Methods for determination and speciation of inorganic and organic selenium in complex matrices involving, inductively-coupled plasma mass spectrometry (ICP-MS), inductively-coupled plasma atomic emission spectrometry (ICP-AES) and high performance liquid chromatography (HPLC) were developed. Selenium species were separated and detected by mixed-mode weak anion exchange and reversed-phase stationary phases with ICP-MS and ICP-AES detection. Selenium-containing nutritional supplements, wheat flour and selenized yeast, were analyzed and the major selenium species were identified and quantified by HPLC-ICP-MS equipped with a dynamic reaction cell (DRC). The quantification response factors for different selenium and sulfur compounds, analyzed by DRC-ICP-MS and ICP-AES were determined. Possible reasons for the obtained results were discussed. The effects of arsenic and selenium added simultaneously to the growth media of Helianthus annus (sunflowers), red sunflowers and Eichhornia crassipes (water hyacinths) were determined with respect to accumulation and metabolism. Total selenium and arsenic in shoots and roots was determined. Quantification of the selenium and arsenic species in E. crassipes by HPLC-ICP-MS and HPLC-ICP-AES was also performed. An accurate and reliable method was developed for the determination of functionalized gold nanoparticles in hepatocarcinoma cell tissues and rat brain material by ICP-MS. A comparison between the gold accumulated in cell and brain tissues treated with neutral, positive and negative functionalized gold nanoparticles was performed.

Speciation of selenium is of interest because selenium is both essential and toxic to humans, depending on the species and amount ingested. Following the discovery in the Clark study that selenium supplementation (in the form of selenized yeast) could reduce the incidence of some types of cancer, selenium-enriched yeast and other materials have been commercialized or proposed as supplements. However, regulation with respect to quality, storage conditions, stability and content of selenium supplements is a problem. The speciation and stability of organoselenium compounds in yeast-based and yeast-free selenium supplement samples have been studied. Enzymatic extraction enabled selenium speciation profiles to be obtained by ion-pair reversed phase high performance liquid chromatography with inductively-coupled plasma mass spectrometry (HPLC-ICP-MS) and gas chromatography with atomic emission detection (GC-AED). Prior to the GC analyses, the extracts were derivatized with ethylchloroformate. Coated fiber solid-phase microextraction (SPME) was used to extract volatile selenium species for determination by GC-AED and GC-mass spectrometry (GC-MS). Total selenium concentrations were obtained by ICP-MS and ICP-optical emission spectroscopy (ICP-OES). Close similarities of speciation were observed between extended storage and brief heating at elevated temperatures of yeast-based selenium supplements. In both cases selenomethionine, the major component in these supplements, was observed to degrade to form S-(methylseleno)cysteine, a sulfur-selenium amino acid, and selenomethionine selenoxide. Selenium concentrations in saliva were determined by electrothermal atomic absorption spectrometry (ET-AAS) and flow injection-atomic absorption spectrometry with hydride generation (FI-HG-AAS). Selenium concentrations were comparable in both methods.

Group III Metal alkyl compounds, such as trimethylindium (TMI), trimethylaluminum (TMA), and trimethylgallium (TMG) are used in metal organic chemical vapor deposition (MOCVD) process to grow semiconductor layers. The analysis of these metal alkyls that are used for growing semiconductor layers is important to ensure product quality. In addition to determining the impurity concentration, its speciation is equally important. This is because volatile impurities are more harmful to the product than the non-volatile ones because they get easily incorporated in the final product. Attempts were made to analyze the samples by inductively coupled plasma-atomic emission spectrometry (ICP-AES) by dissolving them in xylene and kerosene. Different types of calibrations were performed and it was determined that a positive bias exists if non-volatile standards are used for quantification of analytes. This bias is mainly because the impurity analytes are volatile in nature hence more sensitive in nature. A chemometric scheme was developed to remove this calibration bias by analyzing samples at two different spray chamber temperatures. This scheme results in data that are consistent with decomposition experiments where the sample was converted to inorganic form prior to analysis. As verification experiments, exponential dilution results were obtained and they agree with decomposition and chemometric schemes. A graphite furnace was used for the quantification of volatile analytes. Chemical modifiers (ethers with heavy alkyl groups) were used to achieve a temporal separation between the matrix and the analyte. This solved the matrix suppression effect on analytes due to simultaneous vaporization of the matrix and analyte. The concentrations of analytes from different techniques were found to be consistent with each other.

