This thesis focuses on understanding the interactions of cyanine dyes with DNA, through the use of both experimental and computational techniques. Although cyanine dyes are widely used as nucleic acids stains in fluorescence applications, the nature of the association process is not always clear. The cyanine dye PicoGreenRTM (PG) has proven to be extremely useful in monitoring DNA damage but its structure is proprietary, making it impossible to understand its specific interactions with DNA. However, its structure is known to resemble that of another cyanine dye, thiazole orange (TO). We anticipated that the N-propyl pyridinium derivative of TO (PTO) would also intercalate in DNA and that its extra positive charge, relative to TO, would aid in the association. Our studies have focused on the associations of PG, TO and PTO with DNA. Chapter 3 deals with the association of the dyes with single-stranded DNA homopolymers. The combination of spectroscopic techniques and molecular dynamics (MD) calculations provides a unique understanding of the associations of TO and PTO with single-stranded DNA homopolymers. There is highly specific binding of TO and PTO to poly(dG) and poly(dA), while poly(dC) and poly(dT) bind the dyes very weakly and appear to promote dye aggregation. Due to its proprietary structure, PG could not be studied computationally. However, the experimental spectral results suggest that PG associates differently with single-stranded DNA than do TO and PTO. There are two major findings in Chapter 4. Firstly, the dyes associate strongly with double-stranded DNA, as demonstrated by the experimental spectral results and the MD calculations. Experimental evidence supports not only monomeric dye intercalation in DNA, but also dimeric dye intercalation. The results of the MD simulations suggest that TO and PTO bind to DNA without sequence specificity. The second major finding was that a new type of stable dye/DNA complex is formed when single strands of poly(dA) and poly(dT) are hybridized in the presence of PG or PTO, which cannot be obtained by addition of the dye to poly(dA)·poly(dT). For all three dyes, complete DNA renaturation did not occur during thermal cycling of dye/double-stranded calf thymus DNA. These results suggest that intercalating cyanine dyes can interfere with DNA hybridization to double-stranded DNA. Chapter 5 is concerned with a more practical aspect of the association of cyanine dyes with DNA: using thiazole orange to report UVC-induced DNA damage. A variety of spectroscopic techniques, as well as agarose gel electrophoresis, were examined for their ability to detect UVC-induced DNA damage. The most sensitive methodology of all of those tested was fluorescence spectroscopy using TO. All of the spectroscopic techniques involving TO suggest that TO intercalation is susceptible to UVC-induced DNA damage. The computational studies suggest that the presence of cyclobutadipyrimidines, and not 8-oxo-2'-deoxyguanosine, is a factor in the experimentally observed reduction in dye intercalation. These studies have been a proof-of-concept, to demonstrate the usefulness of this fluorescence technique in detecting high levels of DNA damage, comparable to those used in food irradiation.

When a substance is above its critical temperature and pressure it is called a supercritical fluid. Supercritical fluids have the extraction power of solvents while possessing the diffusivity of gases. These properties hold exciting prospects for modern residue analysis methods for pesticides and other environmental pollutants. Three new and novel applications for supercritical fluid extraction are presented in this text. A rapid method for extracting petroleum hydrocarbons from soil is presented as well as analysis by gas chromatography-mass spectrometry and supercritical fluid chromatography. The method gives extraction recoveries greater than 90% with total analytical times of less than 1 hour. Two new methods for extraction and analysis of fourth generation insecticides are also presented. Avermectin, a natural product insecticide, is extracted from soil and animal tissue with recoveries over 80% and with no additional clean-up requirement. Azadirachtin, a biorational larvicide, was extracted from soil and insect specimens with recoveries of 70% and better. Determination of some of the properties of modifiers in supercritical carbon dioxide as they relate to the eluotropic series is also discussed. Overall, a modifiers ability to enhance or decrease supercritical chromatographic capacity factors is dependent on the presence of a primary solvent-solute relationship. The presence or absence of such a relationship is independent of modifier presence.

