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A porous disk device is described that was used for the introduction of sample solutions into a flow system and for on-line extractions. A 0.5$\mu$m 316L stainless steel disk with a 6.35mm diameter and 1.6mm thickness was placed in a Teflon piece of the same thickness and then sandwiched between two blocks of Lucite. Flow channels were made on either side of the disk by cutting oval-shaped holes in Teflon spacers inserted between the disk and the blocks. The disk's void volume was determined with the device placed in a computer-controlled flow manifold. Samples of As(III) were injected, collected in a titration cell and titrated with electrogenerated Ce(IV). The generated titration curves were used to determine the microequivalents of Ce(IV) at the endpoint which were then used with the titration reaction stoichiometry to determine the volume of As(III) injected. The y-intercept of a plot of As(III) volume injected vs. injection times adjusted for flow rates was the void volume of the porous disk. Three determinations using this experiment yielded an average disk volume of 20.60 $\pm$ 0.32$\mu$L. Reproducible injections of a nickel sulfate solution were demonstrated. The device was adapted to perform on-line extractions of caffeine from aqueous solutions into methylene chloride. The aqueous solutions were flowed past the porous disk so that the caffeine was extracted into the CH$\sb2$Cl$\sb2$ contained in the disk. A dispersion of 6.6 was determined for the injection of the plug of solution in the disk into the flow stream and delivery to the detector. A calibration curve of the steady state absorbance vs. concentration of caffeine solutions in $\rm CH\sb2Cl\sb2$ yielded a molar absorptivity coefficient of 6785 M$\sp{-1}$cm$\sp{-1}$. Extraction efficiencies from 0.53 to 2.1%, were obtained for a series of aqueous caffeine solutions ranging in concentration from 0.10002mM to 10.002mM and using a sample volume of 393$\mu$L. The effect of increasing the time that the aqueous caffeine solution was exposed to the $\rm CH\sb2Cl\sb2$ was investigated. The extraction efficiencies increased for hold times up to 180s, after which a decrease in extraction efficiencies was observed. Finally, the feasibility of analyzing real samples containing caffeine was demonstrated through the on-line analysis of a cola sample.

This work includes the investigation and determination of several classes of disinfection by-products from various naturally occurring model compounds using several oxidative and instrumental techniques. Analytical methodology was developed for the determination and identification of nitrosamine species by gas chromatography with time-of-flight mass spectral detection. In addition, this method was successfully evaluated against existing techniques, and applied to real, raw water effluent samples. Identification and determination of various iodinated disinfection byproduct species was accomplished for the further elucidation of potential health risks associated with the municipal disinfected drinking water supply. Finally, products from the oxidation of a model ketoacid (pyruvic acid) by hypochlorous acid were determined to gain a greater understanding of the possible byproducts produced from a treatment facility that utilizes ozonation as primary disinfectant and hypochlorous acid as a secondary oxidizer.

Human serum Transferrin (hTf) is a ∼80 kDa protein, whose function is iron sequestration and transport. The two lobes of the protein (commonly referred to as N- and C-lobes) have a very high degree of structural identity and provide two distinct binding sites for a ferric ion. In addition to iron, serum transferrin also binds a variety of other metals and is believed to provide a route for the in vivo delivery of such metals to cells. In the present study ESI MS is used to investigate interactions between human serum transferrin and two non-ferrous metals (indium and bismuth), conformational changes upon metal binding, as well as characterize human serum transferrin N-lobe (hTf/2N) global dynamics and functionally important local dynamic events. The In-hTf complex was directly detected by ESI MS; the Bi-hTf complex in solution was established by monitoring the evolution of charge state distributions of transferrin ions upon acid-induced protein unfolding in the presence and in the absence of the metal in solution. The large size of Bi3+ ion is likely to prevent formation of a closed conformation (canonical structure of the holo-protein), resulting in a non-native metal coordination which causes anomalous instability of the transferrin-bismuth complex in the gas phase. The apo-hTf/2N and Fe-hTf/2N were used in hydrogen/deuterium exchange (HDX) measurement for characterizing protein dynamics. In this measurement, back-exchange was corrected for every transferrin N-lobe peptic fragment individually. The results showed that iron binding induce more compact conformation and significant decrease of HDX kinetics around hinge regions and Lys206 which is one amino acid from the dilysine-trigger. However, the changes around iron binding sites are not as significant. Our hypothesis is that instead of having frozen states, transferrin has certain frequency of conformational hopping. A new method of rapid detection and identification of disulfide-linked peptides in complex proteolytic mixtures was developed utilizing the tendency of collision-activated peptide ions to lose preferentially side chains of select amino acids in the negative ion mode.