The development of methods for the determination of cadmium and lead at the ultra-trace concentration level has been the object of great attention during the past 20 years. The reason for this interest is their high toxicity even at these low levels, given the fact that once exposure has occurred the elements are accumulated in the body. Several instrumental techniques have been applied to the determination of cadmium and lead in biological and environmental samples, such as urine, blood, hair, foodstuffs, natural and domestic waters, etc. The variation in the complexity of these matrices is quite large, so that measuring the contents of cadmium and lead is a difficult operation because of the concentration of interfering species coexisting in the samples with a low concentration of the analytes. The application of chemical vapor generation procedures to this task offers the possibility to transfer the analyte to a medium free of interferences in a concentration that facilitates a determination by means of spectrometric techniques. In the work described in this dissertation, flow injection methods for the determination of cadmium and lead were devised according to two approaches: the generation of the vapor species with simultaneous transport and detection in an atomization T-cell placed above the burner of a Perkin Elmer 3100 atomic absorption spectrometer; and the generation of the vapor species for their preconcentration on the iridium-coated interior surface of a graphite furnace atomizer, whose temperature was thereafter raised for atomization and detection in two electrothermal atomic absorption spectrometers: a Perkin Elmer 4100ZL, and a Perkin Elmer SIMAA6000. For both approaches, the limits of detection obtained were in the low ng l–1 range. Special attention was given to: (a) the testing of several different designs of gas-liquid separators to maximize the efficiency in the transfer of the analytes from the sample to the atomizer while minimizing transfer of liquid, (b) corroborate the enhanced efficiency reported for several additives such as surfactants and transition metals, and (c) improve the limits of detection in the determination of lead by minimizing contamination of the analytical blank.

Two versions of an enclosed inductively coupled plasma source for atomic emission spectrometry have been developed for the direct analysis of specialty gases: a low-flow enclosed ICP (EICP) discharge sustained inside a spherical quartz container, and an extended, closed-torch ICP (ECT-ICP) based on a conventional ICP torch. The former is optimized for halogens and hydrogen halogenides, and the latter is designed for volatile inorganic hydrides, phosphine in particular. Both techniques offer rapid multielement analysis, wide linear dynamic range, direct sample introduction, minimal sample consumption, and instant atomization of both particulate and gaseous impurities. All parts of the arrangements on are leak-proof and corrosion resistant to ensure safe handling of toxic, corrosive, or otherwise reactive samples and to minimize sample contamination. Four complementary calibration approaches have been studied: gas standard dilution, vapor phase introduction, and exponential dilution, Arsine, silane, and hydrogen sulfide diluted with argon were used as gas standards for As, Si, and S calibration. Volatile compounds (e.g., trimethylaluminum, carbon tetrachloride, iron pentacarbonyl, diethylzinc, trimethylchlorosilane, triethylgermanium chloride, tetramethyltin, tetraethyllead) were introduced into an exponential dilution flask flushed with argon for Al, C, Fe, Pb, Si, Sn, and Zn calibration. Different organometallic compounds can be employed in the exponential dilution technique; however, some of them partially decompose or adsorb to the surfaces before reaching the EICP discharge owing to their reactivity. Indirect semiquantitative calibration based on the ratios of spectral line intensities measured in the EICP and a conventional ICP has been evaluated. The electron and excitation temperatures in both sources were determined and maintained in a certain fashion, so that atomic line intensities in both were correlated. Three chlorine samples were analyzed using an EICP discharge in 100% chlorine. Detection limits ranged from low ppb (Al, As, Fe, Ge, Pb, Sn, Si, Zn) to sub-ppm (C, S) levels. Phosphine samples were diluted 10-fold with argon for the analysis by ECT-ICP, and the detection limits in phosphine were approximately 10 times higher than in chlorine. Hydrogen bromide was analyzed qualitatively.

Speciation has become an important area of research in the fields of food and environmental analysis because the nutritional value and toxicity of an element depend on its chemical form. For that purpose a Gas Chromatography separation device was used coupled with Atomic Emission Detector (GC-AED) that provided detection for the specific element of interest even though when it may be present in very small amounts or co-elute with other components. The effect of hydrogen and oxygen reagent gases on selenium and sulfur detection using the GC-AED was studied, as well as the helium make-up flow gas. The figures of merit for both selenium and sulfur were studied. In theory, the GC-AED response should be independent of the structure of the compound that is analyzed and only depend on the number of atoms of the element on the plasma. The objective was to determine if the AED response was compound independent for Se and S. In the cases where compound independent calibration behaviour was observed, empirical formula determination was made for that element. In order to study amino acids by GC, they need to be derivatized first in order to make them volatile enough for GC. The derivatization procedure chosen was ethylation by ethylchlroformate because is a simple procedure that derivatizes both amino and carboxylic group in one step. The derivatization parameters, such as amount of reactant, best solvent, reproducibility, extraction yield and effect of interferences were studied. Enriched selenium samples, such as yeast, garlic, bacteria, seeds, and nutritional supplements, were studied. The selenium profile in those samples was compared depending on the sample and the total amount of selenium present. The selenium profile was also compared with the sulfur profile for those samples. Also, different extraction procedures were tried, such as hot water, TMAH, and enzymatic extraction. The percentage of selenium as selenomethionine in the samples was calculated either by external calibration or standard addition. Geochemical samples were also analyzed using the GC-AED and selenium-containing compounds were identified in those samples.

