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Lipophilic dye probes are widely used for labelling of cells, organelles, liposomes, viruses and lipoproteins. The lipophilic dye diffuses in the membrane and stains the cell and cells even tolerate the lipophilic dye in high concentration. The fluorescence of styryl dyes increases after insertion into the hydrophobic environment of the lipid membrane compared their fluorescence in the aqueous phase solution. The alkyl chains of the fluorescent styryl dye probe insert into membranes and are used to understand their biophysical properties and their behavior in lipid bilayers. The mechanism of incorporation of the dyes into cell membranes, or vesicle model systems, is not resolved. In this study we used a modified dialkylaminostyryl fluorescent lipid, 4-(4-(dihexadecylamino)styryl)-N-methylpyridinium iodide (DiA), replacing the I- counterion with the Cl- anion to make DiA-Cl increase hydration of the polar head and to enable self-assembling in water and formation of vesicles. Vesicles composed of DMPC (1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine)/DiA, DPPC (1,2-dipalmitoyl-sn-glycero-3- phosphatidylcholine) /DiA, DSPC (1,2-distearoyl-sn-glycero-3- phosphatidylcholine) /DiA, DMPE (1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine)/DiA, DPPE (1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine)/DiA and DSPE (1,2-distearoyl-sn-glycero-3-phosphoethanolamine)/DiA have been prepared in mole ratios between 100/0 to 0/100, in order to investigate the effects of chain length and headgroup type on chain packing and phase separation in these mixed amphiphilic systems, using nanocalorimetry, dynamic light scattering and fluorescence data, as well as confocal laser scanning microscopy (CLSM) and cryo-transmission electron microscopy (Cryo-TEM). In addition, we report the self-assembly of DiA-Cl, to form H-aggregates of lipid bilayers in aqueous solution, beyond a critical vesicle concentration. Lipid bilayers can be fused onto silica nanoparticles (NPs) to form supported lipid bilayer (SLB)-NPs. (SLB)-NPs have a varous interdisciplinary applications from medicine to environmental fields and agriculture sciences. Here, the lipids on the nanoparticles were used for two applications. One was to adsorb polycyclic aromatic hydrocarbons (PAHs) from the environment and the other was as vehicles for foliar delivery of nutrients to plants. Silica SLB nanoparticles can increase the solubility of Benzo[a]Pyrene (BaP) in order to extract the BaP from soil for in situ biodegradation. Initial studies were begun on the effect of foliar application of silica SLBs nanoparticles on plants. The SLBs to be used were prepared using both 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and DiA, in order to determine whether the lipid increased the entry of the silica into the plant leaves and whether the lipids also entered. / Chemistry

Inflammation is implicated in diseases such as hypertension and rheumatoid arthritis (RA). A mechanistic understanding of inflammatory processes as it relates to the disease state and injury needs to be developed. Specifically, the role and modulation of inflammation needs to be assessed, as well as the mechanism that produces arachidonic acid (AA) metabolites (eicosanoids). Eicosanoids are specific biomarkers of inflammation. Their biosynthesis from arachidonic acid can be catalyzed by either free radicals or enzymes such as lipoxygenases (LOX), cyclooxygenase-2 (COX-2) and cytochrome P450. Depending on the pathway or parent molecule, different distributions of eicosanoids are found. The oxidation of AA gives hydroxyeicosatetraenoic acids (HETEs), dihydroxyeicosatetraenoic acids (DHETEs), epoxyeicosatetraenoic acids (EETs), prostaglandins (PGs), isoprostanes (Isops) and thromboxanes (TXs). It is our hypothesis that AA metabolites will help in understanding the progression of inflammatory diseases. To confirm this hypothesis, analytical methods including HPLC-UV and LC-MS were developed. The developed and validated HPLC method was applied to study the effect of acute exercise on prostanoids in hypertensive African American subjects. It was our theory that urinary 6-keto PGF1&alpha and 11-dehydro TXB2 can be used to assess the role of exercise in hypertension. Moreover, we assume that 8-iso PGF2 levels can be used as an indicator to determine the relationship of oxidative stress and endothelial dysfunction in hypertension. The HPLC method involved separating urinary 8-iso PGF2, PGE2, PGD2, PGF2, 6-keto PGF1&alpha and 11-dehydro TXB2 on a SymmetryShield Rp18 column (250mm ¡Á 4.6mm) by an isocratic elution of 17 mM phosphoric acid and acetonitrile in the ratio of 65:35 and at a flow rate of 1.3 ml/min. The wavelength used for detection was 196 nm. Specificity was confirmed by LC-MS. The method was fully validated and was found to be having sufficient sensitivity (limit of quantification - 7.5 ng - 30 ng) for many biological matrices and applications. The accuracy and precision were within bioanalytical method validation limits (90.3 to 112.8 % and RSD < 10%, respectively) and the method was linear over three orders of magnitude. In addition, a HPLC-UV method for the simultaneous determination of urinary creatinine and prostanoids was also developed and validated as it is necessary to monitor creatinine levels in addition to biomarkers when the measurement is done in urine. The method was found to be linear over three orders of magnitude and is sensitive enough for the analysis of creatinine and prostanoids in urine. The advantage of this method was that one can determine the levels of these prostanoids normalized by urinary creatinine in a single analysis and in less than 17 min. The LC- ESI (electrospray ionization) MS method, on the other hand was used to determine the role of HETEs in the initiation, progression and resolution phases of inflammation in RA. It is our assumption that 12/15 HETE can be used as novel targets for the treatment of RA. The separation was performed on a C18 column using a gradient elution of 0.1% formic acid in water and 0.1% formic acid in acetonitrile. The flow rate was 1 ml/min and the run time was 75 mins. The method was found to be specific, sensitive and precise. This LCMS method was also used to develop a retention model for complex regioisomers. Quantitative structure- (chromatographic) retention relationship (QSRR) was used to develop a predictive retention model for fatty acid metabolites. Retention behaviors of the lipid biomarkers were characterized by application of QSRR analysis utilizing Austin Model 1 mode semi-empirical computations. The retention data of these fatty acids were obtained from an RP-HPLC method utilizing a Symmetry C18 column under gradient elution. Molecular descriptors that take into account the polarity; chemical reactivity and hydrophobicity of the analytes were calculated using the semi-empirical AM1 mode. It is our hypothesis that QSRR will give insight into molecular mechanism of separation of lipid biomarkers operating in a given chromatographic system and can predict retention of a new analyte and/or to identify unknown analytes. / Chemistry

