Low levels of biogenic amines occur naturally, but high levels (FDA sets 50 ppm of histamine in fish as the maximum allowable level) can lead to scombroid poisoning. Amines in general are difficult to analyze by Gas Chromatography (GC) due to their lack of volatility and their interaction with the GC column, often leading to significant tailing and poor reproducibility. Biogenic amines need to be derivatized before both GC and HPLC analyses. The objective of this research was to develop a relatively fast, reproducible method to derivatize and quantitate biogenic amines in fish at trace levels using GC/FID. The derivatizing reagent used in the experiments was propyl chloroformate, useful for aqueous samples. To confirm the identity of six derivatized biogenic amines GC/MS was used. To our knowledge no reference spectra for these derivatives has been published. It was concluded that best results are obtained using a Cold-On-Column (C.O.C.) inlet with a short column (15 meters), thick film stationary phase (ZB-5, 1.00μm df), and with recommendations to cut 40 cm from the inlet end of the column every 25 injections when using C.O.C. Duplicate samples of Atlantic Salmon were analyzed on days 0, 3, and 5. Levels of histamine were below 50 ppm for days 0 and 3, but day 5 showed average levels of 160 pm (cadaverine), 1000 ppm (histamine), and 350 ppm (tyramine). Good precision of six amine stardards at 50 ppm was shown: heptylamine 5.2%, putrescine 5.6%, cadaverine 5.0%, histamine 9.9%, tyramine 5.1%, and spermidine 6.2% RSD. / Master of Science

Ammonium ionenes are macromolecules with quaternized nitrogen groups in the main chain. Ionenes are commonly referred to as x,y-ionene, where x and y represent the number of methylene groups between quaternized nitrogens. Synthesis of aliphatic ammonium ionenes has been studied since the early twentieth century; however, absolute molecular weight characterization has only been performed using extensive light scattering and viscosity experiments. Performing aqueous size exclusion chromatography (SEC) on ammonium ionenes provides absolute molecular weight determinations while eliminating the need for separate viscosity and light scattering experiments. We developed a mobile phase composition that provides reliable separation of aliphatic ammonium ionenes using aqueous SEC. For the first time, we report absolute molecular weights of aliphatic ammonium ionenes using this technique. We investigated the influence of charge density and structural symmetry on thermal and mechanical properties of ammonium 6,6-, 12,6- and 12,12-ionenes. Thermal properties were measured using differential scanning calorimetry (DSC) and thermal gravimetric analysis (TGA), and mechanical properties were measured using dynamic mechanical analysis (DMA) and an Instron. Incorporating low molecular weight polymer segments into the main chain of the ionene allows tailoring of polymer characteristics. Poly (tetramethylene oxide) segments decrease hydrophilicity and increase elastomeric character. Linear PTMO based ionenes have been synthesized previously, and we were interested in how branching affected thermal and mechanical properties. We synthesized bis(dimethylamino) poly(tetramethylene oxide) segments, and subsequently, synthesized linear and branched ionenes to study the effects of topology on thermal and mechanical properties. Polymers were analyzed using DMA, DSC, TGA, SAXS, and an Instron. / Master of Science

Peanut skins have long been a waste product of the peanut industry. The aim of this project was to find suitable applications for this rich source of natural bioactive compounds. Solvent extracts of peanut skins and a multistep solvent extraction process to yield oligiomeric procyanidin (OPC) extracts were found to be inhibitory towards three types of yeasts (Saccharomyces cerevisiae, Zygosaccharomyces bailli, and Zygosaccharomyces bisporus). All extracts were devoid of solvents that may have interfered with the results. The OPC extract exhibited the highest inhibitory effect, and was chosen for fractionation. Fractionation was conducted by means of a silica or size exclusion high performance liquid chromatography (HPLC) column. Fractions were then subjected to a yeast growth curve assay to determine the active fractions. The fractions were then characterized by liquid chromatography-mass spectrometry (LC-MS). Negative mode electrospray MS determined the fractions to contain mostly procyanidins but also proanthocyanidins. Since it is possible for multiple compounds to display the same molecular ion, multistep MS and retention time differences were utilized to tentatively identify the compounds based upon their fragmentation schemes. However, co-elution was prominent, thus specific compounds responsible for yeast growth inhibition could not be determined. The yeast inhibition assay demonstrated that the procyanidin dimers up to tetramers had the best anti-yeast capabilities. / Ph. D.

