This dissertation explores a number of different environmentally relevant reactions that lead to the production of perfluorocarboxylic acids (PFCAs), a family of environmental pollutants that does not undergo any further degradation pathways. The compound perfluoro-2-methyl-3-pentanone (PFMP) is a new fire fighting fluid developed by 3M that is designed as a Halon replacement. The environment fate of PFMP with regards to direct photolysis, abiotic hydrolysis and hydration was determined using a combination of laboratory experiments and computational modeling. PFMP was found to undergo direct photolysis giving a lifetime of 4-14 days depending on latitude and time of year. Offline samples confirmed PFCA products and a mechanism was proposed. Polyfluorinated amides (PFAMs) are a class of chemicals produced as byproducts of polyfluorinated sulfonamide synthesis via electrochemical fluorination (ECF). Using synthesized standards of four model compounds, PFAMs were detected and quantified in a variety of legacy commercial materials synthesized by ECF. PFAMs were hypothesized to undergo biological hydrolysis reactions, suggesting their importance as historical PFOA precursors. The PFAMs were also investigated with regards to their environmental fate upon atmospheric oxidation. Using a smog chamber, the kinetics and degradation mechanisms of N-ethylperfluorobutyramide (EtFBA) were elucidated. The lifetime of EtFBA to oxidation by OH was found to be approximately 4 days. Using offline sampling, PFAMs were shown to give PFCAs upon atmospheric oxidation and a plausible mechanism was proposed involving an initial N-dealkylation step followed by loss of isocyanic acid to give a perfluorinated radical. The perfluorinated radical then produces PFCAs by a series of known atmospheric reactions. Finally, the biological hydrolysis of the polyfluoroalkyl phosphate monoesters (monoPAPs) were studied in vitro using a bovine alkaline phosphatase enzyme. Michaelis-Menten kinetic parameters were measured and compared to hexyl phosphate. It was discovered that monoPAPs hydrolyzed on average 100 times faster than hexyl phosphate due to the electron withdrawing fluorine substituents. The results were also used to rationalize the results of a previous in vivo study which suggested monoPAPs were rapidly hydrolyzed in the small intestines of rats following a high dose by oral gavage.

Perfluorinated carboxylates (PFCAs) are persistent and ubiquitous in the environment. Humans are exposed to PFCAs through direct and indirect sources, although the relative importance of each is uncertain. Direct sources of PFCAs have been attributed to two primary fluorochemical manufacturing processes: electrochemical fluorination (ECF) and telomerization. A focus of this thesis was to elucidate an additional direct source of PFCAs resulting from the direct fluorination of polyolefin materials. High density polyethylene bottles with varying levels of fluorination were observed to contain significant amounts of PFCAs, particularly those with carbon chain-lengths ≤ C6, marking an unexplored source of PFCA exposure. PFCAs are also produced indirectly from the biotransformation of fluorotelomer-based compounds, such as polyfluoroalkyl phosphate esters (PAPs) and fluorotelomer alcohols (FTOHs). During this transformation process, two predominant classes of metabolic intermediates are formed: the fluorotelomer unsaturated aldehydes (FTUALs) and the fluorotelomer unsaturated carboxylic acids (FTUCAs). Another focus of this thesis was to examine the reactivity of FTUALs and FTUCAs with endogenous nucleophiles such as glutathione (GSH), select amino acids, and model proteins. FTUALs formed adducts with all nucleophiles examined, where those having shorter carbon chain lengths (i.e. 6:2 and 8:2 FTUAL) were more reactive than longer carbon chains (i.e. 10:2 FTUAL). By contrast, FTUCAs had comparably limited reactivity; although FTUCAs showed mild reactivity with GSH, they did not react with any other nucleophiles. In vitro and in vivo experiments were carried out to determine the extent of protein binding formed from the biotransformation of fluorotelomer-based compounds, including the 8:2 FTOH and the 6:2 PAP diester. A significant portion of these biotransformations yielded covalent protein binding at nmol/mg protein concentrations. Protein adducts were observed predominantly in rat liver and also in plasma and kidney. The formation of reactive intermediates may be toxicologically important through protein deactivation. Cellular toxicity of FTUALs was significantly higher compared to PFCAs and the acid metabolic intermediates (i.e. FTUCAs). The EC50 values calculated from dose-response incubations were dependant on chain length and functional group. The work in this thesis examined an unexplored consequence of indirect exposure to PFCAs, potentially impacting the relative importance of PFCA exposure sources.