The second half of the twentieth century has seen the dawn of the environmental movement. Along with our technological and scientific awareness, has come the realization that our efforts to improve our human condition have caused damage to our environment. In this age of developing environmental enlightenment, one task of analytical chemists has been to assist in the detection and identification of many environmental pollutants. Environmental measurements often involve separation of the analyte from the matrix. Chromatographic separations, especially those involving a gaseous mobile phase (GC), have been widely used. The separation itself does not measure the analyte, however, and chromatographic separation is usually followed with some detection method. One of these methods involves the use of a microwave plasma to break down the analyte into its elemental components and excite those elements with energy such that they emit specific wavelengths of light. Those wavelengths can then be monitored to measure the analyte. The following work describes the use of such a detector, known as an atomic emission detector (AED), for the detection of several pollutants. First, the AED has been implemented for the detection of fluorine and oxygen simultaneously. This is important for the determination of fluoroethers, thought to be a replacement for ozone layer destroying chlorofluorocarbons. Under normal plasma conditions, fluorine etches the tube used to contain the plasma and results in an oxygen response regardless of whether the compound contains oxygen or not. A new plasma was evaluated that uses carbon to protect the walls of the tube from etching and thus greatly reduces the spurious oxygen response. Detection limit and linear dynamic range data is presented. Second, a method was evaluated for the calibration of plasma response with alternative calibrants. Chlorpyrifos was used to calibrate AED response for triademifon, isazofos, and trichlorfon. It was determined that calibration could only be obtained for the isazofos, there being a statistically significant difference between calibration of the chlorpyrifos and the other pesticides. Methods of using large injection volumes with cool-on-column injection was developed to eliminate the need for preconcentration of pesticide samples. Triademifon, isazofos, and trichlorfon were successfully determined at trace levels with no degradation of the chromatographic performance due to the large injection volumes. A method of thermal extraction of priority pollutants was developed. Chlorpyrifos was thermally extracted from soil samples. However, the pesticide showed extraction recoveries that were dependent on the age of the sample. That made calibration of the method impossible. Polychlorinated biphenyls (PCBs) were also used in the evaluation. These showed no time dependence and therefore calibration of the method was possible. Levels of PCBs in a standard reference material (SRM) were determined and compared to certified values. Finally, 1,1,1-trifluoroethyl hydrazine (TFH) was used to derivatize aldehydes for their determination by GC-AED. Good recoveries were obtained. In the determination of aldehydes in ozonated waters, however, the detection limit was found to be much higher than those of competing techniques.

