekarek

Department of Chemistry: Dissertations, Theses, and Student Research

Ultra-thin layer chromatography (UTLC) is a growing field in analytical separations. UTLC is a branch of planar and liquid chromatography that is related to thin layer chromatography. The main advantage of UTLC compared to other techniques is it uses much less material, allowing for faster and more sensitive separations to take place. The UTLC devices fabricated in this project used either silicon oxide or silicon nanopillars deposited on a glass slide using glancing angle deposition (GLAD). Even a thin layer of these nanopillars deposited on a glass slide provide a large surface area for the analyte to be separated. GLAD …

Mass Spectrometry And Nuclear Magnetic Resonance In The Chemometric Analysis Of Cellular Metabolism, Eli Riekeberg

Department of Chemistry: Dissertations, Theses, and Student Research

The development and awareness of Machine Learning and “big data” has led to a growing interest in applying these methods to bioanalytical research. Methods such as Mass Spectrometry (MS), and Nuclear Magnetic Resonance (NMR) can now obtain tens of thousands to millions of data points from a single sample, due to fundamental instrumental advances and ever-increasing resolution. Simple pairwise comparisons on datasets of this magnitude can obfuscate more complex underlying trends, and does a disservice to the richness of information contained within. This necessitates the need for multivariate approaches that can more fully take advantage of the complexity of these …

Ultrafast Affinity Extraction And High-Performance Affinity Chromatography Applications For Measuring Free Drug Fractions: Interactions Of Sulfonylurea Drugs With Normal And Glycated Human Serum Albumin, Bao Yang

Department of Chemistry: Dissertations, Theses, and Student Research

Ultrafast affinity extraction was used with microcolumns containing human serum albumin (HSA) to measure the free fractions of several sulfonylurea drugs in the presence of solution-phase HSA and for the analysis of these drugs at therapeutic levels or determining their binding strengths with this protein. This approach was first used with normal HSA and the drugs glibenclamide, glipizide and glimepiride. This work used both one- and two-dimensional affinity systems based on HSA microcolumns. The first microcolumn was used to extract the free drug fractions in injected drug/protein mixtures. As the retained drug eluted from the first microcolumn, it was passed …

Bioinformatic And Biophysical Analyses Of Proteins, Jonathan Catazaro

Department of Chemistry: Dissertations, Theses, and Student Research

The prevailing dogma in structural genomics is the existence of a strong correlation between protein sequence, structure, and biological function. Proteins with high sequence similarity typically have a similar, if not the same, structure and function. In many cases this logic can fail due to distantly related proteins having very low sequence similarity, a lack of a representative structure, structural novelty, or the absence of a characterized function. Further, the paradigm fails to account for dynamics, which have a significant effect on structural stability and enzymatic efficacy.

Nuclear magnetic resonance (NMR) spectroscopy is uniquely capable of solving the structure, assisting …

Binding Of Oxygen To Human Hemoglobin Within The Erythrocyte Using Icam Spectrophotometry, Kyle K. Hill

Department of Chemistry: Dissertations, Theses, and Student Research

Many of the spectrophotometric techniques used to determine the properties of intracellular human hemoglobin cannot be utilized due to the turbidity of erythrocyte suspensions. An Integrating Cavity Absorption Meter, or ICAM, allows for absorption measurements of strongly scattering samples in the visible-light region of the spectrum. The spectrum of oxygenated hemoglobin within erythrocytes is significantly different from the absorption spectrum of oxygenated hemoglobin in solution. Studies of the oxygen binding to hemoglobin in erythrocytes allowed the four sequential binding constants (Adair constants) to be determined and compared with those of hemoglobin in solution. The Adair constants for hemoglobin in solution …

Applications Of High Performance Affinity Chromatography With High Capacity Stationary Phases Made By Entrapment, John A. Vargas Badilla

Department of Chemistry: Dissertations, Theses, and Student Research

High performance affinity chromatography (HPAC) is a technique that uses a biologically-related agent such as a transport protein or an antibody as the stationary phase in an HPLC system. In recent years, HPAC has been shown to be a valuable bioanalytical tool for studying solute-protein interactions. Human serum albumin (HSA), the most abundant protein in blood (with concentrations of 35 to 50 mg/mL in serum), has been shown to interact with many drugs, affecting their transport, excretion and metabolism. A physical method for immobilizing proteins in HPAC supports has been optimized in this dissertation by using HSA as a model …

