Investigations described in this dissertation found that cleavage at Xxx-Pro bonds depends upon the identity of the Xxx residue, and this finding is immediately applicable to improved peptide sequencing algorithms because enhanced cleavage was evident for Pro-containing peptides when Xxx is His, Asp, Val, Ile, or Leu, and reduced cleavage was evident when Xxx is Gly or Pro. A greater understanding of the mechanism of cleavage at Pro residues was gained in these studies. A non-Pro N-alkylated residue in a peptide directs cleavage C-terminal to the N-alkylated residue; this is dependent upon the conformation of the peptide as cleavage is directed N-terminal to the residue when the D-stereoisomer of the N-alkylated residue is substituted into the peptide. Cleavage rarely occurs C-terminal to Pro residues in peptides, presumably due to steric limitations that are imposed by the 5-membered ring and that hinder formation of necessary intermediates. Dramatically different gas-phase H/D exchange results observed for L-Pro-containing peptides after substitution with the D-stereoisomer of Pro in addition to evidence of Pro-cleavage dependence upon the residue preceding Pro and conformational dependence of cleavage of other N-alkylated residues leads us to conclude that the conformational turn in a peptide backbone and the steric limitations imposed by the Pro ring are the primary factors affecting cleavage at Pro. Fragmentation patterns of peptides containing Pro and other N-alkylated residues support a mechanism of bond cleavage that begins with protonation at a carbonyl oxygen rather than an amide nitrogen. Transfer of the ionizing proton (or its equivalent) to the C-terminal fragment forms "b" ions which may be followed by the transfer of the hydrogen bound to the amide nitrogen N-terminal to the cleavage site for "y" ion formation. The types of experiments designed to investigate Pro-containing peptides also provided new results that answered questions about gas-phase structure of other peptides. Gas-phase H/D exchange of Arg-containing oligoalanines and their derivatives suggests that salt bridge structures may form for these 5-residue peptides. MS3 and gas-phase H/D exchange results of fragment ions from Asp-containing peptides suggest different "b" ion structures formed at Asp-Xxx and non-Asp cleavage positions.

Orientation distributions of redox active films that correlate with an electron transfer behavior were reported for the first time using electroactive total internal reflection fluorescence spectroscopy (EA-TIRF) and electroactive integrated optical waveguide (EA-IOW) absorbance technology developed here. The mean tilt angle and the angular distribution about the mean were recovered using a Gaussian model. Previous anisotropy calculations were only possible using a theoretical approach (Hansen's model). In the work presented here, a novel method was developed that was based on experimental measurements. This method used Fresnel's equations and Snell's law to determine the relative polarized electric field intensity at the interface of the EA-TIRF substrate. Optically transparent semiconductor surfaces of indium tin oxide (ITO) were used as the adlayer for EA-TIRF and EA-IOW devices. The ITO surface morphology, optical and conduction properties were characterized. The ITO was found to have adequate conduction, optical and surface morphology properties for molecular orientation distribution measurements. The results indicated the ITO morphology contributes a minimal degree of error to the calculated distribution. Surface-bound films of model methylene blue were used to characterize the EA-TIRF and EA-IOW techniques prior to the investigation of horse heart cytochrome c. The studies demonstrated potential control of redox active adsorbed films. The mean tilt angle and the angular distribution about that mean were successfully determined. In addition, the studies of the methylene blue films indicated the possibility of orientation-dependent quenching (the loss of an electron from the excited state LUMO to the ITO semiconductor conduction band). Studies of the cytochrome c films indicated anisotropic submonolayers electrostatically bound to the negative ITO surface. Cyclic voltammetry measurements showed the films to be electroactive, exhibiting quasi-reversible electrochemistry. An average tilt angle and the orientation distribution about the angle, as a function of potential, were reported for horse heart cytochrome c. This potential-dependent orientation distribution of submonolayer films is reported for the first time.

An improved spectrophotometric method of determining equilibrium constants is introduced. The advantages offered by photodiode array spectrophotometer are exploited. The most important of these are the ability to precisely measure analyte absorbance at all wavelengths throughout the ultraviolet and visible spectral range and store the data in computer files. A spreadsheet program is used to assemble the spectral data into an organized array and calculate the most probable value of the equilibrium constant for the entire array. Linear least-squares analysis and weighting scheme are employed to calculate the final result. Precision of the equilibrium constant obtained are at least an order of magnitude better than heretofore possible.