This study was directed at developing a predictive kinetic model for ozone consumption by natural organic matter (NOM). The objectives of this research were: (1) to test an existing model, the Staehelin and Hoigne model, for aqueous ozone decomposition in a batch reactor, (2) to develop and test a model for determining the rate of change of aqueous ozone concentration during the ozonation of a simple organic model compound in a batch reactor, and (3) to develop and calibrate a kinetic model for determining the rate of loss of aqueous ozone and a hydroxyl radical probe compound during the ozonation of NOM in batch reactors. In the first stage of laboratory work, experiments were conducted to test the Staehelin and Hoigne model for aqueous ozone decomposition. Two of the rate constants of the Staehelin and Hoigne model were re-estimated in order to improve the agreement between the model predictions and the observed data at neutral pH and under several hydroxyl radical scavenger concentrations. In the second stage of laboratory work, batch ozonation experiments were conducted with the model compound, formic acid. The Staehelin and Hoigne model for ozone decomposition was incorporated in the model development. Natural organic matter, extracted from Forge Pond (Granby, MA) and separated into eight fractions, was ozonated in a batch reactor in the final stage of laboratory experiments. The Staehelin and Hoigne model was included in the model for ozone consumption by NOM. The concentrations of two types of ozone-consuming sites and associated rate constants were estimated for each fraction. The rate constants for all fractions were utilized in estimating average rate constants, which were then used to re-estimate the site concentrations of each fraction. These new estimates of site concentrations were employed in computing composite ozone-consuming-site concentrations for Forge Pond water. It was found that a two-parameter model was quite successful at describing both the residual ozone concentration profiles and the hydroxyl radical profiles (via the probe compound). This firmly established the framework for a central chemical kinetic model that can be used to predict the impact of ozone on micropollutants in waters containing NOM.

The introduction and presence of metals in the marine environment and their reaction and bioaccumulation in organisms and in the marine food web is of environmental importance. The identification and quantification of inorganic and organometallic compounds in marine samples is of importance in studies of the marine food chain and the accumulation of both nutrients and potentially toxic compounds in higher order marine organisms in the marine ecosystem. The goal of this doctoral research was to develop and apply analytical methodology to the sampling, isolation, speciation and determination of metals and organometallic compounds, notably those of the carbon-bonding main group elements such as mercury, tin and selenium in marine samples. The introduction of metals into the marine environment was investigated by analysis of phytoplankton samples cultured by the National Marine Fisheries Service. Procedures for extraction, fractionation and gas chromatographic determination by derivatization and element-specific detection by microwave plasma emission spectrometry (GC-AED) were developed. The biological pathways and transfer of elements along the food chain was investigated on a variety of marine samples including dogfish liver, mussel and lobster heptopancreas. This study was conducted in part at the National Institute of Standards and Technology (NIST), Gaithersburg, MD. Specifically this research centered around providing an alternative procedure to existing methodology for the determination of organomercury compounds in marine samples. Derivatization procedures developed in this research were exhaustively compared with direct gas chromatography of methylmercury chloride, as previously used by NIST. A modified version of the method developed for the analysis of phytoplankton samples allowed both inorganic and organomercury to be simultaneously measured at the μg/g level and below.

Molecular-beam mass spectrometry (MBMS) is a technique that is used to measure stable and radical species within flames, and thus it is a strong tool for understanding the formation and destruction pathways of precursors to PAH and soot. Using MBMS and modeling techniques, three well-chosen premixed flat flames have been characterized. Fuel-lean ethylene flames with and without added allene were mapped to help elucidate the C3H3 self-combination route to benzene formation, and a cyclohexane flame was characterized due to the abundance of cyclohexane within real fuels such as gasoline. Benzene is the precursor to PAH and soot whose formation is the rate-limiting step, and thus knowledge of its formation route is paramount. The flames characterized include an undoped fuel-lean (&phis; = 0.70), C2H4/O2/56.4% Ar flame (30.00±0.01 Torr and ub-300 = 30.6 cm/s), an allene-doped fuel-lean (&phis; = 0.69), 0.19 % C3H4/C2H 4/O2/56.54 % Ar flame (30.00±0.01 Torr and ub-300 = 30.6 cm/s), and a stoichiometric (&phis; = 1.00) cyclohexane/O2/32.5% Ar flame (30.00±0.01 Torr and ub-300 = 35.0 cm/s). Mole fraction profiles of 31, 35, and 70 stable and radical species were measured within the three flames, respectively. They are modeled with overall good agreement between the model and data. Comparison of both fuel-lean ethylene flames shows that benzene was detected in the allene-doped flame but not in the undoped ethylene flame, strongly suggesting the importance of C3 routes to benzene. Reaction path analysis showed that benzene in the allene-doped flame is mainly formed through propargyl self-combination as described by the kinetics of Miller and Klippenstein (2003). Examination of the cyclohexane flame showed high concentrations of benzene. A reaction path analysis showed that benzene is mainly formed instead by the dehydrogenation of cyclohexane. Experiments done at the Advanced Light Source of Lawrence Berkeley National Laboratory show that the isomeric C6H6 composition in the allene-doped flame consisted of 20% fulvene, 45% benzene and 35% 1,5-hexadiyne, while the cyclohexane flame consisted of 99.5% benzene and 0.5% fulvene. This difference in isomeric composition strongly points to the difference in benzene formation pathways in the two fuels.

