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.

The increasing demands for speciation in nutrition, clinical chemistry and toxicology have made the sole determination of the total element in a sample unsatisfactory. It is important to determine the chemical form (species) of the element; most often its oxidation state or its organometallic or complexed nature, since different species of the same element can range from harmless to essential to toxic. In order to address this challenge, a separation technique (gas chromatography, GC) that usually focuses on qualitative analysis was effectively interfaced with an element specific detection technique, atomic emission spectrometry (AED). Likewise liquid chromatography (HPLC) was interfaced to inductively coupled plasma mass spectrometry (ICP-MS) for the characterization of selenium compounds. The consumption of selenium nutritional supplements has been reported to provide anticarcinogenic benefits that are selenium compound dependent. The separation and identification of these selenium species is important in order to understand their activity and metabolism. In this study, methods were developed for the characterization of selenium species in selenized yeast, breath and urine samples. Enzymatic digestion of selenized-yeast samples followed by ethylchloroformate derivatization was used to dissolve amino acids and convert them to volatile compounds suitable for subsequent GC analysis. Volatile selenium species exhaled in breath were trapped on Tenax, then revolatilized by heating and sampled by solid phase microextraction. Gas chromatography with atomic emission detection (GC-AED) or gas chromatography with mass spectrometry (GC-MS) was employed for their detection. Selenium species in urine were characterized by ion-pair chromatography with ICP-MS detection. The monitoring of chromatographic eluents with element specific atomic spectroscopy detectors enabled the determination of selenium species in the enzymatic digests of selenized yeast, breath and urine samples. The analysis of archived selenized yeast samples by GC-AED and GC-MS revealed the presence of a previously unknown aminoacid, S-(methylseleno)cysteine. Low molecular weight organoselenium and mixed organosulfur-organoselenium compounds were identified by GC-AED in exhaled human breath samples from volunteers taking selenium nutritional supplements. Trimethylselenonium ion was identified in the urine of these volunteers by HPLC-ICP-MS.

Interaction of metal ions, both essential and toxic, with macromolecules is important in biological and environmental systems. The availability of trace metals may govern an organism's nutritional strategy in a given environment. Metal ions are rarely found free in most systems because of their active nature. The transportation of contaminants and nutritionally important metal ions in the environment is facilitated by their ability to bind and form complexes with humic substances and other ligands. Capillary electrophoresis-inductively coupled plasma mass-spectrometry (CE-ICP-MS) has been explored to characterize metal ions bound to various macromolecules because of the rapid separation coupled with high separation efficiency, resolving power and small sample size requirements together with sensitive elemental detection capability afforded by this hyphenated technique. Macromolecules examined here include humic substances, selenium compounds and selenoproteins, and some metal transport and storage proteins. Characterization of metal ion complexed humic substances by CE-ICP-MS is a formidable analytical challenge. Particularly of concern are the potential interactions with the CE capillary wall and the poor concentration detection limit inherent with the CE technique. Thus, sample preconcentration by flow field-flow fractionation was employed. Addition of zwitterions to the separation buffer helped to alleviate analyte-capillary wall interactions. A case study of trace metals bound to soil humic acids (HAs) that were successively extracted from the slopes of a local mountain was performed using the developed method. Trace metals complexed with HAs declined with diminishing oxygen-rich moieties in the HA structure. Analysis of selenoproteins extracted from yeast and selenium-containing compounds obtained from selenized supplement tablets by CE-ICP-MS were investigated. It was demonstrated that bare CE capillaries coupled to ICP-MS are suitable for speciation analysis of low molecular weight selenium compounds using a cationic surfactant separation buffer modifier. However, baseline resolution was not always achieved for selenoproteins owing to analyte adsorption onto the capillary wall. Likewise, speciation of some storage and transport proteins had been carried out with uncoated capillary CE-ICP-MS. Very high concentrations of the samples were employed because of low signal sensitivity. Improvement of the interface is necessary in order for kinetic and binding studies to be attempted.