A series of studies was carried out on the direct analysis of solid samples with inductively coupled plasma atomic emission spectrometry (ICP-AES) and mass spectrometry (ICP-MS). Samples were first vaporized by modified electrothermal vaporization (ETV) systems and the vapor was introduced into the ICP. Inert gas flows were used to increase analyte transport efficiency and to reduce matrix effects. / Halogenation reactions were used to improve solid sample vaporization in one of the modified ETV systems. At temperatures ranging from 1000$ sp circ$C to 2400$ sp circ$C pure oxides and carbides were converted to lower boiling point halides and evaporated. Experimental results showed that, with the use of Freon-12 as a gas phase halogenation reagent, nearly 100% vaporization was achieved for $ rm Al sb2O sb3$ (B.P. 2980$ sp circ$C, particle size $<$10 $ mu$m), SiO$ sb2$ (B.P. 2230$ sp circ$C, particle size $<$44 $ mu$m), ZrO$ sb2$ (B.P. 5000$ sp circ$C, particle size $<$74 $ mu$m) and TaC (B.P. 5500$ sp circ$C, typical particle size 5 $ mu$m). The same was true for WC (B.P. 6000$ sp circ$C, particle size $<$10 $ mu$m) if BaCl$ sb2$ was used as a second halogenation reagent. These lead us to believe that we would be able to vaporize samples with almost any inorganic matrix. When the same ETV system was used to analyze four marine sediment reference materials and one coal fly ash reference material, a linear relationship was found between the background corrected peak area and the reference concentration for six of the eight elements monitored.

Derivatized $ beta$-cyclodextrin was used as mobile phase additive for the high performance liquid chromatographic resolution of amino acid derivative enantiomers while using an achiral C$ sb{18}$ stationary phase. Dimethyl-$ beta$-cyclodextrin gave improved enantioselectivity over the underivatized $ beta$-cyclodextrin. The effects of cyclodextrin concentration, pH, and methanol and buffer concentration on enantioselectivity and capacity factor were examined. / $ beta$-Cyclodextrin and two derivatized cyclodextrins were utilized as mobile phase additives for the liquid chromatographic resolution of equilin and estrone as well as estrone from 2-, 4-, and 16$ alpha$-hydroxyestrone. $ beta$-cyclodextrin proved to be suitable in these separations but the modified $ beta$-cyclodextrins provided better resolution. Apparent inclusion complex strengths were calculated for estrone and its derivatives were calculated for each cyclodextrin. / Micellar electrokinetic chromatography using sodium dodecyl sulphate with $ beta$-cyclodextrins was found to give comparable or improved separation of a series of fat-soluble vitamins over reversed-phase high performance liquid chromatographic techniques with cyclodextrins in the mobile phase. / Charged carboxymethyl-$ beta$-cyclodextrin was used in the capillary electrophoretic separation of a series of nine tricyclic antidepressants. Cyclodextrin alone was successful in separating some of the compounds under investigation while complete separation required the addition of a micellar pseudophase. A variety of coatings were investigated to reduce or eliminate the electroosmotic flow.

No description available.

