Isoelectric focusing is one of the most important techniques in protein separations. Preparative-scale isoelectric separations often use buffering membranes (isoelectric membranes), but there are no good known methods for the characterization of their pI values. Therefore, UV-absorbing carrier ampholyte mixtures (UVCAs) have been synthesized, analytically characterized, and utilized for the characterization of the pI value of a buffering membrane. To synthesize the UVCAs, addition of a UV-absorbing electrophile, 3-phenoxypropyl bromide (PhOPrBr), to a pentaethylenehexamine (PEHA) carrier ampholyte backbone, resulted in an intermediate that was subsequently reacted with increasing amounts of acrylic acid (up to 8 equiv) and itaconic acid (up to 2 equiv) via Michael’s addition. The intermediates and final products were characterized by 1H-NMR and full-column imaging capillary isoelectric focusing techniques. An optimal blended mixture of selected UVCAs was first desalted and purified by isoelectric trapping and its composition verified by full-column imaging isoelectric focusing. The mixture of UVCAs possessed a broad pI distribution from approximately pH 3 – 10. By isoelectric trapping, the mixture was separated into two subfractions with a polyacrylamide-based isoelectric membrane of known pI as the separation membrane and poly(vinyl) alcohol-based buffering membranes as the restriction membranes. The pI of the most basic UV-active carrier ampholyte in the anodic fraction was determined to be 4.4 and the pI of the most acidic UV-active carrier ampholyte in the cathodic fraction was determined to be 4.4, confirming that the pH of the polyacrylamide-based isoelectric membrane was pH 4.4.

Isoelectric focusing is one of the most important techniques in protein separations. Preparative-scale isoelectric separations often use buffering membranes (isoelectric membranes), but there are no good known methods for the characterization of their pI values. Therefore, UV-absorbing carrier ampholyte mixtures (UVCAs) have been synthesized, analytically characterized, and utilized for the characterization of the pI value of a buffering membrane. To synthesize the UVCAs, addition of a UV-absorbing electrophile, 3-phenoxypropyl bromide (PhOPrBr), to a pentaethylenehexamine (PEHA) carrier ampholyte backbone, resulted in an intermediate that was subsequently reacted with increasing amounts of acrylic acid (up to 8 equiv) and itaconic acid (up to 2 equiv) via Michael’s addition. The intermediates and final products were characterized by 1H-NMR and full-column imaging capillary isoelectric focusing techniques. An optimal blended mixture of selected UVCAs was first desalted and purified by isoelectric trapping and its composition verified by full-column imaging isoelectric focusing. The mixture of UVCAs possessed a broad pI distribution from approximately pH 3 – 10. By isoelectric trapping, the mixture was separated into two subfractions with a polyacrylamide-based isoelectric membrane of known pI as the separation membrane and poly(vinyl) alcohol-based buffering membranes as the restriction membranes. The pI of the most basic UV-active carrier ampholyte in the anodic fraction was determined to be 4.4 and the pI of the most acidic UV-active carrier ampholyte in the cathodic fraction was determined to be 4.4, confirming that the pH of the polyacrylamide-based isoelectric membrane was pH 4.4.

Polyelectrolytes of opposite charge in aqueous solution can undergo a liquid-liquid phase separation known as complex coacervation. Complex coacervation of ampholytic proteins with oppositely charged polyelectrolytes is of increasing interest as it results in a protein rich phase that has potential applications in food science, protein therapeutics, protein purification, and biocatalysis. However, many globular proteins do not phase separate when mixed with an oppositely charged polyelectrolyte, and those that do phase separate do so over narrow concentration, pH, and ionic strength ranges. Much of the work that has been done on complex coacervates looks at polymer-polymer systems. While there have been some initial studies showing that proteins can undergo complex coacervation, the major design factor studied to date has been overall protein charge. The tools of genetic engineering, which allow the precise tuning and placement of charge have not been used to more fully understand the design criteria for protein complex coacervation. In this dissertation, we developed a model protein library based on green fluorescent protein (GFP) to study the impact of protein net charge and charge distribution on protein phase separation with polyelectrolytes. We developed a short, ionic polypeptide sequence (6-18 amino acids) that can drive the liquid-liquid phase separation of globular proteins. We characterize the phase behavior of the protein library with a homopolymer and diblock copolymer of similar chemistry to elucidate how protein design impacts macro- and microphase separation. In these phase characterization studies, differences in the nature of phase separation as well as the salt stability of the protein coacervates with the different polymer species are identified. We finally used this model protein library to study the effects of the protein design and phase separation behavior for coacervate-based applications including intracellular protein delivery, purification, and protein stabilization.

Department of Chemistry / As an alternative energy source, lithium ion batteries have become increasingly important with a wide range of applications in industry, and many international companies are investing in this big project. This study was aimed at the development of safer lithium-ion power sources by using new organic additives to overcome the possible safety problems. In this study, the conformations and energies of several synthesized boronates were investigated through computational study using density functional theory (DFT) with the Becke’s three-parameter hybrid method utilizing the Lee-Young-Parr correlation functional (B3LYP). After initial energy optimization using Møller-Plesset Perturbation theory (MP2), the conformational preferences and energetics in vacuo were investigated using DFT calculations and the 6-31G(d,p) basis set. Subsequently, cyclic voltammetry and electrochemical impedance spectroscopy were used to characterize the compounds in terms of their usefulness as electrolyte additives. At least two of these show excellent promise for use in lithium-ion batteries.