Chemometric And Bioinformatic Analyses Of Cellular Biochemistry, Bradley Worley

Department of Chemistry: Dissertations, Theses, and Student Research

The amount of information collected and analyzed in biochemical and bioanalytical research has exploded over the last few decades, due in large part to the increasing availability of analytical instrumentation that yields information-rich spectra. Datasets from Nuclear Magnetic Resonance (NMR), Mass Spectrometry (MS), infrared (IR) or Raman spectroscopy may easily carry tens to hundreds of thousands of potentially correlated variables observed from only a few samples, making the application of classical statistical methods inappropriate, if not impossible. Drawing useful biochemical conclusions from these unique sources of data requires the use of specialized multivariate data handling techniques.

Unfortunately, proper implementation of …

Analysis Of Free Solute Fractions And Solute-Protein Interactions Using Ultrafast Affinity Extraction And Affinity Microcolumns, Xiwei Zheng

Department of Chemistry: Dissertations, Theses, and Student Research

This dissertation describes the use of a high-performance affinity chromatography method based on ultrafast affinity extraction and microcolumns to study biological interactions.

In the first project, a new method was created and based on ultrafast affinity extraction to determine both the dissociation rate constants (k_d) and association equilibrium constants (K_a) for drug-protein interactions in solution. Various conditions to optimize the use of ultrafast affinity extraction for equilibrium and kinetic studies were considered.

The objective of the next portion of this dissertation was to develop a chromatographic approach to measure free drug fractions in more …

Chromatographic Analysis Of Drug-Protein Interactions During Diabetes And Characterization Of Human Serum Albumin Through Multidimensional Mass Spectrometry, Ryan E. Matsuda

Department of Chemistry: Dissertations, Theses, and Student Research

Diabetes is a metabolic disease that can lead to the non-enzymatic glycation of serum proteins such as human serum albumin (HSA). Previous studies have indicated that glycation can affect the structure and function of these proteins. This dissertation describes the development of tools and techniques based on high performance affinity chromatography (HPAC) and multidimensional mass spectrometry to analyze the effects of glycation on the function and structure of HSA.

A major portion of this research involved the utilization of HPAC to examine the effect of glycation on the binding of three second-generation sulfonylurea drugs and one third-generation sulfonylurea drug. These …

The 8-Silyloxyquinoline Scaffold As A Versatile Platform For The Sensitive Detection Of Aqueous Fluoride, Xinqi Zhou

Department of Chemistry: Dissertations, Theses, and Student Research

As one of the most essential elements in nature, fluoride has been well known and extensively studied since the 16^th century. Since then the two-edged nature of fluoride in biological systems has been well described. Specifically, moderate fluoride exposure can aid in tooth and bone development, while fluoride overexposure results in the depilating disease known as fluorosis. Because of the side effects upon overexposure of fluoride, there is a need to develop fast and straight forward methods to sensitively and selectively detect and quantify fluoride concentration in drinking water. With this goal in mind and by taking advantage of …

Toward An Expanded Role For Collision-Induced Dissociation In Glycoproteomic Analysis, Venkata Kolli

Department of Chemistry: Dissertations, Theses, and Student Research

This thesis begins with an overview of the state-of-the-art in tandem mass spectrometry (MS/MS) analysis of glycopeptides. In this introduction, the primary focus is on utilization of different ion dissociation techniques for MS/MS to obtain structural information of N-glycopeptides. This includes a discussion of the importance of complementary MS/MS methods to attain complete structural characterization of N-glycopeptides. Emerging methods involving the use of a single ion dissociation technique for complete glycopeptide connectivity analysis were also presented. Next, the application of collision-induced dissociation (CID) to provide both amino acid sequence and monosaccharide connectivity for model N-glycopeptides was discussed in detail. Implementation …

Analysis Of Drug Interactions With Lipoproteins By High Performance Affinity Chromatography, Matthew R. Sobansky

Department of Chemistry: Dissertations, Theses, and Student Research

High density lipoprotein (HDL), low density lipoprotein (LDL), and very low density lipoprotein (VLDL) are lipoproteins previously shown to bind many basic and neutral hydrophobic drugs in serum. These interactions impact the distribution, delivery, metabolism, and excretion of drugs and are important in determining drug activity, pharmacokinetics, and toxicity in the human body. Information about drug-lipoprotein interactions and the strength of these interactions can be useful in determining the distribution of drugs following administration.