Methods for selenium speciation using chromatographic and atomic spectrometric methods were developed. The objectives of the study were to quantify low concentrations of selenium and identify the different Se species present in some clinical and environmental samples. A 10 to 1000-fold improvement in detection limit was observed. The application of an inductively coupled plasma optical emission spectrometry (ICP-OES) instrument (a PerkinElmer Optima 4300DV) with flow injection hydride generation (FI-HG) sample handling (a PerkinElmer FIAS 400) as a method for the determination of selenium was described. The flow injection signals were processed by version 3.0 of PerkinElmer's Winlab32 ICP software that allows time-based transient signals to be processed, as well as the normal wavelength-based transients obtained with steady-state sample introduction. The FI-HG-ICP-OES was coupled to online preconcentration. A silica-based Discovery SAX resin was used as a preconcentration material for selenite. The method was used to determine percent recovery of low ppt levels of Se in different water samples such as spring water, river water, lake water, and seawater. The use of the Multi Mode Sample Introduction System (MSIS™) was also evaluated for the determination of selenium. The efficiency of the HG procedure improved the detection limit by 100 fold when compared to that of conventional nebulization. The procedure also allowed better tolerance to interfering ions. The Se uptake of Brassica napus was investigated. Different Se species were added to the soil and hydroponic media. Selenomethionine and the "newly identified selenoaminoacid", S-(methylseleno)cysteine were identified in the plant extracts by gas chromatography with atomic emission detection (GC-AED). Ion exchange chromatography with inductively coupled plasma mass spectrometry detection (IC-ICP-MS) was developed to identify the Se species in total parenteral nutrition (TPN) and the urine of neonates. Since the identity and amount of the selenium used in supplementation is known, identifying the resulting metabolites can give a new aspect on the selenium metabolic pathway in neonates. Preliminary determination of selenite by GC-AED was investigated. Inorganic Se in the form of selenite (SeO32-) was converted to piazselenol. The use of both 2,3-diaminonaphthalene and 4-chloro- o-phenylendiamine as derivatizing agents was investigated.

The parameters affecting the generation of stibine (SbH3) from homogeneous borohydride solutions and immobilized borohydride were evaluated for a flow injection system coupled to a quartz tube atomic absorption spectrometer. The stibine release and transport efficiency were affected by the design of the hydride generator and the vapor generation conditions. A new method for the determination of antimony by quartz tube atomic absorption spectrometry with flow injection chemical vapor generation from a tetrahydroborate-form anion exchanger was developed. Flow injection hydride generation parameters were optimized. Several samples could be injected before the column was reloaded with borohydride. Interferences from transition and hydride forming elements and signal suppression due to high ionic strengths were eliminated. The developed method was successfully applied for the determination of antimony in spiked natural waters. Several synthetic organic cation exchangers, inorganic ion exchangers and organic sorbent ion exchangers were evaluated as selective sorbents for inorganic antimony preconcentration without prior complexation. Neither the organic cation exchangers, nor the organic sorbent ion exchangers gave good results. Only one of the inorganic ion exchangers evaluated gave satisfactory results. The different parameters affecting the generation of arsine, bismuthine and hydrogen selenide from immobilized borohydride were evaluated by using a flow injection system coupled with a quartz tube atomic absorption spectrometer. Hydrides from these elements were generated from immobilized borohydride in the presence of other hydride-forming elements. In all cases, multiple injections were made before the column needed to be reloaded with borohydride. A new method for the simultaneous determination of antimony, arsenic, bismuth, selenium, tin and mercury by flow injection chemical vapor generation atomic emission spectrometry with tetrahydroborate immobilized on a strong anion exchange resin was developed for the first time. Both flow injection and vapor generation parameters were optimized. Simultaneous vapor generation, good sensitivities and low detection limits were achieved. The developed method was successfully applied to the determination of antimony, arsenic, bismuth, selenium, tin and mercury in natural water samples and a standard reference material with satisfactory results. Preliminary results for chemical vapor generation of manganese, zinc, nickel, cobalt, iron and lead were obtained.

Abstract not available

Abstract not available