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Diethylaminoethyl cellulose was titrated with acid and base in various concentrations of NaCl. The pK values were determined from titration curves using a graphical technique. Two titratable groups were observed from the acid titration curve, a major and a minor group. The minor group, which represented 20% of the acid titration, was barely detectable in the base titration curve and had a salt-independent pK of 6.0. The pK of the major group was strongly salt-dependent and was approximately a linear function of log N NaCl. An equilibrium shift caused by the counter ion may explain the salt dependence. Hydrogen bonding may explain the presence of the minor group. The salt dependence of the pK of ion exchange celluloses may explain pH shifts and artifactitious peaks occasionally observed in column chromatography of proteins.

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Due to the rising health care costs and with the advent of biogenerics, there is a growing demand to develop new and reliable techniques to characterize proteins and biopharmaceuticals. In addition, characterization aids in understanding the intricate relationship between a protein's structure and its function. To address this challenge, two protein structural parameters, 1) amino acid surface area and 2) amino acid microstructure, were chosen to be investigated. Two chromatographic techniques, 1) ion exchange chromatography (IEC) and 2) immobilized metal affinity chromatography (IMAC), were used to characterize the above-mentioned protein structure parameters. The model protein chosen for our work is T4 lysozyme. The protein consists of 164 amino acids with molecular weight ~ 18 kD. SYBYL 7.1 software was used to generate in silico point mutants. Two categories of protein variants (point mutants) were generated using site-directed protein mutagenesis. The goal for generating point mutants was to obtain mutants that vary in the two structural parameters. The first category point mutants vary in the surface accessibility of a surface accessible histidine residue. The second category point mutants predominantly vary in protein net charge and the amino acid microstructure. In total, seventeen point mutants were generated: 1) category I consists of seven variants that vary predominantly in their histidine surface accessibility, and were obtained by replacing a charged amino acid residue at different locations on the surface of the protein molecule, and 2) category II consists of ten variants that vary in both net charge and charge distribution were obtained by replacing charged and neutral amino acid residues at different locations (different microenvironments) on the protein surface. PCR technique was used to generate the point mutants. Gene and protein sequencing were employed to confirm the veracity of point mutation. CD and Lysozyme activity assays were performed to determine whether or not the 3D structure of all the protein variants was intact. Zonal analysis was used to obtain the binding strength values of all seventeen variants in IMAC with copper as the immobilized metal ions, and gradient elution method was used to obtain the relative retention times (rRT) values of all the variants in IEC. The seven lysozyme variants generated in category I each contains one surface histidine residue. In IMAC, there is a correlation between the surface accessibility of the lone surface histidine and the protein's binding strength with R²⁺= 0.76. In IEC, the correlation between the protein's microstructure, which predominantly consists the surface accessibility of the histidine residue, and the protein's retention times was R²⁺= 0.95. However, there were few outlier variants (e.g. variant K83H) which did not follow the correlations. The variations presented by few outlier variants can be attributed to the presence of intramolecular bonds, which restrict the mobility of the amino acid side chains and subsequently hinder the specific interaction between the amino acid residue and chromatographic media. For category II variants, short and medium range charge perturbations around the sole histidine residue in T4 lysozyme were engineered within 15 Ã distance of histidine. There was a strong correlation (R²⁺ = 0.96) between the theoretical (DeltaDeltaGElec) values, calculated using simple Coulomb's law, and the experimental (DeltaDeltaGB) values, which were obtained by measuring the protein binding strength values using IMAC. Similar correlation (R²⁺= 0.93) was obtained between the change in net charge (-2 to +2 units) and the relative retention times in IEC. Similarly, there were few variants (e.g. S136K, R76D) that did not follow the trends. The deviations of the few outlier variants can be attributed to the presence of unique microstructure effects around the histidine residue. These microstructure effects were quantified in IMAC as (DeltaDeltaGMicro), and in IEC they were quantified by the change in rRT values. In summary, all seventeen variants had different binding strengths and rRT values indicating the variation in the protein structure around the histidine residue. Our work reveals that it is possible to capture the microstructural effects of a protein through the combination of protein molecular modeling and simple chromatographic experiments. / Ph. D.

