(Uncorrected OCR) Abstract Porphobilinogen (PBG) synthase condenses two molecules of aminolaevulinic acid (ALA) to form PBG in heme biosynthesis. The enzyme activity is sensitive to inhibition by heavy metals such as lead. It can act as a biological indicator of chronic lead POis~r\g to identify the risk group, especially in children, so that early treatment can be given to prevent possible permanent damages. A reversed-phase ion-pair HPLC analytical method for the assay of the PBG synthase activity based on detection of PBG production has been validated. A Hypersil CN column (150 x 4.6 mm; 5 urn) was employed together with a mixture of acetonitrile-40 mM phosphate buffer at pH 2.4 with 5 mM 1-heptanesulphonic acid (8:92, v/v). UV detection was performed at 240 nm. PBG was eluted as a spectrally pure peak resolved from its impurities in the methanol-inhibited enzyme reaction. The method was sensitive with a limit of quantitation of 2 ~M. The within-run and between-run precisions were 8.2% and 13.8% respectively. The recovery was 93.4 �7.1% (n=6). The preliminary reference range of the PBG synthase activities in the local pediatric population were from 21.5 to 26.3 ~mol/L RBC/min. Bland and Altman statistical analysis showed that the HPLC assay and the colorimetric assay could not be used interchangeably. The HPLC assay was an alternative way to assess the PBG synthase activities in the human erythrocyte samples. IV / abstract / toc / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / abstract / toc / Medical Sciences / Master / Master of Medical Sciences

published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

The goal of this project is to establish an efficient and economical industrial process for extraction of a Kaufmann and Tobschirbel orbitide (KT) mixture from flaxseed oil. KTs occur at a low level in flaxseed oil and must be concentrated at least 600 fold to produce a useful commercial concentrate. KT peptides are more polar than most lipids may be separated using solid- or liquid- phase extractants. Extraction protocols were investigated to determine a better approach for KT peptide extraction. Commercial solid-phase extraction methods would require the adaptation of bench-scale silica flash column chromatography. The first approach was to develop methods for separation of peptides using only silica, ethyl acetate and ethanol. Ethyl acetate is known to remove both oil and peptides from silica. Therefore, the ability of low temperature to decrease the peptide elution from silica was studied. The other method utilized liquid-liquid extraction. In order to measure the success of an extraction an analytical method was required to evaluate the separation of peptides from oil. An analytical procedure was developed that readily determined the relative concentration of peptide and lipid. Aqueous and anhydrous ethanol partitioning was used to extract the KT mixture from flaxseed oil. Ethanol solutions between 50 and 100% in water (v/v) were mixed with flaxseed oil. The oil and peptide content of the extracts were determined using 1H-NMR. Liquid-liquid extraction using 70% aqueous ethanol at volume ratio (solvent to oil) of 0.25:1 produced a mole ratio of 2:1 (KTs to oil) making it the optimal solvent for KT extraction. In the second part of this project, the scale of liquid-liquid extraction was increased through several 10 to 30-fold steps to establish a potential industrial extraction process for recovery of the KT mixture. The feasibility of processing the solvent containing mixed peptides was investigated. Multiple evaporation and adsorption methods were also tested, including falling film evaporation, rotary evaporation, a combination of rotary evaporation and freeze drying, and a combination of rotary evaporation and spray drying. Various experimental methods to enrich and isolate KTs from water-rich fraction were performed. At the end of this project, 3328.89 g of KT mixture was produced that was suitable for commercial purposes. The increase of extraction scale was 140,000 fold.