Volatile organic compounds (VOCs), among them non-methane hydrocarbons (NMHCs) and low molecular weight carbonyl compounds (aldehydes and ketones), affect the oxidative capacity of the atmosphere and thus pollutant lifetimes and global climate. VOCs in the surface oceans may be transported into, or derived from, the atmosphere. This thesis describes the development and optimization of chromatographic and preconcentration methods to determine volatile organic compounds (VOCs) in surface seawater and marine air, and their use to explore VOC distribution and fluxes at the sea-air interface. It includes the first measurements of many carbonyl compounds in temperate and subarctic marine waters and the first estimates of fluxes of several aldehydes from the ocean surface into the marine atmosphere. Sea surface air, size-fractionated marine aerosols, and surface ocean water dissolved organic matter were simultaneously sampled in the Nordic seas. Nineteen C2-C7 NMHCs were quantified in the air samples. Site-to-site variability in NMHC concentrations was high, suggesting variable, local sources. The aerosols consisted mainly of inorganic marine material, but a culturable bacterium identified as Micrococcus luteus was also isolated from the 9.9 – 18 micrometre fraction, suggesting organic matter may be transferred from the surface oceans to the atmosphere by marine aerosols. Lastly, a number of VOCs, including acetone, were detected in the seawater samples using solid-phase microextraction (SPME), leading to the subsequent development of an SPME application for carbonyl compounds in seawater. A mobile, economical and solventless method for the detection and quantification of carbonyl compounds in seawater, a matrix of global importance, was developed. The compounds were derivatized using O-(2,3,4,5,6-pentafluorobenzyl)-hydroxylamine (PFBHA)and then pre-concentrated by SPME for gas chromatography with mass spectrometric (GC/MS) or flame ionization (GC-FID) detectio / Les composés organiques volatils (COV), une classe de composés qui inclut les hydrocarbures non méthaniques (HCNM) et les composés carbonylés (aldéhydes et cétones) de faibles masses moléculaires, affectent la capacité oxydante de l'atmosphère et, par consequent, le temps de résidence des polluants de même que le climat à l'échelle globale. Les COV présents dans l'eau de surface océanique peuvent être transferés de l'océan vers l'atmosphère ou vice versa. Cette thèse décrit le développement et l'optimisation de méthodes chromatographiques et de pré-concentration pour la détermination des COV dans l'eau de mer et dans l'air marin, ainsi que leur utilisation dans l'exploration de la distribution des COV et leurs flux à l'interface air-mer. Les premières déterminations de la concentration de plusieurs composés carbonylés dans l'eau de mer tempérée et subarctique, de même que les premières estimations du flux de plusieurs aldéhydes de l'océan vers l'atmosphère marin sont aussi incluses. L'air de surface, de l'aérosol marin distribué en taille et de la matière organique dissoute présente dans l'eau de mer de surface ont été échantillonnés simultanément dans les mers nordiques. Dix-neuf hydrocarbures non méthaniques de C2 à C7 ont été quantifiés dans les échantillons d'air. Une grande variabilité dans les concentrations de HCNM mesurées d'un site à l'autre indique la présence de sources locales et variables. L'aérosol était composé principalement de matière inorganique d'origine marine, mais une bactérie cultivable, Micrococcus luteus, a été isolée à partir de la fraction des particules mesurant de 9,9 à 18 micromètres, ce qui suggère que le matière organique peut être transferée de l'océan à l'atmosphère par l' aérosol marin. De plus, certains COV, dont l'acétone, ont été détectés dans les échantillons d'eau de mer grâce à la technique de micro-extraction sur phase solide (

In an industrial society it is critical that techniques be developed for the measurement of chemical species in the environment, in humans and as both intended and unintended products of manufacturing. Initially, these techniques were developed around sophisticated instruments and often involved complex procedures. It is obviously advantageous if the cost of analyses can be reduced and the experimental procedures simplified, while still maintaining the quality of the data collected. Furthermore, it is often desirable to have measurements performed rapidly, with on-site measurement sometimes deemed useful or even essential. All of these desirable outcomes may, in some cases, be obtained by miniaturisation. The interest in miniaturisation has led to rapid growth of the field of microfluidics, an area of study which involves using small volumes of liquids, often with detection systems specifically tailored to these reduced volumes. Microfluidic systems must have some way of moving liquids through various stages of chemical or physical processes. One particularly interesting pumping method involves the use of centrifugal force, which eliminates the need for pumps and minimises connections to the platform on which the analysis is done. Up to this point, centrifugal systems have generally been constrained to a limited number of sequential analytical steps as liquid could only flow in the direction demanded by the applied centrifugal force.In this thesis, a variety of liquid manipulation techniques on centrifugal microfluidic platforms were developed and characterised. These techniques were used to miniaturise standard classical analytical methods and implement them on centrifugal microfluidic platforms with the goal of monitoring environmentally important