Anion exchange methods were used to measure the percent of weakly bound and strongly bound copper in estuarine waters and agricultural drainage. Copper bound to an ion exchange resin has been operationally defined as bioavailable copper, or copper that is available for uptake by aquatic organisms. Initial laboratory testing indicated that the percent of copper bound to the resin did not differ between a freshwater solution (pH 6.5 buffered with 30 mg/L as calcium carbonate) and a saline solution (25 ppth salinity at pH 8). Adding 20 mg/L natural organic matter (NOM) to each solution lowered the amount of copper sorbed to the resin by the same amount in saline and fresh water. Varying salinity from 16 to 30 ppth did not effect % bioavailability. This indicates that the ions typically found in seawater do not compete with copper for ion exchange onto the resin. Varying initial copper concentration from 195 to 495 ppb for the given NOM content of 12.5 mg/L did not effect % bioavailability, indicating that as the copper concentration of this water is increased over the range studied, the fraction of potentially toxic copper is constant. Decreasing the pH of the system from 8.5 to 4 and the NOM from 12.5 to 0 mg/L statistically increased % bioavailability in both cases. However, copper retention onto the resin at pH values near 4 was hindered, possibly because resin functional groups become inefficient at low pH values. MINEQL+ predicted that the resin was not retaining approximately 30% Cu⁺² at a pH of 4. Copper retention to the resin was also not as high as expected at NOM contents between 0 and 3 mg/L at pH 8.1. MINEQL+ indicated that the water studied at pH 8.1 was comprised primarily of strongly bound copper-organic ligand species and Cu(OH)₂. Experimental results suggested that the resin retained only 50% Cu(OH)₂, which is a potentially bioavailable copper complex. However, MINEQL+ computer modeling indicated that the resin was accurately retaining potentially bioavailable copper in waters with a pH from 5.5 to 8.5, containing 3 to 12.5 mg/L NOM. After assessing the characteristics of copper retention by the resin, it was applied to an estuarine system and agricultural drainage to measure bioavailable copper. The results indicated that in copper-containing agricultural runoff and groundwater the percent bioavailable copper was approximately 15% of the dissolved copper. This percentage was relatively constant over the range of copper concentrations observed. In estuarine systems receiving agricultural runoff, the percent bioavailable copper was about 11%. This research was supported by a grant from the Virginia Pesticide Control Board. / Master of Science

Recent studies funded by the US Department of energy have shown that coal and coal byproducts contain elevated contents of Rare Earth Elements (REEs), making them a potential resource for these critical materials. The approach employed in this research focused on the concentration and extraction of REEs from fine coal refuse derived from various preparation plants in the Appalachian coal basin of the United States. Initial efforts in this research focused on the identification and characterization of REEs in various fine coal refuse streams from nine distinct industrial preparation plants in Appalachia. The average REE content in these materials was determined to be approximately 200 ppm, but the REE content showed a strong correlation to the aluminum content, suggesting that the REEs are closely associated with the clay minerals present in the refuse. Given the relatively low REE concentrations, initial efforts sought to concentrate the REEs through decarbonization and dispersive liberation steps. In these tests, high-shear agitation in the presence of a polyelectrolyte, followed by sedimentation was able to isolate the REE-enriched fine clay particles from siliceous gangue minerals. Following the dispersive liberation step, all samples were found to have an REE content greater than 300 ppm, a benchmark used for many initial exploratory studies. In one case, the REE content was increased by more than 125%. Subsequent extraction tests initially utilized a direct ion-exchange leaching approach with ammonium sulfate as lixiviant. In all cases, the simple ion-exchange leaching process failed to recover significant quantities of rare earth elements, ultimately suggesting that the REEs in fine coal waste may be passivated or bound in a colloidal phase. To access this colloidal phase, several alternative approaches were evaluated, including leaching with alternative ion-exchange lixiviants, reductive leaching, gas-purged leaching, and others. The approach that showed the most promise was strong alkaline pretreatment, followed by ion-exchange leaching with ammonium sulfate at pH 4. A combination of strong alkali and high-temperatures treatment successfully liberated the REEs, converting them to a form amenable to ion-exchange leaching. The highest REE recovery achieved with this method was determined to be 39%. Lastly, bench-scale solvent extraction tests were used to further concentrate REEs in the leach solution and demonstrate