Solvated transition metal solvent cluster ions were generated by electrospray ionization and their spectroscopy and photo dissociation dynamics have been studied by laser photofragment spectroscopy. Solvation of Co 2+ by water and methanol has been examined by studying Co2+ (H2O)n and Co2+(CH3OH) n (n = 4–7). An electrospray ionization reflectron time-of-flight mass spectrometer (ESI-RTOFMS) was developed to produce gas phase transition metal solvent cluster ions. This system couples with a Nd:YAG pumped dye laser to produce a unique mass spectrometry-laser photodissociation system. This instrument was compared to ESI-mass spectrometers in the literature. It shows a mass resolution (m/Δm) of 250 and a detection limit of 480 pmol. The instrument compares favorably to existing analytical instrumentation and several improvements are suggested. Hydrated cluster ions, Co2+(H2O)n (with n = 4–7), have been generated by electrospray ionization and studied by laser photofragment spectroscopy. The similarity between the spectrum of gas-phase Co2+(H2O)6 and the absorption spectrum of aqueous cobalt (II) suggests that Co2+(H2 O)6 (aq) is responsible for the room-temperature solution absorption spectrum. The observed photodissociation spectrum of Co 2+(H2O)4 is similar to new bands that appear in aqueous cobalt(II) at high temperatures and have been assigned to Co 2+(H2O)4 (aq) in the literature. The hexahydrate dissociates by loss of one or two water molecules, whereas the heptahydrate dissociates by loss of two or three water molecules. In both cases, loss of two water molecules is the preferred dissociation pathway. The tetrahydrate dissociates either by simple loss of water or by charge separation to form CoOH+(H2O)2 and H3O +, with charge separation being the preferred dissociation channel. Methanolic cluster ions, Co2+(CH3OH)n (with n = 4–7), show behavior similar to that of the hydrated clusters. The gas phase spectra display similar shifts from the solution absorption spectra, but absorb more strongly than the corresponding hydrated clusters. The hexamethanol cluster dissociates via loss of one or two methanol molecules; the heptamethanol cluster dissociates via loss of one, two or three methanols. The tetramethanol cluster primarily dissociates by a charge separation mechanism similar to that observed in the hydrated clusters, forming Co(OCH3) +(CH3OH)2 and H+(CH3OH); dissociation by simple loss of methanol is a minor channel.

The formation of specific phase-separated morphologies is central to achieving high performance polyurethanes. Polyurethanes are composed of various structural elements possessing a mixture of different functional groups, molecular weights, and sequence lengths. The chemistry, the phase behavior, and the kinetics of phase evolution will influence the type of phase-separated morphology formed. In fact, the phase behavior also depends upon the chemical structure and the molecular weight distribution of the components. Despite the importance of chemical structure, it is still not understood quantitatively. In addition, little is known about how the developing structure organizes into different phase-separated morphologies. The work herein addresses these issues. The molecular weight distributions, end groups, and linkages of polyurethane structural elements were quantitatively determined. The structural elements included polyether and polyester macrodiols, polyurethane prepolymers, and polyurea hard segments. Under homogeneous conditions, the molecular weight distribution formed obeys a Schultz-Flory distribution; although when toluene diisocyanates are used as the diisocyanate the effect of change in reactivity narrows the distribution. Under heterogeneous conditions, the phase separation of water causes a change in the local stoichiometry and narrows the distribution further. In the presence of typical polyurethane side reactions, the distribution is broadened. The formation of allophanate linkages was most prevalent in PPG prepolymers prepared at reaction temperatures of 145°C. Infrared spectroscopy was used to study the crystallization behavior of semicrystalline polyurethanes and the reaction and morphological evolution of polyurethane foams. Hydrogen bonding between urethane groups was shown to influence all aspects of the crystallization behavior, including the initial state, nucleation and growth rates, and the final morphology. Hydrogen bonding proves to be less crucial in the onset of phase separation in polyurethane foams. The most crucial parameter was shown to be hard segment anisotropy. Foams prepared from diisocyanates yielding highly anisotropic hard segments phase separate at lower reaction conversion, with a faster rate, and to a higher degree of phase separation and perfection.

