A range of fluorescence fluctuation analysis methods were developed and applied to labels commonly used in biological samples. Various super-resolution techniques were demonstrated in vitro and in vivo using objective-type Total Internal Reflection Fluorescence Microscopy (TIRFM). An experimental Fluorescence Correlation Spectroscopy (FCS) setup was developed along with associated post-acquisition data algorithms. The technique was applied to investigate the stoichiometry of a protein subunit of ribonucleotide reductase (RNR). FCS method was adapted with an Electron Multiplying Charge Coupled Device (EMMCD) detection scheme and applied to inorganic Quantum Dots (QDs) diffusing in solutions of different viscosities. Super-resolution Optical Fluctuation Imaging (SOFI) algorithm was implemented in ImageJ software and conclusive results obtained on reference samples of QDs and combed DNA. Potential applications of temporal correlation analysis to the study of diffusive processes and single particle tracking were also discussed. A new super-resolution technique applicable to multiple adjacent fluorescent molecules called Direct Object Resolution by Image Subtraction (DORIS) was developed and tested with QD complexes. The method enables one to accurately map the position of two emitters displaying intermittency in their fluorescence emission and separated by a distance below the diffraction limit, without the need of complex instrumentation or analysis. The technique relies on the subtraction of the Point Spread Function (PSF) of each single fluorescent probe, and is in theory applicable to any blinking or flickering dye. The principle was first demonstrated on simulated data and experimentally on QDs coupled by 100-basepair double-stranded DNA constructs. Super-resolution by image subtraction was further applied in vivo in S. pombe cells, where distances between clustered fluorescent fusion proteins were accurately determined. The selective activation of photoswitchable probes mEos3 was exploited to optimise the DORIS subtraction process and provided a simple method to determine the relative positions of closely spaced emitters within an aggregate, as encountered in association sites or multimeric complexes.

The National Air Emission Monitoring Study (NAEMS) project was funded by the Agricultural Air Research Council (AARC) to evaluate agricultural emissions nationwide. Utah State University (USU) is conducting a parallel study on agricultural emissions at a Cache Valley poultry facility. As part of this parallel study, samples of animal feed, eggs and animal waste were collected weekly from three manure barns (designated: manure barn, barn 4 - manure belt and barn 5 - high rise) from May 2008 to November 2009. These samples were analyzed to determine ammonia content, total Kjeldahl nitrogen content and ammonia emission. The yearly average calculated NH3 values for manure barn, barn 4 and barn 5 were determined in units of mg NH3/gmanure as: 1.1 ± 0.2, 0.6 ± 0.1 and 0.8 ± 0.1, respectively. The yearly average calculated TKN values in units of % N were determined as: 2.0% ± 0.3, 1.6% ± 0.3 and 1.9% ± 0.3 for manure barn, barn 4 and barn 5, respectively. The yearly average of NH3 emission in units of mg NH3/bird-day was determined to be 440 ± 180 mg NH3/bird-day for barn 4, and 540 ± 190 mg NH3/bird-day for barn 5. The ammonia and organic amines emissions in ambient air at a Cache valley confined poultry facility were measured by using a sulfuric acid trapping solution in an impinger train followed ion chromatography (IC) detection. The yearly average concentrations of ammonia in ambient air at the barns were calculated at 11.9 ± 2.9 ppm at the manure belt barn and 12.7 ± 3.1 ppm at the high rise barn. No organic amines were detected in the collected ambient air samples by the ion chromatography method. Because there were no amines detected by the IC method, limits of detection of organic amines in air were studied. The results showed that the organic amines in the manure must occur at a minimum concentration of 1 ppm in order to have sufficient vapor pressure so that enough is transported to the impingers for trapping and subsequently be detected by the IC.