compounds such as aqueous sulfide. A two-phase liquid displacement pumping technique and a pneumatic-centrifugal pumping technique are demonstrated and presented. The developed pneumatic-centrifugal system was used to significantly increase the toolbox of capabilities for centrifugal microfluidic platforms, simultaneously enabling critical microfluidic operations such as valveless liquid transfer, metering, liquid flow switching, agitative micromixing, and liquid recirculation. This technique is based on contactless implementation of pneumatic pressure using compressed air on a continually rotating centrifugal microfluidic platform, thereby enabling complete liquid flow control by combining the effects of pneumatic pressure and centrifugal force.This new type of pneumatically enhanced centrifugal microfluidic platform greatly simplifies the fabrication process by minimising valving requirements, as well as improving efficiency by performing analyses in a highly automated manner. The pneumatic approach was applied to an on-disk calibration and spectrophotometric measurement using the method of standard additions. Similarly, another pneumatically enhanced platform was developed for performing liquid-liquid extractions between an aqueous phase and an organic phase, demonstrating that these centrifugal platforms are not only capable of performing complex multi-step reactions, but also multi-cycle reactions and processes. Finally, an application-specific pneumatically enhanced centrifugal platform was developed for the spectrophotometric determination of aqueous hydrogen sulfide.All of the developed analytical methods only required small sample and reagent volumes, are highly automated and convenient, and have the potential to be performed in a field environment without the need for highly trained personnel. / Dans notre société industrielle, la conception de techniques pour la quantification d'espèces chimiques dans l'environnement, les humains et les dérivés de la production manufacturière est primordiale. Au départ, ces techniques avaient été élaborées à partir d'instruments sophistiqués et se basaient sur des procédures complexes. Il serait donc avantageux de pouvoir réduire les coûts d'analyse et simplifier les procédures expérimentales, tout en maintenant un niveau élevé de la qualité des données recueillies. De plus, il est souvent souhaitable de pouvoir effectuer ces mesures rapidement, et si possible sur le site où l'échantillon à analyser est recueilli. Toutes ces caractéristiques bénéfiques des méthodes analytiques peuvent être obtenues, dans certains cas, à travers la miniaturisation. L'intérêt pour la miniaturisation a mené à une croissance rapide des systèmes microfluidiques, un domaine d'études qui se concentre sur l'utilisation de petits volumes de liquide et des systèmes de détection spécialement adaptés à ces volumes réduits. Tout système microfluidique doit intégrer une méthode de transfert des liquides à travers différentes étapes de traitements chimiques ou physiques. Une méthode de pompage particulièrement intéressante utilise la force centrifuge, ce qui permet d'éliminer l'utilisation de pompes ou connections externes au système où s'effectue l'analyse chimique. Jusqu'à présent, les systèmes employant la force centrifuge ont été limités par le nombre d'étapes analytiques consécutives, le liquide ne pouvant se déplacer que dans une seule direction définie par la force centrifuge appliquée.Pour cette thèse, plusieurs techniques de manipulation des liquides sur un système microfluidique à base de force centrifuge ont été dévelopées et caractérisées. Ces techniques ont été utilisées pour miniaturiser les méthodes analytiques classiques pour ensuite les intégrer à des plateformes microfluidiques à base de force centrifuge, l'objectif final étant la surveillance d'espèces chimiques dans l'environnement. Une technique de pompage par déplacement de deux phases liquides et une technique de pompage pneumatique à base de force centrifuge sont démontrées. La technique pneumatique à base de force centrifuge qui a été développée augmente de façon significative les capacités de la boîte à outils des systèmes microfluidiques à base de force centrifuge. Ce nouveau système permet d'effectuer simultanément des opérations essentielles dans les systèmes microfluidiques telles que le transfert de liquides sans valves, les dosages, la commutation du débit des liquides, les micromélanges par agitation ainsi que la recirculation des liquides. Cette technique se base sur l'application sans contact d'une pression pneumatique en utilisant de l'air comprimé sur un système microfluidique à base de force centrifuge en rotation constante. Ceci permet un contrôle complet du débit des liquides en combinant les effets de la pression pneumatique et de la force centrifuge. Le processus de fabrication de ce nouveau système est grandement simplifié par l'ajout du système pneumatique car cela diminue le nombre de valves à intégrer dans le système. De plus, son efficacité est accrue grâce à la possibilité d'effectuer des analyses de façon automatisée. Cette approche pneumatique a été appliquée à des mesures spectrophotométriques par la méthode des additions connues effectuées directement sur le disque. Dans le même ordre d'idées, un autre système employant la fonction pneumatique a été développé pour effectuer des extractions liquide-liquide entre une phase liquide et une phase organique. Ceci a démontré que la plateforme centrifuge est capable non seulement d'effectuer des réactions chimiques complexes en plusieurs étapes, mais aussi de répéter les cycles de réactions et autres processus.