Investigations of new applications of the inductively coupled plasma (ICP) for analytical atomic emission spectroscopy are performed. Research efforts are focused in three major areas: emissions below 185 nm, analysis of wear metals in lubricating oils and use of the ICP as a selective detector for high performance liquid chromatography (HPLC). A unique plasma coolant tube containing a side arm which allows direct observation of the discharge is used to investigate emissions in the vacuum ultraviolet (VUV) spectral region between 120 and 185 nm. Emissions from elements which do not emit radiation in the visible region are observed. Oxygen emissions at 130 nm, nitrogen at 149 and 174 nm and carbon at 155 and 165 nm make up the background spectrum. These elements are present as impurities in the argon gas used to sustain the ICP discharge. Fifteen emission lines from bromine are observed. Those at 153 and 163 nm are the most intense. Sulfur also has fifteen emission lines and chlorine has nine in this region of the spectrum. The VUV region is found useful for observation and potential analysis of many elements. A heated sample introduction system attached to a Babington nebulizer is investigated as a means to aerosolize lubricating oils for introduction into the ICP. This allows direct analysis of wear metals in oil samples without requiring the usual sample dilutions. Several commercial brands and weights of motor oil are spiked with iron in order to evaluate this system. Heating the oil as it enters the nebulizer is found to increase the nebulization efficiency as much as sixtyfold in some cases. Differences in nebulization efficiency due to viscosity are almost entirely eliminated through the application of heat. A linear calibration curve extending three orders of magnitude from a detection limit of one ppm iron is determined. The ICP is used as a selective detector for HPLC. Nucleotides separated by anion exchange chromatography are determined in the ICP by observing phosphorus emissions. Methanol and acetonitrile used for reverse phase HPLC are successfully run in the IPC. The method is evaluated by using the ICP to determine phosphorus in compounds separated by using reverse phase conditions. The HPLC is used to separate organic interferences from several silicone samples using reverse phase conditions allowing the ICP to accurately analyze silicon content.

No description available.

A sol-gel stationary phase was developed for in-situ infrared (IR) detection of analytes on thin-layer chromatography (TLC) plates. These sol-gel-based TLC plates have improved optical properties compared with conventional TLC plates in IR spectroscopic analysis. Samples can be analyzed in transmission geometry, requiring no special attachments. The sol-gel-based TLC plates demonstrate significantly better light throughput and a wider spectral range than conventional TLC plates analyzed in diffuse reflectance geometries.The sol-gel precursor, methyltrimethoxyorthosilicate (MTES), was templated with cetyltrimethylammonium bromide (CTAB) and urea in order to form a porous sol-gel. Aerosol deposition was used to apply the sol-gel solution onto either glass slides or silicon wafers within an enclosed chamber. Many variables were studied to determine their effect on the quality of the sol-gel stationary phases, including the ratio of MTES:methanol:water:CTAB:urea:HCl:, gelation times and temperatures, and deposition rate. Sol-gel films prepared using MTES/methanol/water/CTAB at ratios of 1 : 20 : 7 : 0.2 containing 5 wt% urea (relative to MTES) and pH 1.5 were crack-free, mechanically stable, and uniform in appearance. The films were tens of microns thick with a highly interconnected porous structure.For chromatographic separations, the films exhibited good solvent migration velocity and could be repeatedly washed and reused for TLC separations without showing degradation in the separation. Several different classes of compounds, including polyaromatic hydrocarbons and dyes, were successfully separated. Theoretical plate values measured on the MTES-based sol-gel films were comparable to those obtained on commercially available TLC plates.