Studying the intrinsic properties of molecules in the gas-phase is advantageous, since it reduces the complexity present in solution that arises from interactions between the molecule of interest and other species present in the local environment, including those with the solvent itself. In this report, the photophysical properties of gaseous cationic rhodamine B (RBH+) were determined and photodissociation reaction kinetics and power dependence of three prototropic forms of fluorescein; the cation ([F + H]+), monoanion ([F - H]-), and dianion ([F – 2H]-2), each of which possesses their own distinct spectral properties, were measured. The analyte ions of interest were formed via electrospray ionization, mass-selected and stored in a quadrupole ion trap mass spectrometer which has been customized to enable gas-phase spectroscopic studies. Knowledge of the intrinsic photophysical properties of such chromophores in the gas-phase will enable a better understanding of how the local environment of the molecule alters its properties.

The advantage of one-bead-one-compound combinatorial libraries is that hundreds of thousands to millions of compounds can be rapidly synthesized and screened simultaneously. The beads supporting the compounds of interest are then isolated and analyzed to decipher the structure of the desired compound. Many methods are currently used to allow deconvolution of the compound on the individual beads. Herein is described a novel method to encode TentaGel beads using absorption of different ratios of lanthanide ions. The encoding process is completed in parallel with the synthesis of the library of compounds. Once the desired beads are identified, the lanthanide ions can be released from the bead and analyzed using Inductively Coupled Plasma-Mass Spectrometry (ICP-MS).

Master of Science / Department of Chemistry / Daniel A. Higgins / Lithography techniques have been widely used to fabricate optical, electronic and optoelectronic devices with sub-micron scale spatial resolution. In the most common lithographic procedures, a light sensitive polymer, called a photoresist, is exposed and developed to form a binary relief pattern on a substrate. The finest features are produced by X-ray or electron-beam methods, both of which are very expensive to employ. Less expensive methods use ultraviolet (UV) light to expose the photoresist through a photomask. The resolution in these methods is somewhat lower and is governed by diffraction of light by the photomask, the quality of the photomask, and by any chemical/physical development steps subsequently employed. Due to the above limitations, we have been investigating direct-write, ablative multiphoton lithography as an alternative method for preparing high-resolution patterns. With this method, near-IR light from an ultrafast pulsed laser source is focused into a polymer film, leading to depolymerization and vaporization of the polymer. Arbitrary binary patterns can be produced by raster scanning the sample while controlling exposure of the film to the laser. Importantly, high-resolution etching of the polymer film is achieved without the use of a photomask and without chemical development steps. While arbitrary patters are easily prepared, it is also possible to prepare three-dimensional (i.e. grayscale) surface relief structures. In this study, ablative multiphoton photolithography is used to prepare binary and grayscale structures in thin films of PEDOT:PSS, an electrically conductive organic polymer blend. A simple kinetic model is proposed to explain the etching process. Data on the power-dependence of polymer etching can be fit to this model and is used to determine the order of the nonlinear optical process involved. The etch depth as a function of laser focus is also investigated and shown to follow the same kinetic model. The results show that three-dimensional (grayscale) patterns can be prepared by modulating either the laser power or the laser focus. Images of several binary and grayscale structures prepared by this method are presented.

Doctor of Philosophy / Department of Chemistry / Stefan Bossmann / Estrogen-derived DNA adducts in human could be the initiating step of breast and prostate cancer, as the scientific literature suggests. Previous studies demonstrated that 4-hydroxy-estrone (estradiol)-1-N3Adenine and 4-hydroxy-estrone (estradiol)-1-N7Guanine were the most abundant adducts found in urine of human subjects. Sensitive detection of these adducts in urine samples could lead to better breast and prostate cancer risk assessment. The standard adducts were synthesized and characterized by NMR and mass spectrometry. Since these adducts are not fluorescent at room temperature an aminomethyl (-CH2NH2) linker was introduced at the C-17 position for derivatization with fluorescence label. This linker allowed to attach highly fluorescent water soluble quantum dots (QDs) for indirect adduct detection. A direct gram-scale synthesis of highly fluorescent, photostable water soluble QDs was executed by developing a new class of 4,4’-bipyridinium salt based twin ligands with 85% and 15% of carboxylic acid and maleimide termini, respectively. These ligands not only stabilized the QDs in water but also provided versatile linkers for two labeling strategies. The twin ligands were afforded by a facile synthesis through SN2 nucleophilic substitution reaction. Labeling of adducts was achieved via a covalent coupling between the (-CH2NH2) linker and the carboxyl (-COOH) terminal ligand on the QDs. However, ELISA experiments utilizing an IgM antibody didn’t reveal any measurable signal from adduct-QD complexes suggesting that one QD is bound to a large number of adducts through –COOH terminal ligands present on QD surface. To explore the binding capabilities of QDs in more detail, a maleimide terminal ligand (a twin partner on the QDs) was synthesized to explore the thiol (-SH) functionality of thiopyrene. Preliminary ELISA showed that these QDs gave detectable fluorescent signal originating from the [pyrene-S-QD] ̶ 8E11 monoclonal antibody (mAb) complex when QD was selectively excited at 470 nm. This clearly indicates that it is necessary to develop a strategy for a distinct 1:1 labeling procedure between QD and the adduct of interest. In addition, IgG (instead of IgM) antibodies should be developed for biosensor application. The latter could afford binding of mAb in upright position, leading to an increase in surface density of mAb and better detection limit.