Mass spectrometry has enjoyed enormous popularity over the years for studying biological systems. The theme of this dissertation was to develop and use mass spectrometry based tools to solve five biologically oriented problems associated with protein architecture and extend the utility of these tools to study protein polymer conjugation. The first problem involved elucidating the false negatives of how proteins with few basic residues, forms highly charged ions in electrospray ionization mass spectrometry (ESI MS). This study showed that the unfolding of polypeptide chains in solution leads to the emergence of highly charged protein ions in ESI MS mass spectra, even if the polypeptide chains lack a sufficient number of basic sites. In the second problem, a new technique was developed that can monitor small-scale conformational transitions that triggers protein activity and inactivity using porcine pepsin as a model protein. This work allowed us to revise a commonly accepted scenario of pepsin inactivation and denaturation. The physiological relevance of an enzyme-substrate complex was probed in our third problem. We observed by ESI MS that pepsin forms a facile complex with a substrate protein, N-lobe transferrin under mildly acidic pH. The observed complex could either be a true enzyme-substrate complex or may likely results from an electrostatically driven association. Our investigation suggested that the enzyme binds nonspecifically to substrate proteins under mild acidic pH conditions. The fourth problem dealt with the investigation of conformational heterogeneity of natively unstructured proteins using a combination of spectroscopic techniques and ESI MS as tools. It was observed that four different conformations of alpha-synuclein coexist in equilibrium. One of these conformations appeared to be tightly folded. Conclusions regarding the nature of these states were made by correlating the abundance evolution of the conformers as a function of pH with earlier spectroscopic measurements. The final problem was aimed at monitoring conformational transitions in polypeptide and polymer segments of PEGylated proteins using PEGylated ubiquitin as a model system. This studies suggested that for a PEGylated protein, polypeptides maintain their folded conformation to a greater extent whiles the polymer segments are bound freely to the protein.

Cellular Retinoic acid binding protein I (CRABP I), a member of intracellular lipid binding proteins (iLBPs), binds physiologically to a mostly hydrophobic ligand, all-trans Retinoic Acid (RA). The binding site is inside an internal cavity, inaccessible in a static apo-protein conformation. Binding of RA to CRABP I does not result in significant changes of the protein tertiary structure, suggesting significance of dynamics in the ligand recognition and binding processes. One of the proposed scenarios for the protein-ligand binding process invokes the notion of a flexible portal region adjacent to the binding site, while another model suggests that the requisite dynamic events are induced by dimerization of the apo-protein in solution. In this work RA binding to CRABP I is studied in dilute solutions (low micro-molar range), where no dimer and/or oligomer formation occurs. Modulation of backbone dynamics within various segments of the protein by its ligand is assessed using a combination of hydrogen exchange, electrospray ionization mass spectrometry and collision-induced dissociation of protein ions in the gas phase. Consistent with the portal model of ligand entry, several protein segments are flexible in the absence of the ligand. At the same time, the two segments containing arginine residues forming a salt bridge with RA form the least flexible region in the apo-form of the protein. Although the presence of RA in solution reduces flexibility of all protein segments, the largest effect is observed within four strands that form one of the two β-sheets enveloping a cavity, which houses the ligand-binding site. These results are consistent with a model in which ligand binding occurs through a partially unstructured state of the protein with unobstructed access to the ligand-binding site. This intermediate (whose core is formed by the two stable arginine-containing strands) corresponds to a relatively low-energy local minimum on the apo-protein energy surface and is frequently sampled under native conditions. Intermediate states were visualized under various mildly denaturing conditions and characterized by HDX/CAD ESI MS. The similar amide backbone protection pattern suggests that the intermediate states bear significant resemblance with the activated intermediate state.

Metal complexes play fundamental roles in synthetic, biological, and catalytic processes, and when doing so they are often present at very low concentrations. Determining the coordination structure of complexes at trace levels or in complicated mixtures, however, is a very difficult task. This dissertation focuses on the investigation of a new methodology, based on ion-molecule (I-M) reactions and mass spectrometry (MS), to determine the coordination structure of metal complexes sensitively. This work describes the study of the gas-phase reactivity exhibited by a variety of model transition metal complexes with different reagent ligands in a quadrupole ion trap mass spectrometer. A series of tetradentate and pentadentate ligands containing N, O, and/or S donor atoms were synthesized and complexed with late first-row transition metals. Noticeable differences in reactivity were found as the ligand field around the central metal ion was changed. The electronic structure of the metal center was also found to exert a remarkable effect on the gas-phase reactions of metal complexes, and in fact, complexes with different geometric structures could be distinguished. Angular overlap model (AOM) and density functional theory (DFT) calculations were used to explain the experimental observations. The analytical utility of the I-M reactions was also investigated. Fluctuations in reagent and buffer gas pressures and vacuum system temperature were examined as possible error sources affecting the conclusions drawn from the I-M reaction procedure. Reagent gas pressure and temperature were found to be the most important factors affecting the reproducibility of the experimental data. Because the analytical information derived from these I-M reactions relies on complexation of a reagent ligand, steric factors that might affect these reactions were also evaluated. In summary, I-M reactions can distinguish complexes with different coordinating functional groups and geometries, and these reactions have some potential for providing coordination structure information for complexes present at trace levels.