Investigation and control of alkylsilane stationary phase structure in reversed phase liquid chromatography is presented. Raman spectroscopy is used to probe the alkyl chain conformational order and interchain coupling as a function of various chromatographic conditions. A new method is further developed to fabricate alkylsilane stationary phases with controlled surface coverage. The alkyl chain conformational order and interchain coupling of a series of high-density docosylsilane (C22) bonded stationary phases is shown as a function of temperature, surface coverage, polymerization method, common solvents and solutes. The conformational order of C22 stationary phases is compared to that of octadecylsilane (C18) stationary phases to understand the chain length effect on stationary phase structure. The conformational order information as indicated by Raman spectral order indicators for a C22 phase are correlated with the capacity factor and separation efficiency for each solute studied to gain insight into the retention mechanism. These studies help to understand the origin of stationary phase shape selectivity and the separation process in general. Based on these results, the molecular pictures at the stationary phase/solvent interface are proposed. The effect of pressurized solvent environments on two C18 phases is studied to obtain direct evidence for changes in stationary phase structure due to pressure. These changes are compared to effects of solvation relative to air in the same solvents. In addition, Raman spectral order indicators are identified for perdeuterated alkyl-containing system. This study provides a foundation for studying stationary phase structure in complex systems comprised of long alkyl-containing solutes.A further development of a new method is presented as well for synthesizing alkylsilane stationary phases with precisely controlled surface coverage by using a displaceable surface template monolayer of n-alcohol. A mechanism for this process is proposed based on the studies of n-alcohol concentration and chain length effect on the stationary phase surface coverage. The utility of these new stationary phases as chromatographic support is demonstrated. The shape selectivity for these new phases is comparable to or better than similar phases prepared by conventional methods.

Burkholderia pseudomallei is the causative agent of Melioidosis, an endemic disease in South East Asia, and is classified as a category B biological agent. Currently, there is no licensed vaccine for this disease; the mortality rate is high due to the incorrect diagnosis and the pathogen insusceptibility to general antibiotics. A mass spectrometry based proteomic approach has been applied in order to identify the proteins that are responsible for pathogenicity.Methods were developed for the proteomic analysis of Burkholderia species using B. vietnamiensis G4, an opportunistic pathogen as the model organism. Both gel-based (LC-MS/MS) and gel-free MudPIT (LC/LC-MS/MS) approaches have been applied for the analysis of the proteins extracted from four different cellular fractions of these bacteria. More than 1200 proteins were identified from these analyses, including many proteins previously identified as virulence factors of these bacteria. Similar methodologies were applied to build a proteome map of non-pathogenic B. thailandensis E264 to use as a reference for the pathogenic studies. Additionally, proteomes of two B. thailandensis strains isolated from two geographical locations were compared to investigate the differences in protein expression of these organisms.Proteins identified from pathogenic B. pseudomallei were compared with the non-pathogenic B. thailandensis and opportunistic pathogen B. vietnamiensis proteins. Many species specific proteins were identified from this proteomic analyses; those proteins can be used as antigen targets to selectively identify these pathogenic bacteria in a complex biological matrix using affinity capture methods.Ticks are vectors that can transmit disease causing pathogens one host to another. Knowing the pathogen reservoir is important in order to control disease spread in the environment. Application of mass spectrometric methods to identify the host blood components from tick vectors was investigated using tick nymphs which feed only once in their life cycle. Using mass spectrometry based proteomics; host specific proteins like hemoglobin and immunoglobulin were identified from a single tick nymph analysis. Additional studies have examined the fatty acid profiles of rabbit and sheep blood fed tick nymphs using SPALDI mass spectrometry. Different fatty acid profiles were obtained for these tick nymphs, but further investigations are required to validate these findings.

A hydrophilic interaction chromatography (HILIC) technique has been developed and validated for determination of common denaturants (denatonium benzoate, crystal violet and methylene blue) in denaturated alcohol formulations. Among the three different polar stationary phases (i.e., aminopropyl, cyanoethyl and silica) studied the cyanoethyl phase provided much stronger retention for the organic cations. It was shown that high efficiencies were reached only with anionic ion-pairing reagent that reduces the interactions with the silanol groups. The anion ion-pairing strength under HILIC conditions was: acetate < formate << trifluoroacetate < perchlorate. This study also investigated the effect of various experimental factors on the retention of the cyanoethyl stationary phase, such as acetonitrile content, pH, ionic strength, and ion-pairing anion concentration in the mobile phase. The separation of three denaturants was achieved in about 8 min with a mobile phase containing 60% (v/v) acetonitrile and 10 mmol/l HClO4. The proposed method was validated and applied to the determination of danaturating agents in various Lithuanian denaturated alcohol formulations.