A study of six liquid chromatographic column packing materials was conducted. The materials investigated were: SI-60, SI-100, ALOX T, RP-2, RP-8, and RP-18 (E. M. Laboratories, Elmsford, N.Y.). The column packing materials had an average particle diameter of 10 µm and were slurry packed into stainless steel columns that were 3 mm i.d. and 250 mm in length. Two series of probe compounds were utilized to investigate the differences in selectivity exhibited by the column packing materials in the normal phase mode and in the reversed phase mode. A column oven which controlled temperature to within ± 0.2 °C over the range of 0°C to 100°C was utilized throughout the work. Four different mobile phases were used, two for the normal phase work and two for the reversed phase work. The effect of temperature was investigated in both the normal phase mode (20°C - 50°C) and in the reversed phase mode (20°C - 80°C). The results of the selectivity study indicate that the column temperature can have a pronounced effect in normal phase liquid chromatography. As temperature increased, k' values went through a minimum in some cases, indicating a change in retention mechanism. As the temperature is increased, the retention of the compounds may be increased, decreased, or remain constant. RP-2 is a versatile column packing material which is useful in both the normal phase mode and the reversed phase mode. In the normal phase mode it behaves as a low surface area adsorption column and of the three adsorbents, (SI-60, SI-100, and RP-2), it has the widest scope. In the reversed phase mode it also has the widest scope of the three reverse phase columns studied (RP-2, RP-8, and RP-18). In reversed phase liquid chromatography, increases in retention as temperature was increased were not observed and plots of log k' vs. temperature in °C resulted in straight lines with very similar slopes. Column temperature is however an important operating parameter which is useful in optimizing separations. Temperature was found to have little effect on the efficiency of the columns but was found to have a significant effect on the scope of the column packing materials in both the normal phase mode and in the reversed phase mode. When utilizing high surface area column packing materials or column packing materials with a high percentage of hydrocarbon bonded phase, temperature control is essential in order to obtain the best reproducibility. / Doctor of Philosophy

Gaseous phase is a critical state of matter around us. It mediates between the solid crust on earth and inter-stellar vacuum. Apart from the atmosphere surrounding us where compounds are present, natively, in a gaseous phase, they are also trapped within soil and dissolved in oceanic water. Further, those that are less volatile do enter the gaseous phase at high temperatures. It is this gaseous phase that we inhale every second. It is thus critical that we possess the tools to analyze a mixture of gaseous compounds. One such method is to separate the components in time and then identify, primarily based on the retention times, also known as gas chromatography. This research focuses on the development of gas detectors and their integration, in different styles, primarily for gas chromatography. Utilizing fabrication techniques used in semiconductor industry and exploiting scaling laws we investigate the ability to improve on conventional gas separation and identification techniques. Specifically, we have provided a new spin to the age-old thermal conductivity detector enabling its monolithic integration with a separation column. A reference-less, two-port integration architecture and a one-of-its-kind released resistor on glass are some of its salient features. The operation of this integrated device with a preconcentrator and in a matrix array was investigated. The more unique contribution of this research lies in the innovative discharge ionization detector. An ultra-low power, sensitive, easy to fabricate detector, it requires more investigation for a thorough understanding and will likely mature to replace the thermal conductivity detector, as the detector of choice for universal detection, in time to come. / Ph. D.

Advances in thin layer chromatography (TLC), including smaller more uniform particles, use of a scanning spectrophotometer (densitometer), and sample application devices, led to the development of the High Performance Thin Layer Chromatography (HPTLC) technique. HPTLC allows quantitative as well as qualitative results of much smaller amounts. in some cases down to the picogram level. With these advancements, the limiting factor in detection of smaller concentrations has become the plate itself, and more specifically the preparation of the absorbent and binder and the layering process. This research evaluated HPTLC plates from several manufacturers for significant differences between manufacturers and between plates of each manufacturer. Several concentrations of three drugs of abuse were applied, developed, and quantitated. Both Rf and peak area were statistically evaluated to look for any effect of manufacturer, specific plate for that manufacturer, specific drug, concentration, and/or cross nested effects. Significant differences were found between manufacturers for both Rf and peak area with E. Merck and Baker plates having the best overall results. All manufacturers were found to have some plates with obvious visual surface defects that were not suitable for use. The major source of variation for all manufacturers was the plate to plate variation rather than track to track deviations on any given plate. / Master of Science