Doctor of Philosophy / Department of Chemistry / Takashi Ito / This dissertation describes the fabrication and characterization of nanoscale structures in commercially available polymers via multiphoton ablation and bottom-up self assembly techniques. High-resolution surface imaging techniques, such as atomic force microscopy (AFM) and chemical force microscopy (CFM) were used to characterize the physical features and chemical properties, respectively, of these nanoscale structures. Fabrication using both top-down and bottom-up methods affords flexibility in that top-down allows random, user-defined patterning whereas bottom-up self assembly produces truly nanoscale (1-100nm) uniform features. Multiphoton induced laser ablation, a top-down method, was used to produce random sub-micron scale features in films of poly(methylmethacrylate) (PMMA), polystyrene (PS), poly(butylmethacrylate) (PBMA) and poly[2-(3-thienyl)ethyloxy-4-butylsulfonate] (PTEBS). Features with 120-nm lateral resolution were obtained in a PMMA film which was concluded to be the best polymer for use with this method. It was also found that etching resolution was highest for polymers having high glass transition temperatures, low molecular weights and no visible absorption. Bottom-up self assembly of polystyrene-poly (methylmethacrylate) (PS-b-PMMA) diblock copolymer and UV/acetic acid treatment produced nanoscale cylindrical domains supported by a substrate. AFM imaging at the free surface showed metastable vertical PMMA domain orientation on gold substrates. In contrast, horizontal orientation was obtained on oxide-coated silicon regardless of surface roughness and annealing conditions. The horizontal domain orientation on silicon substrates was ideal to probe simultaneously the difference in surface charge and hydrophilicity of the two distinct nanoscale domains of UV/AcOH treated PS-b-PMMA films. CFM on UV/acetic acid etched PS-b-PMMA revealed the presence of –COO- groups which were found to be more abundant inside the etched trenches than on the unetched PS matrix as shown by ferritin adsorption onto etched PS-b-PMMA. Lastly, the PS-b-PMMA was cast as a free-standing monolith at the end of a quartz micropipette. AFM revealed circular PMMA dots at the free surface, indicating alignment parallel to the long axis of capillary. Ion conductance within nanochannels indicated surface –charge governed ion transport at low KCl concentrations and flux of negatively-charged sulphorhodamine dye demonstrated the permselective nature of nanochannels.

Doctor of Philosophy / Department of Chemistry / Christopher T. Culbertson / High-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) are two well-established analytical separation techniques that are continuously being adapted for performing distinctive separations and analyses of multitudes of complex and/or unique samples. Since their introduction, these techniques have been pivotal in the discovery, analysis, and understanding of a variety of samples and still prove to be key analytical tools for biological investigation. Using these techniques, one can obtain a wide-range of valuable sample information from the hydrophobicity and molecular weights to size and charge distributions. Furthermore, these techniques allow for sample analysis, purification, and collection for additional sample analysis, such as mass spectrometry analysis. My doctoral dissertation encompasses the full scope of these two techniques and novel approaches for the investigation of distinct, relevant samples. Described herein is the fabrication of glass microfluidic devices used for CE and their diversity for numerous investigations. Chapter 2 shows that the resolution of the photomasks used in microchip fabrication does not alter the separation efficiency of the devices, as the separations remain diffusion-limited. Using an in-house built capillary electrophoresis system, wheat proteins were separated more than 25% faster than previously reported in literature, and the electropherograms used for sample varietal identification. The fabrication of a robust, portable CE system capable of performing biological analysis in microgravity and hypergravity environments is also discussed. The need for and features necessary to achieve a reliable, robust, automated system is further described in Chapter 4. Isolation and analysis of the pea aphid (Acyrthosiphon pisum) salivary secretions was completed for the first time using HPLC. By altering the aphid environment and the sample treatment parameters, sample concentrations were increased above the limit of detection. Coupled with mass spectrometry, identification of pea aphid salivary proteins such as exopeptidase, angiotensin converting enzyme, and Buchnera proteins has been achieved. Finally, a simplified contact conductivity detection system for the detection of jurkat cells was developed that surpasses current, complex optical systems. The experiments described in this dissertation demonstrate novel approaches for the preparation, separation, analysis, and identification of a wide variety of common, and uncommon, samples.