The research presented in this dissertation uses high performance affinity chromatography (HPAC) and packed columns to study binding of the drug propranolol to immobilized lipoproteins such …

Syntheses Of Aminyl Diradicals And Nitroxide Tetra- And Octaradicals, Arnon Olankitwanit

Department of Chemistry: Dissertations, Theses, and Student Research

This dissertation comprises of two main research projects: aminyl diradicals and calix[4]arene nitroxide tetra- and octaradicals. The design, multi-step synthesis and characterization, including NMR, EPR spectroscopy, magnetic measurements and X-ray crystallography of precursors and target compounds will be discussed.

Aminyl radicals, nitrogen-centered radicals, are typically short-lived and considered as reactive intermediates. Because of their unique magnetic properties, aminyls are attractive building blocks for high-spin di-and polyradicals. The challenge is in the design and synthesis of aminyls with increased stability. In this chapter, we have prepared triplet (S = 1) ground state aminyl diradicals, which are derivatives of aza-m …

The Development And Applications Of Nmr Metabolomics Analysis Of Bacterial Metabolomes, Steven M. Halouska

Department of Chemistry: Dissertations, Theses, and Student Research

Metabolomics is a relatively new field that involves the study of metabolic responses that are occurring within a biological system. Metabolite profiles of an organism, tissue extract, and biofluids are important indicators to determine the physiological state of a biological profile. Comparison of such profiles from different phenotypes can be used to identify specific metabolic changes leading to the understanding of metabolic pathways, disease progression, drug toxicity and efficacy, and cellular responses to different intracellular and extracellular conditions. Metabolomics investigations often use sophisticated analytical techniques such as NMR spectroscopy to provide an unbiased and comprehensive approach to evaluate metabolic perturbation …

Utilizing Nmr Spectroscopy And Molecular Docking As Tools For The Structural Determination And Functional Annotation Of Proteins, Jaime Stark

Department of Chemistry: Dissertations, Theses, and Student Research

With the completion of the Human Genome Project in 2001 and the subsequent explosion of organisms with sequenced genomes, we are now aware of nearly 28 million proteins. Determining the role of each of these proteins is essential to our understanding of biology and the development of medical advances. Unfortunately, the experimental approaches to determine protein function are too slow to investigate every protein. Bioinformatics approaches, such as sequence and structure homology, have helped to annotate the functions of many similar proteins. However, despite these computational approaches, approximately 40% of proteins still have no known function. Alleviating this deficit will …

Affinity Chromatography In Environmental Analysis And Drug-Protein Interaction Studies, Efthimia Papastavros

Department of Chemistry: Dissertations, Theses, and Student Research

This dissertation will examine the use of novel affinity sorbents to extract emerging contaminants from water. These contaminants include carbamazepine, an anti-epileptic drug which is resistant to natural degradation in the environmental and to drinking water treatment procedures. This drug has been found in fish, drinking water, estuarine and coastal waters, and river sediment and has been used as a general marker of contaminants in wastewater. Carbamazepine was one of the most commonly detected compounds in surface-water and groundwater samples in a recent reconnaissance study of untreated drinking water sources in the U.S. Besides using this drug as a representative …

Development And Optimization Of Organic Based Monoliths For Use In Affinity Chromatography, Erika L. Pfaunmiller