Raman spectroscopy is normally a non-destructive, highly selective technique that has become an ubiquitous tool for analytical chemists. One of the primary limitations of Raman spectroscopy, however, is the relatively low cross-section of the technique. With signal enhancements relative to normal Raman scattering as high as 1011, the ultra-trace detection of adsorbates down to the single molecule level has been achieved with SERS. Despite the dramatic improvement in the sensitivity and the high selectivity afforded by the SERS method, the acceptance of SERS as a general analytical tool has been hindered by a lack of stability and reproducibility in the substrates. This lack of stability has been particularly troublesome because unstable substrates exhibit reduced shelf lives as well as a reduced ability to monitor processes that occur under non-ideal conditions such as high temperature or harsh chemical environments. In this thesis, two different works are reported that address the two major hurdles facing the SERS field in the development of a stable and reproducible SERS substrate. First, the development of a SERS-active substrate that exhibits improved temporal and thermal stability and is capable of in-situ high temperature measurement of analytes adsorbed on the surface is presented. The substrates are prepared by depositing an ultra-thin layer of alumina by Atomic Layer Deposition (ALD) onto silver island films grown by thermal evaporation. We demonstrate the application of alumina-coated substrates to the measurement of the dehydration of trace amounts of calcium nitrate tetrahydrate as a function of temperature. As a development of the above mentioned work, the combination of a silver/gold layered architecture obtained by thermal evaporation with an ultra-thin alumina overlayer to generate a re-usable SERS substrate that is simple, relatively inexpensive and stable is reported. The relative thicknesses of the silver and gold and the alumina overlayer was optimized to deliver the maximum SERS enhancement and optimal stability when the substrate was subjected to high temperature. Utilizing the method of thermal desorption of the analyte, the substrate surface is regenerated and able to be reused multiple times with little reduction in SERS activity. Second, in the development of a reproducible SERS substrate, the application of monodisperse silver nanocube colloidal substrate in microfluidic SERS is demonstrated. In static SERS experiments, one often has to search for “hot spots”, which are positions of a drastically increased SERS signal compared to the rest of the probe volume, in an inhomogeneous solution. To overcome this problem and prevent the decomposition and or fragmentation of SERS substrate and analyte, respectively, the implementation of flow cell is a promising way. At the beginning analyte, colloidal solution and aggregation agent were brought into a mixing chamber, where they were thoroughly mixed before being directed to a sample cell for detection. With this method, a relatively high amount of sample volume is necessary. In addition to the advantages listed above, efforts have been made to reduce the required amount of the sample solution by the design of low-cost poly (dimethylsiloxane) chips via soft lithography technique. The sample solution is passively pumped through the microfluidic channel, where an optical detection window is implemented for acquisition of a SERS spectrum.

Photon-based tandem mass spectrometry provides a versatile ion activation strategy for the analysis of polypeptides, proteins, and lipids. 351-nm ultraviolet photodissociation mass spectrometry (UVPD-MS) is a facile and selective tandem dissociation technique used to elucidate chromophore-modified peptides within large mixtures. A bis-aryl chromogenic chemical probe was utilized to target solvent exposed primary amine residues within native protein states. Collision-induced dissociation (CID) was employed to indiscriminatly characterize the complete proteolytic digest while chromophore containing peptides were selectively dissociated with 351-nm UVPD; thus streamlining the identification of targeted peptides with structurally informative residues. Protein amine residue reactivities were then compared with predicted solvent exposures to elucidate protein tertiary structures, their mechanistic properties, and ligand-binding interactions. High-energy 193-nm UVPD is a fast, high-energy tandem mass spectrometry method and frequently generates fragment ions typically inaccessible to CID-based methods. Native mass spectrometry was coupled to top-down 193-nm UVPD for the gas phase characterization of non-covalent protein-ligand and protein-protein complexes. This method yielded a unique array of fragment ions for a comprehensive analysis of protein structures. UVPD of non-covalent complexes generated many polypeptide backbone fragments to characterize the primary sequence of proteins. Furthermore, top-down UVPD engendered cleavages with intact electrostatic interactions; this provided insight into the binding interfaces within protein-ligand complexes and the higher order structural architectures of oligomeric complexes. High-resolution 193-nm UVPD was paired with high performance liquid chromatography (LC) for the streamlined structural analysis of amphiphilic glycolipids within complex mixtures. For all glycolipids, UVPD provided the most comprehensive structural analysis tool by affording a diverse array of fragment ions to characterize both hydrophobic and hydrophilic moieties. UVPD based LC-MS separations of gangliosides shed light on the ceramide lipid bases, glycan moieties, and their isobaric structural variants. UVPD activation of lipid A and lipooligosaccharides (LOS) compounds generated a mixture of C-C, C-O, and C-N fragment ions to illustrate the hydrophobic acyl structures, while cleavages within the glycosidic, and cross-ring cleavages allowed the determination of acylation patterns. Novel LC-MS separation strategies were developed to elucidate and structurally characterize complex mixtures of lipopolysaccharide containing compounds. / text