that mixed rare earth concentrates can be successfully produced from fine coal refuse. / Master of Science / Since the introduction of personal electronics, rare earth elements (REEs) have become essential raw materials for modern life. They are used in many common household goods such as cell phones, computers, and flat screen TVs. They are also vital components in various industrial, medical, and military applications. Currently, the majority of the world's supply is obtained from China, which has raised concerns on the vulnerability of the supply chain and the potential impacts of supply disruption on clean energy technologies. In light of this risk, the US Department of Energy has classified a number of REEs as critical elements and has subsequently funded research to investigate ways to diversify the supply chain through alternative resources. The approach employed in this research seeks to extract and recover REEs from fine coal refuse. This industrial waste is a byproduct of the coal mining and beneficiation processes. Given the long legacy of coal mining in the Appalachian region, hundreds of millions of tons of fine waste are currently being stored in surface impoundments, and millions of tons of additional fine coal waste is being produced each year from active mining and beneficiation operations. By valorizing this waste material through REE recovery, mining companies will be incentivized to reprocess existing impoundments, ultimately promoting superior economic and environmental outcomes. Despite their name, rare earths are not "rare" from the standpoint of raw abundance; however, their scarcity is derived from the complexity of the extraction and separation processes. In China, the majority of the heavy rare earth elements are produced from ion-exchangeable clays. These clays have REEs weakly attached to the surface, so that they can be readily recovered by washing them with a salt solution that remove the positively charged rare earth ions from surface. The technical approach employed in this project sought to replicate this process for the clay materials found in fine coal refuse. Additional steps were needed to properly concentrate, activate, and extract the REEs; however, the end-to-end processing tests confirmed that mixed rare earth concentrates can be produced from fine coal wastes consisting primarily clay minerals.

This dissertation investigates three major problems in IC, ranging from the practical to the theoretical. The first is of interest to the field of quantitative analysis. Since ion chromatography is primarily a quantitative technique, the linear range of the detector is of particular interest. The linear range was determined to be nearly five orders of magnitude for several common ions. The probes used were fluoride, chloride, sulfate, sodium, and potassium with concentrations ranging from 10 ppb to 1000 ppm. This portion of the dissertation describes the first attempt made to elucidate the linear detection range in ion chromatography using micro-membrane suppression. The Iinearity experiments demonstrate the versatility of ion chromatography with respect to quantifiable concentrations. However, a major problem is encountered when attempting to determine a trace ion concentration in the presence of a high concentration of a similarly charged ion (matrix ion). The second part of this dissertation offers a solution to this problem. In ion chromatography, the large excess of a matrix ion affects not only the ions expected in that region of the chromatogram, but also destroys the chromatographic exchange process. One objective of this study was to develop a rapid quantitative method to determine 5 ppm chloride in the presence of 100,000 ppm sulfate. A standard anion exchange column will completely lose resolution between chloride and sulfate at a sulfate concentration of approximately 2500 ppm. The apparatus designed and built by the author uses two analytical ion—exchange columns coupled in series by a high pressure switching valve. ln order to complete the system, the author designed and built y a high pressure conductivity detector cell to monitor the conductivity of the effluent from the first column. The system works by overloading the first column and then allowing the effluent to pass onto the second column. When the trace chloride in on the second column, the first column is switched out of line. This process allows the trace chloride to be separated from the residual sulfate. The column switching study brought to light several theoretical questions about the inner workings of the packing materials used in ion chromatography. These questions include the effects that flow rate and temperature have on capacity and efficiency in ion chromatography separations. Using chloride and sulfate as probes, the chromatographic efficiency was shown to decrease from 30° to 60° C. Even more surprising was the result that sulfate retention increased with increasing temperature while chloride retention decreased under the same conditions. Detailed studies as well as possible explanations are included in the third part of this dissertation. / Ph. D.