The development of rapid clinical and inline measurement techniques for the analysis of complex chemical systems has become an area of great interest in analytical research. Ideal protocols make quick and accurate measurements while using inexpensive reagents and low-cost instrumentation. There are several measurement challenges that can inhibit many of the current spectroscopic techniques from effectively quantifying certain sample matrices. Highly scattering media such as suspensions, tissue, blood, serum, and other biological fluids are difficult to analyze using conventional optical strategies. Extensive preparatory and separation techniques are used to reduce the complexity of the matrix. The research illustrated in this thesis is centred on the application of hydrogel sensors together with ultrasound spectroscopy as tools for overcoming these challenges. In this thesis, specially designed chemical sensors are used to bind target analyte compounds, stimulating a change in the acoustic properties of the sensor. We have developed two sensors based on hydrogel chemistry in order to measure vitamin D status. Frequency analysis of ultrasonic waves that are passed through the sample is made to characterize the acoustic signature of the sensor. A multi-component linear regression model is constructed to correlate the resonance frequency change of the sensor with the addition of analyte. The first hydrogel sensor uses native vitamin D binding protein (Gc-globulin, VDBP) as the recognition element for vitamin D3. This novel approach to hydrogel sensor construction consists of multiple protein units chemically bound to chains of cellulose. The cellulose structures are further cross-linked with one another to form a three-dimensional hydrogel network. These sub-micron hydrogels are dispersive in aqueous media, permitting their use in biological samples. Sensor resonance frequency information can be obtained by passing ultrasound through the Gc-globulin hydrogel solution and interpreting the signal changes. When binding events occur between Gc-globulin and vitamin D3 in solution, a shift in resonance frequency occurs and the response is observed in the transmitted ultrasonic wave. Using this approach, quantification of vitamin D3 is achieved between 2-10 nM with a 0.87 nM standard error of estimation. The second protocol described in this work uses dispersive acrylamide-based sub-micron hydrogel molecules. These sensors are synthesized in solution with the target analyte to create molecularly imprinted recognition sites. After polymerization, the target molecule is removed though dialysis and the recognition pockets are retained. These pockets provide a recognition site for the hydrogel sensor, binding to target analyte compounds present in the solution. Aliquots of the analyte are administered into the sensor solution, which induces physical changes in the hydrogel sensor, resulting in a shift in sensor oscillation frequency. Using this technique, quantification of vitamin D3 is achieved for 25-150 nM solutions with a 12.3 nM standard error of estimation. The quantitative analysis of the two aforementioned sensors in complex biological media is demonstrated. Measurements were made in unfiltered human serum to demonstrate the feasibility of the hydrogel sensors in real-world applications. Serum samples were collected from multiple donors to demonstrate the minor impact of the sample matrix on the quantification abilities of the sensors. The demonstration of ultrasound sensitive hydrogels as an effective method for quantifying chemical compounds in liquid media is presented in this thesis. The ultrasound method is promising for rapid laboratory analysis of patient vitamin D status. Short measurement times, reduced instrumentation cost, and minimal sample preparation makes ultrasound an excellent candidate for point-of-care analysis. / Le développement de techniques d'évaluations cliniques et en ligne associées à l'analyse de systèmes chimiques complexes est devenu un domaine de grand intérêt en recherche analytique. Un protocole idéal pourrait effectuer des mesures rapides et précises en utilisant des réactifs peu dispendieux et à faible coût d'instrumentation. Hors, de nombreux obstacles liés aux techniques de spectroscopie actuelles peuvent nuire à la quantification efficace de certaines matrices d'échantillons. Par exemple, des milieux diffusants à grande variation tels que du sang, du sérum, des tissus, des suspensions