Optimization of capillary gas chromatography-Fourier Transform Infrared (GC-FTIR) spectrometry has been accomplished by studying various columns, lightpipe designs, FTIR detectors and spectroscopic parameters. For adequate separation of complex samples the efficiency of WCOT (Wall Coated Open Tubular) fused silica capillary columns was found to be unmatched by packed columns or wide bore glass capillaries. A consequence of using more narrow bore columns, however, is lower sample capacity and less IR detectability. Two 6 cm light pipes and a more narrow 40 cm lightpipe were compared with respect to both optical throughput and eluent band broadening. FTIR spectroscopic parameters such as mirror velocity and number of scans coadded were examined in order to achieve an optimum signal to noise ratio. The complexity of a particular sample has been shown to dictate certain spectroscopic parameters. The sensitivity differences of two liquid-nitrogen cooled FTIR detectors used in the GC-FTIR experiments have been determined with reference to using them with either a long or short lightpipe. The capability of optimized capillary GC-FTIR has been demonstrated in the analysis of model compound mixtures and comp lex petroleum products including test aviation jet fuels. Major attention was given to the detection and identification of aliphatic and aromatic components in the highly complex samples. Computerized library search routines have been used to tentatively identify eluting components by infrared spectral matching to quality vapor phase library file spectra. Gas chromatography-mass spectrometry (GC-MS) data are also included for the same jet fuel samples to directly compare extent of information provided and relative spectroscopic sensitivities. / Master of Science

Supercritical fluid chromatography (SFC) has been gaining much exposure over recent years. The characteristics inherent to SFC give it capabilities in separating nonvolatile, thermally labile compounds inseparable by GC, and with greater efficiency per unit time than HPLC. As in the past with the more conventional chromatographic methods, the need for higher information detectors, such as IR, exists for SFC. The interface of a Fourier Transform Infrared (FTIR) spectrometer to a packed column (analytical scale) SFC is demonstrated. Characterization of mobile phases for SFC-FTIR is presented. Also, parameters for the optimization of spectral sensitivity, such as mobile phase transparency and flow cell design, are discussed. Demonstrations are made via model mixture separations. Finally, the application of this technique to the analysis of free fatty acids is presented. Identification of carboxylic acids of carbon number 6-18 is made on several standard mixtures and on free fatty acids extracted from various complex samples. The spectral identification of the compounds as free fatty acids is presented, and spectral evidence for dimerization of the acids in CO 2 and the coelution of saturated and unsaturated carboxylic acids is shown. / Master of Science

A method using gas chromatography for the analysis of carbon dioxide, carbon monoxide, and nitric oxide in diesel exhaust was developed. A gas chromatograph containing a liquid phase column in series with a molecular sieve column, each of which eluted into thermal conductivity detectors, was utilized. Activation of the molecular sieve column was achieved by heat-treating and purging with nitric oxide. The chromatograph was calibrated by introducing sample mixtures of known concentration and measuring the responses. The exhaust gases of a diesel engine were analyzed while the engine operated at constant speed and load. Engine speed was kept at 1400 rpm while several different loads were applied. The results of these tests indicated that carbon dioxide, carbon monoxide, and nitric oxide concentrations all increased with load in the load range studied. Carbon monoxide exhibited a greater dependence on load than did the other compounds. Additionally, water was injected into the intake air stream to study its effect on nitric oxide concentration. Nitric oxide levels were reduced by 15% when a water/fuel mass ratio of 0.75 lb/lb was used. / Master of Science

1. The sensitivity obtained even with the free compounds (except morphine) make gas chromatography a good technique for trace analysis of drugs in biological samples. 2. For many of the drugs studied the detector response curves are not linear with sample concentration but the calibration curves are reproducible if care is exercised. This makes quantitative analysis feasible down to 50-100 nanogram levels with proper technique. 3. Long column life is needed for routine analyses. Those columns employed in this work were used continuously for over seven months with no appreciable deterioration. / Master of Science