Department of Chemistry: Dissertations, Theses, and Student Research

Affinity chromatography is an important and useful tool for studying biological interactions, such as the binding of an antibody with an antigen. Monolithic supports offer many advantages over traditional packed bed supports in affinity chromatography, including their ease of preparation, low back pressures and good mass transfer properties. Monoliths can be broken down into two basic categories: organic (polymer) and inorganic (silica) monoliths. There are many varieties of polymer based monoliths; however, a large focus has been on co-polymers of glycidyl methacrylate (a functional monomer) and ethylene dimethacrylate (a cross-linking agent). The solvents of choice for making this type of …

Preparation And Characterization Of Biomimetic Hydroxyapatite-Resorbable Polymer Composites For Hard Tissue Repair, Kristopher R. Hiebner

Department of Chemistry: Dissertations, Theses, and Student Research

Autografts are the orthopedic “gold standard” for repairing bone voids. Autografts are osteoconductive and do not elicit an immune response, but they are in short supply and require a second surgery to harvest the bone graft. Allografts are currently the most common materials used for the repair of segmental defects in hard tissue. Unlike autografts, allografts can cause an undesirable immune response and the possibility of disease transmission is a major concern. As an alternative to the above approaches, recent research efforts have focused on the use of composite materials made from hydroxyapatite (HA) and bioresorbable polymers, such as poly-L-lactide …

Characterization Of Novel Macrocyclic Polyether Modified Pseudostationary Phases For Use In Micellar Electrokinetic Chromatography And Development Of A Chemiluminescence Presumptive Assay For Peroxide-Based Explosives, Raychelle Burks

Department of Chemistry: Dissertations, Theses, and Student Research

This work describes the first use and characterization of macrocyclic polyether (MP) modified sodium dodecyl sulfate (SDS) pseudostationary phases (PSPs) for use in micellar electrokinetic chromatography (MEKC), as well as the development of a presumptive chemiluminescence assay for peroxide-based explosives. In MEKC separation and detection, resolution is optimized by using various PSPs or by altering the properties of a single PSP using different class I or II modifiers. Class I modifiers target the PSP through direct interaction with micelles, while class II organic modifiers operate by altering the BGE. The of MPs 18-crown-6, 15-crown-5, and 12-crown-4 were used to modify …

Optimization And Implementation Of Entrapment: A Novel Immobilization Technique For High-Performance Affinity Chromatography, Abby J. Jackson

Department of Chemistry: Dissertations, Theses, and Student Research

The proper use of high performance affinity chromatography (HPAC) in the study of biologically-related systems requires that careful attention be paid to the nature in which the affinity ligand is incorporated into the stationary phase.  Ideally, the behavior of the immobilized ligand should mimic the behavior of the ligand in its natural environment.  Retaining the soluble form of the ligand, by avoiding covalent immobilization completely, is one effective way to retain the activity of the ligand.  Previously, noncovalent immobilization techniques that do not modify the ligand of interest have included physical entrapment onto low performance supports.

This dissertation introduces an …

Ultrasonic Activation Of Triacetone Triperoxide, Latravia R. Dobson

Department of Chemistry: Dissertations, Theses, and Student Research

Triacetone triperoxide (TATP) is an organic peroxide that has received widespread attention in recent years. TATP is a ketone peroxide with a high active oxygen content. It is relatively shock sensitive, with explosive decomposition easily initiated, and is therefore considered a primary explosive. However, TATP is also a powerful explosive possessing about 83 % of the power of TNT. TATP can be prepared quickly and easily from inexpensive household chemicals in the absence of any specialized facilities, making it the explosive of choice for modern day terrorists. TATP poses a major challenge for security and law enforcement services around the …

Chromatographic Studies Of Drug-Protein Binding In Diabetes, Kathryn (Krina) S. Joseph

Department of Chemistry: Dissertations, Theses, and Student Research

Drug-protein binding can have a dramatic impact on the distribution and metabolism of a drug. This manuscript describes the use of high-performance affinity chromatography to examine the binding of various compounds to human serum albumin (HSA) in normal and diabetic disease states. The first study examined the use of four coumarin compounds as possible alternatives to warfarin as a probe for Sudlow site I on HSA. High-performance affinity chromatography and immobilized HSA columns were used to compare and evaluate the binding properties of these probe candidates. It was found from this group that 4-hydroxycoumarin was the best alternative to warfarin …