New methods for the determination of cadmium and lead by flow injection chemical vapor generation atomic absorption spectrometry with tetrahydroborate immobilized on an anion-exchanger were developed. Both flow injection and hydride generation parameters were optimized. The method has less suppression by some coexisting elements than the generation by the reaction with tetrahydroborate in aqueous solution, and smaller amounts of reagents are required. The developed method was successfully applied for the determination of trace amounts of cadmium and lead in environmental and biological materials. A method for the determination of trace concentrations of cadmium, by electrothermal atomic absorption spectrometry with flow injection chemical vapor generation from a terahydroborate-form anion-exchanger with in-atomizer trapping on a Zr-Ir coated graphite tube atomizer, was developed. Both electrothermal atomization and flow injection hydride generation parameters were optimized. A detection limit of 2 ppt was obtained. The method was successfully applied to the analyses of various types of environmental and biological samples. The cold vapor generation of mercury from tin chloro anion complexes immobilized on an anion exchanger was successfully developed for the first time. A purer tin(II) chloride reagent was obtained by passing solution through the anion-exchange column. The method was applied to the determination of trace amounts of total and inorganic mercury in different types of sample matrices with satisfactory results. A procedure for the speciation of mercury from the immobilized tetrachlorotin(II)- and tetrahydroborate-form anion-exchange columns was developed. Better tolerance of some coexisting elements was found compared to that of conventional cold vapor generation techniques. The method was applied for the speciation of mercury in canned fish and seafood samples and in some standard reference materials. A mercury speciation procedure for environmental and biological samples based on in-atomizer trapping on a gold-coated graphite tube atomizer and electrothermal atomic absorption spectrometry was developed. Both types of immobilized reducing agent gave satisfactory results. A new method for preconcentration of trace inorganic mercury in water samples as the anionic-chloro complex on anion-exchanger with reductive elution with tetrahydroborate solution was developed. The detection limit was 0.8 ppt. The method was applied to the analysis of natural water, spring water, drinking water, tap water and seawater samples. Cadmium and lead were determined in Typha angustifolia and Lemna minor in support of phytoremediation studies. Typha angustifolia is a suitable plant for the phytoremediation of lead contaminated soil. The interaction of cadmium and lead on the phytoextraction by Typha angustifolia in contaminated soil was investigated. The methods developed required only a small sample size from Lemna minor plants, so that multiple samples from the same plant could be taken.

The new stationary phases developed in HPLC need to be characterized to benefit from selectivities that may be offered by the newly developed columns. Such selectivities could be valuable in environmental analyses, drug development and other applications. This research was involved in the exploration of metal chelates as possible test probes in the characterization of columns. The metal complexes with aromatic rings gave satisfactory results and offered an opportunity of characterizing columns at longer detection wavelengths that may reduce interferences usually observed at the commonly used shorter wavelengths. Characterization of columns was performed on both silica-based and zirconia-based phases by measuring the efficiency, hydrophobicity, steric selectivity and shape selectivity of the columns. The Diamondbond C18 column was found to display characteristics similar to that of silica-based columns when compared to ZirChrom-PBD. It was also observed that reporting the steric selectivity of columns, additional information may be obtained by including the relative retention factors and resolution of analytes. Such information has not been previously reported. The selectivity of the Diamondbond C18 and silica-based columns was also compared in the determination of metal complex isomers. In this study it was observed that the DiamondBond C18 had better selectivity at high pH and temperature than the silica-based columns. At high pH and elevated column temperatures the DiamondBond C18 separated the isomers of N, N' 2,3 butylenebis(trifluoroactylacetoinato) copper(II) and palladium(II), whereas the silica-based columns could only separate the Cu(II) isomers. The interaction of the DiamondBond C18 with the metal complex isomers is discussed as a function of both pH and temperature. The application of beta-diketone ligands in derivatization Cr(III) ions in water and urine samples was also explored. The benefit of employing larger ligands at longer wavelengths is highlighted. The chromatography of metals complexed with β-diketones, bidentate salicylaldime, tetradentate salicylaldimies, non-fluorinated β-ketoamine and fluorinated β-ketoamine ligands was explored using both silica-based and zirconia-based columns. Chromatographic trends observed by varying bridging groups on the metal complexes are reported for both the silica-based and zirconia-based columns. Lastly, the newly developed silica-based and zirconia-based columns were evaluated in the separation of arsenic and selenium species. The behavior of several silica-based columns with tetrabutylammonium cationic-pair reagent in separating arsenic and selenium species has been compared at mid and low pH. The possibilities of employing modified silica-based and zirconia-based columns in the separation of arsenic and selenium species at high pH has been discussed.