This thesis describes new analytical methods for trace or ultra-trace analysis by molecular absorption and emission spectroscopy. The initial part of the thesis is devoted to an introduction to molecular electromagnetic absorption spectroscopy and molecular fluorescence. The principles, advantages and limitations of spectrophotometric and fluorimetric measurement are discussed. The concepts of enzymes and their applications in analytical chemistry are also expounded. Two organic compounds, 5-( 4-arsonophenylazo )-8-(p-toluenesulfonamido)quinoline (APTSQ) and 5-(p-methoxyphenylazo)-8-(p-toluenesulfonamido) quinoline (MPTSQ), have been synthesised and used as new chromogenic anellor f1uorogenic reagents. Five specific, highly sensitive, simple, precise and inexpensive novel analytical methods have been developed: (1) A spectrophotometric method is described for the determination of cobalt. The maximum absorbance is at 582 nm with a molar absorptivity of 1.18 x 1 as I mor' cm-1 . Beer's law is obeyed for cobalt concentrations in the range 0-0.5 J.l.g mr'. (2) A fluorimetric method for the determination of cobalt is proposed. The fluorescence intensity is measured at Aex 287 nm and Aem 376 nm. The response is linear up to 25 ng mr' and the detection limit is 0.002 ng mr'. The mechanism of the fluorescence reaction has been investigated. (3) A flu~ri~etric method is proposed for the d~ter~i~~tion of H202. The response IS linear up to 12.2 ng mr H:t>2. The detection limit IS 0.16 ng mr'. (4) An enzymatic assay for glucose by spectrofluorimetry is described. The fluorescence intensity is proportional to the concentration of glucose up t0180 ng mr'. A detection limit of 5.4 ng mr' was obtained and allowed the determination of glucose in an extremely small amount of serum (O.5J.1.I) and urine (1 J.l.1). (5) A fluorimetric method for the determination of iron is proposed, based on the reaction between iron(III) and MPTSQ in the presence of cetyltrimethylammonium bromide. The fluorescence intenSity (Aex=317 nm, A.em=534 nm) is linear up to 170 ng mr' with a detection limit of 0.12 n9 mr'. An investigation of the mechanism of the fluorescence reaction has been made. The applications of the proposed methods for the determination of the concerned analytes at low levels in biological, environmental, pharmaceutical or beverage samples are also reported.

This thesis is principally concerned with spectroscopic and computational studies of artists' pigments. Manuscripts, art and archaeological artefacts were examined by Raman microscopy, identifying the pigments and drawing conclusions for historical and conservation purposes. Studies of Anglo Saxon and later manuscripts have shown the Insular palette triumvirate, assumed to be orpiment, red lead and verdigris, to contain red ochre and vergaut, but no verdigris. This remains unchanged until the introduction of lazurite in c. 920 AD and vermilion in the 12th century. Lazurite has been erroneously identified on the Lindisfarne Gospels, by the technique of Roosen-Runge. Raman microscopy shows the blue pigments to be exclusively indigo, casting doubt on analyses performed using Roosen-Runge's technique. The Islamic manuscript palette was found to be remarkably consistent across a substantial geographical area over an extended period. It is also very similar to that of early Western manuscripts. Comparison of these results with existing literary sources has shown the latter to be highly inaccurate. The palette of William Blake was examined and compared to results of analysis by False Colour Infrared Photography (FC-IP). The FC-IP technique was determined to be inappropriate for pigment identification. Two significant artefacts were shown to be modern forgeries: a rare Assyrian fresco contains a modern green pigment, and the world famous Vinland map was found to have significant quantities of anatase in the yellow lines. Density Functional Theory methods were applied to the mechanism of decay isomerisation of As4S4, which was partially clarified, and to the geometries of R2SeX2 (R = CF3, CF2H, CFH2, CH3, CH2CH3, CH(CH3)2, t-Butyl, X=F, Cl, Br, I, At). The most stable geometry was found to be determined by the polarity of the Se-X bonds and the steric and electron-withdrawal effects of the R-group on the C-Se bond strength.

<p> This dissertation details original work focused on the DNA adduct N2-(1-carboxyethyl)-2'-deoxyguanosine (CEdG). This DNA adduct results from the spontaneous reaction of DNA with the endogenous and exogenous formed, carbohydrate-derived, reactive carbonyl species, methylglyoxal. Using <i>in vitro</i> steady state kinetics, we have shown that CEdG in template DNA leads to DNA miscoding effects when the model replicative polymerase, exonuclease-free Klenow fragment (KF-) is used. The development, validation and application of a novel stable isotope dilution, triple quadrupole mass spectrometric method for the quantitation of CEdG is also detailed. This method was used to quantitate CEdG in urine from diabetic rats, urine from human patients, human tumor and adjacent biopsy tissue, diabetic animal tissue and DNA treated with methylglyoxal. Finally, we detail the adaptation, validation and application of a novel, commercially-available microfluidic HPLC-chip for increased sensitivity in the quantitation of CEdG and also apply it to the quantitation of the RNA analogue, CEG. Combined, these studies establish CEdG as a potential biomarker for glycation and point to a viable avenue for connecting chronic glycolytic flux with genetic instability. </p>

