No description available.

No description available.

No description available.

No description available.

No description available.

A broadband coupling approach applied to a single mode, sol-gel, planar integrated optical waveguide (IOW) was used to create a multichannel attenuated total reflection (ATR) spectrometer. Initial attempts to create an achromatic coupling element for sol-gel waveguides, based upon previous work applied to vacuum deposited glass devices, did not lead to an easily achievable design. Instead a simplified, non-achromatic approach based upon impinging an incident light beam with a large numerical aperture onto an incoupling prism was used. This simplified broadband coupling approach was used to create a sol-gel IOW-ATR spectrometer that transmitted light down to at least 400 nm, and produced a measurable bandwidth of ~ 250 nm; both phenomena are marked improvements upon the capabilities of previously reported devices. An experimental demonstration of this device proved it capable of measuring the visible spectrum of a thin film of horse heart cytochrome c adsorbed to the sol-gel surface at a submonolayer coverage. The broadband spectral capabilities of this sol-gel device were also used to experimentally validate a new method for determining the angular orientation of molecules bound to an arbitrary waveguide surface. In addition to the sol-gel IOW work, the simplified broadband coupling approach was applied to a previously reported multilayered electroactive waveguide device, which was used to collect electrically modulated, broadband spectra for thin films of cytochrome c, as well as a dicarboxyferrocene moiety. Both of these IOW-ATR spectrometers represent improved tools for probing the near-surface chemical environments of molecular assemblies.

Calibration powders were developed using combinations of milk ingredients for the purpose of calibration of infrared milk analyzers. They were shown to be capable of producing calibrations very similar to conventional calibration milks available commercially. A subsequent collaborative study was carried out involving nine laboratories to assess the performance of the preformulated powders in industrial quality control, payment and dairy herd analysis laboratories. The calibration powders were shown to produce consistent calibrations within laboratories and between laboratories and met AOAC specifications in terms of accuracy and repeatability. The reconstituted solutions were shown to be stable for up to 6 h at 40$ sp circ$C and could be stored under refrigerated conditions and used for repeated analyses for up to 21 days without apparent deterioration in calibration performance. It was concluded that the calibration powders perform as well as conventional calibrants, were more consistent overall and suitable for any calibration application. The calibration powders have the stability and performance characteristics to serve as reference standards for monitoring instrument performance and would be a useful tool for accrediting payment and dairy herd analysis laboratories.

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19F NMR studies of proteins provide unique insight into biologically relevant phenomena such as conformational fluctuations, folding and unfolding, binding and catalysis. While there are many advantages to the use of 19F NMR, experimental challenges limit its widespread application. The focus of this thesis has been to address some of these limitations, including resonance assignment and perturbations arising from fluorine probes, and to develop more robust methods of studying protein topology by 19F NMR. 19F NMR experiments designed to measure local hydrophobicity and exposure were developed and evaluated in two systems, Fyn SH3 and calmodulin, labeled with 3-fluorotyrosine. Paramagnetic effects from dissolved oxygen, solvent isotope shifts from deuterium oxide, and 1H-19F NOEs were each sufficient in establishing relative solvent exposure, while the combination of effects from oxygen and deuterium oxide were able to delineate local hydrophobicity and solvent accessibility of 19F probes. Two NMR based resonance assignment protocols were developed using 13C, 15N-enriched 3-fluorotyrosine and 3-fluorophenylalanine, separately biosynthetically incorporated into calmodulin. In the first approach, isotopic enrichment facilitated two-dimensional heteronuclear experiments based on INEPT and COSY magnetization transfer schemes to correlate the fluorine nucleus to sidechain and backbone 1H, 13C, and 15N atoms, providing complete spectral assignment. The assignment of 3-fluorophenylalanine resonances was achieved using 19F-, and 15N-edited homonuclear NOE experiments to connect the fluorine nucleus to intraresidue and neighboring 1H and 15N resonances. While both strategies were successful, the NOE-based method was vulnerable to alternate relaxation mechanisms, including chemical shift anisotropy and chemical exchange. Structural perturbations arising from uniform incorporation of 3-fluorophenylalanine in calmodulin was thoroughly investigated using 19F and 1H-15N NMR spectroscopy, 15N spin relaxation and thermal denaturation via circular dichroism spectroscopy. While stability was unaffected, NMR experiments revealed increased protein plasticity, minor conformers and line broadening. The merit of fractional fluorine labeling in reducing such disruptions was demonstrated, and labeling levels of 60-75% provided an optimal balance between native-likeness and the usual advantages of 19F NMR in our system. The 19F NMR techniques developed here are broadly applicable and will expand the utility of 19F NMR in studies of protein systems.