Methods for the determination and speciation of selenium and arsenic in complex matrices involving, inductively coupled plasma mass spectrometry (ICP-MS) coupled with high performance liquid chromatography (HPLC) was developed. Selenium species were separated and detected by fluoro-acid ion-pairing chromatography with ICP-MS detection and the arsenic species were separated and detected by anion-exchange chromatography with ICP-MS detection. Selenium-containing nutritional supplements, including commercially available selenium supplements, yeast samples, mushroom and garlic samples were analyzed and the major selenium species was identified and quantified by HPLC-ICP-MS. A novel amino acid, S-(methylseleno)cysteine containing a selenium-sulfur group was identified in selenized yeast. Thermal stability studies of the selenized yeasts and selenium-enriched yeast tablets suggest that S-(methylseleno)cysteine is formed at higher temperatures. In Brassica juncea (Indian mustard) plant shoots and roots various selenium species were identified and quantified, after growing the plants in selenium containing [Se(IV) and Se(VI)] hydroponic media for 40 days. The identification of various organoselenium species in the plant shoots and roots will help elucidate the selenium metabolic pathway in selenium-accumulators. A method was developed for the determination of total selenium in selenium-enriched yeast materials by electrothermal atomic absorption spectrometry (ETAAS) with direct slurry sampling. After establishing that organoselenium species are borohydride active in a flow-injection hydride-generation system, a procedure was developed to determine selenomethionine in yeasts and water samples. Total selenium in enzymatic extracts of yeasts was also determined. An ultrasound-assisted procedure was developed for the extraction of arsenic species from soil standard reference materials, spiked soils and plant material. The extracted arsenic species were separated by anion-exchange chromatography prior to determination by plasma source mass spectrometry. Various arsenic species were identified and quantified in the soil standard reference materials and hydroponically grown Brassica juncea shoots and roots.

Due to its versatility, mass spectrometry (MS) has been recognized as an excellent tool for the analysis of peptides and proteins. The structural information that this technique has provided for metalloproteins so far, however, has not extended much to coordination structure. Metal catalyzed oxidation (MCO) reactions in conjunction with MS analysis have been utilized to determine the metal-binding sites of proteins. This method involves first selectively oxidizing the protein by generating reactive oxygen species (ROS) in the immediate vicinity of the metal. These ROS can quickly react with the amino acids that are part of the metal-binding site. The oxidatively modified residues are then identified using a combination of proteolytic enzymes and the peptide sequencing ability of MS. The initial focus of the dissertation was on finding the most specific MCO reaction conditions to site-specifically oxidize the amino acids bound to Cu and Fe in several proteins with known binding sites to test the viability of this approach. Relatively general reaction conditions involving ascorbate as a reducing agent and O2 and/or H2O2 as oxidizing agents have been found. The utility of this MCO/MS approach was assessed by the application of the method to β-2-microglobulin (β2m), which has unknown Cu-binding sites. The MCO/MS approach allowed us to gather information on Cu coordination under conditions that would make such determinations very difficult by other techniques. Interestingly, our results show that different conformers of β2m have different Cu coordination environments, and these results may provide insight into the Cu-induced amyloidogenesis of this protein. The MCO/MS procedure relies on collision-induced dissociation (CID) of oxidized peptide fragments. Hence, a better understanding of the dissociation patterns of oxidatively modified peptides has also been sought to facilitate identification of the oxidized residues. In general, the dissociation patterns of oxidized peptide ions are found to depend on peptide composition, charge state, and type of modification. More specifically, oxidation of methionine and cysteine residues, but not histidine residues, has a dramatic effect on the dissociation of peptide ions when the charge state of the peptide is less than or equal to the number of basic residues in the peptide.