Doctor of Philosophy / Department of Chemistry / Takashi Ito / We have investigated the orientation of cylindrical domains in thin films of a polystyrene–poly(methylmethacrylate) diblock copolymer (PS-b-PMMA) on planar substrates having different surface roughnesses and hydrophilicities. The research in this dissertation covers the substrate surface modifications, the enhancement of the diblock copolymer film coating, and the refinement of the treatments leading to nanoporous material. Treatment of the substrate with organic mercaptans forming self-assembled-monolayer (SAM), leading to various hydrophilicities of the surfaces, was inconclusive as far as orienting the PMMA domains in the PS matrix due to thermal instability of some thiols. This directed us to a different approach involving substrate roughness. PS-b-PMMA films of 20~200 nm thick were prepared via spin-coating on silicon, gold or indium tin oxide (ITO) substrates, and annealed in vacuum at 170 °C for 60 hours to induce the formation of cylindrical PMMA domains. Atomic force microscopy (AFM) images indicated the domain orientation at the free surface. In PS-b-PMMA films much thicker than the domain periodicity (L0), the domains were oriented perpendicularly to the free surface regardless of underlying substrates, reflecting the balanced interactions of PS and PMMA blocks at the polymer–vacuum interface. In films having thickness similar to L0, vertically oriented domains were observed on the Au and ITO surfaces that are covered with nanoscale grains, whereas horizontal domains were observed on the smooth Si substrates. In particular, the cylindrical PMMA domains were efficiently perpendicularly aligned when the grain size nearly was equal to L0. The perpendicular domain alignment induced by the substrate roughness was corroborated using cyclic voltammetry (CV) for gold substrates coated with PS-b-PMMA films whose PMMA domains were removed by UV and subsequent acetic acid treatments. The CV data also suggested that the PMMA domains were successfully removed, leaving a nanoporous stable PS matrix on the substrate.

Master of Science / Department of Grain Science and Industry / David L. Wetzel / Isogenic waxy wheat lines differ from their non waxy (normal) parents in functionality, end use, and chemical (i.e. amylopectin/amylose, lipid) contents. Other investigations of waxy and parent wheats involved the carbohydrate and protein fractions. The goal of this work is to apply chemical images to discriminate between the waxy and parent wheats and define the contribution of contrasting lipid profiles. Recent waxy topics include current interest in plant breeding activity to develop new lines that incorporate desirable traits with advantageous success in baking and milling, and the differences needed in milling techniques for waxy versus normal wheats that may be associated with lipids. From our empirical preliminary success in sorting parent wheat kernels from waxy wheat full null specimens by nearIR chemical imaging it was anticipated that using fundamental vibrational spectra in the mid infrared would provide the chemical basis of discrimination. FT-IR microspectroscopic in situ probing and imaging of kernel frozen sections was applied to genetically pure, well documented isogenic breeding lines. With the use of high spatial resolution, elucidation of fundamental vibrations of mid IR provides chemical manifestation of the genetic expression that differentiates waxy wheats from their parent wheats. Comparison between numerous contiguous pixels, typically 3,000 for each type, establishes a consensus and a mean spectrum with characteristic bands for waxy and parent. Extractions with solvents of differing polarity were employed to aid in lipid extraction in situ and kernel extracted endosperm. Differences between kernel sections of waxy and parent are observed using FT-IR microspectroscopic imaging. However, revealing lipid class contribution to the molecular bands required infrared analysis after selective extraction. Triple mass spectrometry of lipid molecule ions was used for compositional analyses to enhance lipid class profile distinction. A normal and waxy advanced breeding line wheat were also analyzed via the same methods. It was noted that digalactoslydyglyercides are the most abundant lipids in all samples, however the relative lipid profiles of normal wheat versus waxy wheat differ as well as tetraploid versus hexaploid. It is observed that in the endosperm of all parent wheat versus waxy wheat specimens analyzed, all waxy wheat specimens contained higher lipid content. Methods were also applied to partial waxy isogenic cultivars to determine detection limits that correspond to the degree of waxy genetic expression.