et d'autres liquides biologiques sont difficiles à analyser en utilisant des stratégies optiques conventionnelles. De nombreuses techniques de préparation et de séparation sont utilisées pour réduire la complexité de la matrice. La recherche illustrée dans cette thèse se concentre sur l'application de capteurs hydrogels allant de pair avec la spectroscopie ultrasonore comme outils pour surmonter ces obstacles. Dans cette thèse, des capteurs chimiques spécialement conçus sont utilisés dans le but de lier des composés faits d'analytes spécifiques. Cela provoque un changement des propriétés acoustiques des capteurs en question. Nous avons développé deux capteurs à base d'hydrogels afin de mesurer les niveaux de vitamine D. Une analyse de fréquence des ondes ultrasonores transmises à travers l'échantillon est effectuée afin de caractériser la signature acoustique des capteurs.Le premier capteur hydrogel utilise la protéine porteuse de vitamine D (Gc-globulin, VDBP [Vitamin D Binding Protein]) comme composant de reconnaissance de la vitamine D3. Cette nouvelle méthode de synthèse de capteurs hydrogels se compose de plusieurs unités protéiques liées chimiquement à des chaînes de cellulose. Les structures de cellulose sont réticulées entre elles pour former un réseau d'hydrogels tridimensionnel. Ces hydrogels submicroniques sont dispersifs dans des milieux aqueux, ce qui permet leur utilisation dans des échantillons biologiques. Des informations de fréquence de résonance du capteur peuvent être obtenues par la transmission d'ultrasons à travers la solution d'hydrogels VDBP et l'interprétation des variations du signal. Lors de la liaison entre la VDBP et de la vitamine D3 en solution, un changement dans la fréquence de résonance se produit et la réponse en fréquence est observée dans l'onde ultrasonore transmise. En utilisant cette méthode, la quantification de vitamine D3 est atteinte entre 2-10 nM avec une erreur type d'estimation de 0.87 nM. Le deuxième protocole décrit dans cette thèse utilise des hydrogels submicroniques dispersifs à base d'acrylamide. Ces capteurs sont synthétisés en solution avec l'analyte pour créer des sites de reconnaissance à empreinte moléculaire. Après polymérisation, la molécule cible est éliminé par dialyse et les poches de reconnaissance sont conservées. Ces poches deviennent des sites de reconnaissance pour le capteur hydrogel dont l'empreinte a été prise, et elles peuvent repérer les molécules cibles en solution. Des aliquotes d'analyte sont ajoutées à la solution de capteurs, ce qui provoque des changements physiques dans le capteur hydrogel et un changement dans la fréquence de résonance du capteur. En utilisant cette méthode, la quantification de vitamine D3 est atteinte pour des solutions de 25-150 nM avec une erreur type d'estimation de 12.3 nM. L'analyse quantitative des deux capteurs mentionnés précédemment est démontrée dans des milieux biologiques complexes. Les mesures ont été effectuées dans du sérum humain non filtré afin de démontrer la possibilité d'utilisation des capteurs hydrogels dans des situations réelles. Les échantillons de sérum utilisés ont été prélevés sur plusieurs donneurs dans le but de démontrer que la matrice de l'échantillon affecte peu les capacités de quantification des capteurs.

The two molecules nitrogen dioxide and chlorine dioxide present an intriguing array of energy levels for the spectroscopist to unravel. When considered as a rigid molecular frame the molecules possess rotational levels characterized by the usual asymmetric rotor theory. Nitrogen dioxide has been discussed from this point of view and complete structural parameters have been determined. However, these relatively simple rigid rotor levels are split by the effects of the magnetic moments of the unpaired electron and the nuclear spin. Therefore, the microwave spectrum is more complex than expected from the rigid rotor point of view. Ordinarily, one would expect a removal of the two fold degeneracy due to the electron spin and the 2I + 1 fold degeneracy from the nuclear spin. This arises because of the magnetic interaction between these spins and the field generated by the rotating nuclear frame. For nitrogen dioxide there is a six fold splitting of each rotational level and one would expect a group of transitions as depicted in Figure 3. Since analogous remarks hold for chlorine dioxide we shall only mention that eight fine structure levels will result from these interactions.