The kinetic nature of flow injection (FI) experiments has led to the development of analytical methods that rely on the concentration gradients that are formed as a result of the hydrodynamics of the flowing stream. The concentration-time profile of the flow injection peak contains a wealth of chemical information, most of which are ignored in the typical FI experiment based on peak height. The objective of this work was to study a number of novel ways in which concentration-time data obtained from a variety of FI experiments can be used for analytical determinations and for the determination of fundamental reaction parameters. Time intervals were used as quantitative analytical parameters for slow and fast reactions. The Belousov-Zhabotinskii oscillating chemical reaction was generated in a flow injection system and monitored under conditions of stopped flow. The injection of an analyte into the slowly reacting system altered the behavior of the reaction. Times between events on the absorbance-time profile were used in a new kinetic method of analysis. The time interval between doublet peaks was the analytical parameter used in a study of FI doublet peaks. Mixing devices used in the experiment were compared and various aspects of the theory behind flow injection peak-width methods were discussed. Results from FI doublet peak determinations were presented and the overall precision of the method was evaluated. Formation quotients and reaction rate constants were calculated from sets of data points taken from the concentration-time profile of the FI peak. A flow injection system incorporating a well-stirred tank was used to create the concentration gradients from which the data were obtained. Two iterative methods were used to calculate formation quotients from absorbance-time data. The well-stirred tank model was modified for the situation of removal of sample by reaction. Novel ways to determine rate constants from absorbance-time data were based on the derived equations.

The purpose of this research is to improve the performance of Inductively Coupled Plasma Optical Emission Spectrometry (ICP-OES) by way of a study of the sources of limiting performance. This is demonstrated with the use of practical diagnostics and new ICP-OES instrumentation having a cross-dispersed echelle spectrometer and a matched segmented-array charge coupled device detection system. The main finding of this work is that the advanced detection system can be coupled with multivariate data processing to reduce analytical precision and detection limit values to their fundamental limits. Practical diagnostic protocol enables elucidation of the factors governing these important figures of merit in ICP-OES. A key diagnostic method used in this work is the signal to background ratio - relative standard deviation of the background (SBR-RSDB) approach developed by Boumans for detection limit studies. This methodology serves as the framework to compare detection limits and understand the physical basis for improvements or differences. The SBR-RSDB approach was used to show how multivariate methods take advantage of the simultaneous multielement spectral registration of the new instrumentation. The combination of simultaneous spectral readout and multivariate processing yields detection limits which are reduced to the photon shot noise limit, even when source flicker noise is dominant in the background signal. Other demonstrated advantages of multivariate methods are the capability for spectral interference correction and automatic background correction superior to conventional methods. The SBR-RSDB approach was central to understanding the performance differences between axial and radial viewing of the inductively coupled plasma. Real-time internal standardization is demonstrated to improve precision values to the photon shot noise limit. For certain elements, a barrier to the achievement of shot noise limited precision was identified and linked to differences in the noise signatures of the reference and analyte signals. The magnitudes of the noise in the signals were strongly related to excitation energy for atomic lines. An explanation in terms of the influence of incompletely vaporized droplets in the observation volume is proposed which is consistent with the observed results.

The overall objective of this thesis has been to improve the performance characteristics of analytical atomic spectrometers using on-line flow based front-end chemistry and various separation schemes with minimum or no physical modification of the instrument. The underlying philosophy was to develop methods which were rapid, and easy to operate using the minimum amounts of sample and reagent and which generated the minimum amount of waste and were amenable to automation. A method for the on-line preconcentration of copper was developed. This method uses a precipitation and dissolution approach and the manifold was directly coupled to an atomic absorption spectrometer. Using a novel semi-automated flow injection manifold design an order of magnitude improvement in detection limit over conventional aspiration was obtained. Using a precipitation reaction, potentially hazardous material was removed on-line from samples before their introduction into the spectrometer. There was a kinetic limitation of the reaction involved in this method, but using a recirculating loop manifold this was overcome and the method was successfully applied to the on-line determination of various trace metals in solutions containing high amounts of silver. A flow injection method for the rapid on-line determination of trace metals in solutions containing high concentrations of sodium nitrate was developed. This method was developed as fast diagnostic procedure using special in-line mixers to dilute the samples on-line and thereby reduce the effect of the matrix. The method was successfully applied to samples obtained from the Royal Canadian Mint. Using flow injection sample introduction and inductively coupled plasma-mass spectrometry detection the determination of mercury was achieved using a gas/solid reaction. The approach was to generate a cold vapor of mercury, by reaction with a reductant, and trapping it on a gold/platinum gauze and releasing it by controled heating and passing it to the ICP-MS for detection and quantification. This method was successfully applied to a variety of water samples. Matrix effects were reduced and sensitivity and detection limit were improved. A study of solid/liquid reactions for the on-line matrix separation and preconcentration of trace elements in sea water was carried out. The method involved on-line adjustment of the pH of the sample solutions and then passing the sample over an ion-exchange chelating resin. The analytes were retained on the resin and the matrix was washed away to waste. The analytes were removed from the resin with acid and introduced into the ICP-MS for detection and quantification. This sample cleanup procedure was successfully applied to the determination of several trace elements in spiked sea water standard reference materials.