Transferrins constitute a class of metalloproteins that sequester and transport iron in vertebrates. Understanding the metal-binding properties of transferrin is essential for studies of iron metabolism and metabolic disorders. However, dynamics of these systems is poorly understood, since they are not amendable to analysis by NMR due to the high spin of the metal ion and the large molecular weight of the protein (35–80 kDa). In this work we successfully developed and utilized an ESI MS based approach to study metal-release properties of the human serum transferrin N-lobe (hTF/2N), as well as the intact transferrin from human (hTF). Both composition and conformation of a protein complex was monitored under a variety of conditions that are designed to mimic extracellular and endosomal environments. It was determined that iron dissociation occurs in vitro at typical endosomal pH 5.5 when physiological chelators (e.g. citrate) are present at endogenous levels. In the absence of such chelating agents, detectable iron release occurs at much lower pH (<4.5) and is accompanied by unfolding of the protein. ESI MS data also indicated the transient nature of the anion in the holo-protein. Furthermore, studies of hTF revealed that the conformation of the iron-free N-lobe is stabilized when the C-lobe contains iron, confirming the existence of an interlobe interaction within the protein. Another surprising discovery was that the apo-N-lobe is significantly less flexible compared to the apo-C-lobe. These results provide strong support for the earlier suggestions that hTF interacts with its receptor (TfR) primarily through the C-lobe both at the cell surface and inside the endosome. We also successfully extended the above ESI MS approach to characterize interaction of intact transferrin (80kDa) with metals of medicinal relevance, such as In3+ and Bi3+. The results of our studies have revealed that indium-hTF complex exhibits the same pattern of conformational and metal release properties as iron-hTF. Because of conformational similarities between indium-hTF and iron-hTF, it is possible that indium-hTF is recognized by transferrin receptor protein (TfR) at the cell surface and is transported into the cells by the same mechanism of receptor mediated endocytosis as iron. On the other hand the results of our bismuth studies suggest that hTF adopts a semi-open conformation upon bismuth uptake. This conformation is different from that of iron-hTF, therefore it is likely that bismuth transport does not occur via the similar mechanism.

The study of hydride generation as an analytical technique has occupied a section of analytical chemistry for a considerable period of time. Applications of the technique have built upon the existing background of chemistry with few forward steps. The progress of analytical chemistry requires that the fundamental chemistry be studied so that it may then be applied within the understood limitations. Hydride generation is a technique that is heavily influenced by a variety of factors including but not limited to pH and oxidation state. The required oxidation state for hydride generation of arsenic and antimony is +3 and depending upon sample preparation, adjustment of the oxidation state is required. Bromide has been identified as a reductant for pentavalent arsenic and antimony and can be applied to reduce selenium to the hydride forming state of Se IV. Experiments are then described that demonstrate the limitations and application of bromide as a reductant for the simultaneous reduction of arsenic, antimony and selenium to the optimum hydride forming state, prior to hydride generation. The post-column chemistry of arsenic is examined with the application of microwave-assisted chemistry to the digestion of organoarsenical species. A mixture of bromide and bromine is applied under conditions that are also successful for the post column digestion of organoselenium compounds. The significance of the post column digestion is that it will assist in the development of simultaneous determination of arsenic and selenium species. The post-column chemistry of antimony has thus far been limited to the addition of borohydride under conditions that compromise the sensitivity of the species studied and is limited to those species that are borohydride active. There are several reports in the literature of unidentified antimony species that were not identified by the technique of hydride generation. Therefore, the application of photo-oxidation to those species would enable their subsequent determination by hydride generation. Two small projects with graphite furnace atomic absorption spectrometry are also discussed. The first project describes the development of a method for the determination of gold in cell structures. The second describes the measurement of bismuth in transferrin protein.

The main theme of this research is to understand the structure-property evolution during crystallization of a semicrystalline thermoplastic polymer. A combination of techniques including rheology, small angle light scattering, differential scanning calorimetry and optical microscopy are applied to follow the mechanical and optical properties along with crystallinity and the morphology. Isothermal crystallization experiments on isotactic poly-1-butene at early stages of spherulite growth provide quantitative information about nucleation density, volume fraction of spherulites and their crystallinity, and the mechanism of connecting into a sample spanning structure. Optical microscopy near the fluid-to-solid transition suggests that the transition, as determined by time-resolved mechanical spectroscopy, is not caused by packing/jamming of spherulites but by the formation of a percolating network structure. The effect of strain, Weissenberg number (We ) and specific mechanical work (w) on rate of crystallization (nucleation followed by growth) and on growth of anisotropy was studied for shear-induced crystallization of isotactic poly-1-butene. The samples were sheared for a finite strain at the beginning of the experiment and then crystallized without further flow (Janeschitz-Kriegl protocol). Strain requirements to attain steady state/leveling off of the rate of crystallization were found to be much larger than the strain needed to achieve steady state of flow. The large strain and We>1 criteria were also observed for morphological transition from spherulitic growth to oriented growth. An apparatus for small angle light scattering (SALS) and light transmission measurements under shear was built and tested at the University of Massachusetts Amherst. As a new development, the polarization direction can be rotated by a liquid crystal polarization rotator (LCPR) with a short response time of 20 ms. The experiments were controlled and analyzed with a LabVIEW™ based code (LabVIEW™ 7.1) in real time. The SALS apparatus was custom built for ExxonMobil Research in Clinton NJ.