Forensic scientists attempt to solve crimes through examination of the evidence found at crime scenes. By determining the relative concentrations of certain elements present in bullets, a profile of an individual sample can be developed that will allow investigators to match ammunition from a crime scene with that from a known source. The removal of a substantial proportion of the lead matrix is required for the determination of trace elements in bullets by inductively coupled plasma mass spectrometry (ICP-MS). Several flow injection methods for the selective removal of lead from bullets have been developed. On-line solid-phase extraction with the commercially available resin Pb-Spec® removed the matrix quantitatively. Pb-Spec incorporates an immobilized crown ether, giving the resin selectivity for lead. The analytes were not retained, but passed through the column for quantification by ICP-MS. The method was validated by analyzing NIST SRMs 2415 (battery lead), 2416 (bullet lead), and 2417 (lead-based alloy). The injection of large sample volumes into a flow injection ICP-MS system produces transient peaks with flat tops. The signal at peak maximum is directly proportional to analyte concentration. Sample throughput can be improved by making replicate measurements during the residence time at peak maximum instead of requiring multiple sample injections. A successful procedure utilizing Pb-Spec for the removal of matrix lead from a bullet sample was developed, and validated by the analysis of SRMs. On-line precipitation and filtration of the matrix was examined. The bullet sample was combined with sulfuric acid, and the conditions for maximum precipitation were obtained. The resultant lead sulfate was filtered on a column packed with macerated glass wool. The formation of sulfates by the analytes was not favored, so they were detected by ICP-MS. The potential for lead preconcentration by Pb-Spec was investigated with environmental water samples, and the retention characteristics were examined in detail. The feasibility of incorporating a crown ether-containing supported liquid membrane into a flow injection manifold was investigated for the removal of high concentrations of lead from solutions. Experimental and theoretical results demonstrated that the time scale of mass transfer was too slow for flow injection applications.

The development of nephrocalcinosis, the progressive formation and deposition of oxalate crystals in the kidneys, in premature infants is a relatively common and potentially serious condition. Although hyperoxaluria and hypercalciurea are both associated with the pathogenesis of nephrocalcinosis, it is believed that hyperoxaluria may be more important in enhancing the risk of calcium oxalate precipitation. Therefore, dietary measures to decrease urinary oxalate excretion in preterm infants may lead to decreased incidence of nephrocalcinosis. Total parenteral nutrition (TPN) solutions can act as a direct source of oxalate introduction into premature infants since they contain ascorbic acid (vitamin C) which is known to oxidize to oxalate in-vitro. Since increased urinary oxalate excretion has been reported in infants receiving TPN is was of clinical interest to be able to determine whether the infusion of TPN solutions into premature infants resulted in an unnecessary oxalate burden. A robust ion chromatographic method with suppressed conductivity detection was developed for the reliable determination of oxalate at part-per-million (ppm) levels in TPN solutions. An eluent system based on sodium carbonate and sodium bicarbonate was developed, permitting the separation and selective detection of oxalate from the major anionic components of the TPN matrix. The interference effect of method induced oxalate formation from ascorbate, which is known to hinder the determination of oxalate in TPN by currently available methodologies, was shown to be absent. The application of uncertainty in measurement was applied to ensure that a low level of experimental uncertainty was carried through to the obtained data. The effect of non-linear analyte response that is commonly reported when using carbonate and bicarbonate eluents with suppressed conductivity was evaluated. The deviation of calibration data from a linear response was found to be only very slight over the oxalate concentrations studied (0.2 ppm–30 ppm). The application of the developed method to the preliminary study of oxalate formation in TPN subjected to phototherapy conditions was successfully demonstrated.

A method was developed for the determination of total arsenic in rice grain by microwave-assisted digestion inductively coupled plasma mass spectrometry. Standard calibration solutions were matrix-matched with respect to acid concentration and carbon content post-digest. The importance of eliminating the drying step during sample preparation procedures was investigated. The method was validated with spikes containing standard arsenate solutions into the rice matrix, and with certified reference material SRM1568a (rice flour) from NIST. The method was successfully applied to a commercially available rice sample. Four arsenic species [arsenate (As(V)), arsenite (As(III)), dimethylarsinic acid (DMA) and monomethylarsonic acid (MMA)] were extracted from rice grains by microwave-assisted extraction and separated with high performance liquid chromatography inductively coupled plasma mass spectrometry. The method includes a novel sample clean-up step involving a dialysis procedure to decrease the amount of large starch molecules in the injection solution, in order to minimize poor resolution of chromatographic peaks and maximize column life. The method was validated with spikes of standard arsenic solutions, added to the rice matrix before the extraction procedure. Literature reference values for arsenic species quantification in SRM1568a (rice flour) were also compared. This method was successfully applied to a commercially available rice sample. A study into improvements in reverse phase-HPLC separations of arsenic species was conducted. For the first time, a Sunfire C8 column from Waters (Milford, CT) was employed for the separation of arsenic species in rice extracts. This column was compared to a Symmetry C8 column with respect to total elution time, detection limits, interference effects, and column life, and evaluated with respect to peak resolution, shifts in retention times, and peak symmetry.