<p>Over half of the biosolids produced in wastewater treatment facilities in the United States are land-applied as a nutrient-rich soil amendment. However, these biosolids are not regulated for chemicals of emerging concern that are often present at high concentrations in biosolids. The overall goal of this dissertation is to evaluate the specific chemical fates of these compounds in biosolid-amended soil, including their persistence, degradation pathways, and phytoaccumulation potential. </p><p>As a first step toward this goal, the fate of select brominated flame retardants (BFRs) when exposed to sunlight was examined to evaluate their photodegradation pathways and to identify degradation products that may be used as markers of environmental degradation in future studies. In Chapter 2, the photodegradation of three polybrominated diphenyl ether (PBDE) congeners (i.e., the nonabrominated congeners BDE 206, 207, and 208) was examined individually in three different solvents exposed to natural sunlight. Rapid degradation of nonaBDEs was observed coincident with formation of octa- and heptabrominated PBDEs. The photodegradation pathways of each nonaBDE congener were consistent among the different solvent matrices tested; however, mass balances were found to vary with solvent type. The octabrominated congener, BDE 202, and the ratio of BDE 197 to BDE 201, were identified as degradation products that can serve as environmental markers of debromination. Additional photodegradation studies were conducted with two new BFRs used in replacements for pentaBDE mixtures: 2-ethylhexyltetrabromobenzoate (TBB) and di(2-ethylhexyl)-tetrabromophthalate (TBPH). Both TBB and TBPH underwent photolysis more slowly than nonaBDEs and primarily formed debrominated products. This study is the first to report on the photodegradation of TBB and TBPH via debromination reactions and suggests that these replacement flame retardants may be more photolytically persistent than higher brominated PBDE congeners.</p><p>Chemical analysis of biosolids collected from wastewater treatment plants (WWTPs) can help determine whether these flame retardants are migrating from the indoor environment to the outdoor environment, where little is known about their ultimate fate and effects. In Chapter 3, concentrations of a suite of flame retardants and the antimicrobial compound triclosan were measured in opportunistic samples of municipal biosolids and the domestic sludge Standard Reference Material (SRM) 2781. Grab samples of biosolids were collected from two WWTPs in North Carolina and two in California. Biosolids samples were also obtained during three subsequent collection events at one of the North Carolina WWTPs to evaluate fluctuations in contaminant levels within a given facility over a period of three years. The biosolids and SRM 2781 were analyzed for PBDEs, a suite of alternate brominated and chlorinated flame retardants, and triclosan. PBDEs were detected in every sample analyzed, and &#931;PBDE concentrations ranged from 1750 to 6358 ng/g dry weight (dw). Additionally, the PBDE replacement chemicals TBB and TBPH were detected at concentrations ranging from 120 to 3749 ng/g dw and from 206 to 1631 ng/g dw, respectively. Triclosan concentrations ranged from 490 to 13,866 ng/g dw. The detection of these contaminants of emerging concern in biosolids suggests that these chemicals have the potential to migrate out of consumer products and enter the outdoor environment. Furthermore, land application of these contaminated biosolids may result in soil contamination and enhance the bioaccumulation and long-range transport potential of these compounds. </p><p>In order to fully evaluate the benefits and impacts of biosolids land application, a comprehensive view of the behaviors of chemicals of emerging concern in biosolids is needed. In Chapter 4, the fates of a suite of flame retardants and triclosan in soil were evaluated in a greenhouse experiment utilizing three biosolid amendment levels (control, low, and high) and two vegetation treatments (unplanted and planted with alfalfa (<italic>Medicago sativa</italic>)). BDE 47, BDE 209, TBB, TBPH, and triclosan declined significantly in the high biosolid-amended vegetated soil between Days 0 and 28 (p < 0.05), and then reached a plateau between Days 28 and 90 during which no further significant loss from soil was observed. In contrast, no significant losses of those analytes were observed from soil at the high biosolids amendment in non-vegetated pots. The percent of a given analyte lost from the vegetated soil at the high amendment between Day 0 and the plateau ranged from 43.1% for TBPH to 60.9% for triclosan and was significantly and negatively related to the log octanol-water partition coefficient (log K_OW) of the analyte (p = 0.0103, R<super>2</super> = 0.9178) and marginally significantly and positively related to the log of water solubility (p = 0.0686, R<super>2</super> = 0.7213). Alfalfa root and shoot tissues were monitored for the analytes of interest but no clear evidence of phytoaccumulation was observed. Methyl triclosan formation was observed in the biosolid-amended soils during the study period, indicating that biotransformation played a role in the observed dissipation of triclosan. The results of Chapter 4 demonstrate that PBDEs, selected alternate BFRs and triclosan are highly recalcitrant in biosolid-amended soils but capable of undergoing dissipation in the presence of alfalfa and, in the case of triclosan, biotransformation. </p><p>In conclusion, this dissertation provides a comprehensive view of the fates of flame retardants and triclosan in biosolid-amended soil, identifying markers of degradation that can be used in complex real-world scenarios, developing methods for measurement of a diverse suite of analytes in biosolids and plant tissues, and demonstrating the persistence of these compounds in biosolid-amended soil.</p> / Dissertation

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