The purpose of this research was to design a simple, novel interface for on-line coupling of Supercritical Fluid Extraction (SFE) with High Performance Reversed Phase Liquid Chromatography (HP-RPLC), and to explore its ability for quantitative analysis of analytes in different matrices. First, a simple interface was developed via a single one six-port injection valve to connect the SFE and LC systems. A water displacement method was utilized to eliminate decompressed CO2 gas in the solid phase SFE trap and connection tubes. To evalute this novel hyphenated system, spiked polynuclear aromatic hydrocarbons (PAHs) in a sand matrix were used as target analytes with the achievement of quantitative results. Also PAHs in naturally contaminated soil were successfully extracted and quantitatively determined by this hyphenated system. Compared to the EPA method (Soxhlet extraction followed by GC-MS), on-line SFE-LC gave precise (4-10% RSD) and accurate results in a much shorter time. Based on this hyphenated technique, a method for the extraction and analysis of hyperforin in St. John's Wort was developed under air/light free conditions. Hyperforin is a major active constituent in the antidepression herbal medicine-Hypericum Perforatum (St. John's Wort). Hyperforin is very sensitive to oxygen and light. There is no way to date to determine whether any degradation occurs during the sample-processing step in the analytical laboratory. On-line coupling of SFE-LC with UV absorbance/ electrospray ionization mass spectrometry (SFE-LC-UV/ESI-MS) provided an air/light free extraction-separation-detection system, which addressed this issue. Mass spectral data on the extract confirmed the presence of the major degradation compounds of hyperforin (i.e. furohyperforin and two of its analogues). Thus, the degradation process must have occurred during plant drying or storage. The feasibility of quantitative extraction and analysis of hyperforin by on-line SFE-LC was made possible by optimizing the extraction pressure, temperature, and modifier content. High SFE recovery (~90%) relative to liquid-solid extraction was achieved under optimized conditions. We then extended the interface's application to an aqueous sample by using a liquid-fluid extraction vessel. Quantitative extraction and transfer were achieved for the target analytes (progesterone, phenanthrene, and pyrene) spiked in water, as well as in real samples (urine and environmental water). During each extraction, no restrictor plugging was realized. Extraction temperature and pressure were optimized. Different amounts of salt were added to the aqueous matrix to enhance ionic strength and thus extraction efficiency. Methanol and 2-propanol were used as CO2 modifiers. Two modifier modes were compared, e.g. dynamically mixing modifier with the CO2 extraction fluid, and pre-spiking modifier in the extraction vessel. Surprisingly, we found pre-spiking the same amount of modifier in the vessel enhanced the recovery from ~70% to ~100% for progesterone, phenanthrene, and pyrene due to a "co-extraction effect". The last phase of our work explored the disadvantages/limitations of this hyphenated technique through the analysis of more highly polar phenolic compounds in grape seeds. Five types of SFE trapping adsorbent materials were evaluated in an effort to enhance the collection efficiency for the polar components. Pure supercritical CO2 was used first to remove the less polar oil in the seeds. Then methanol-modified CO2 was used to remove the polar components (e.g. phenolic compounds). Catechin and epicatechin (90%) were exhaustively extracted out of the de-oiled seed after 240 minutes with 40% methanol as modifier. Both singly linked (B-type) and doubly linked (A-type) procyanidins were identified by LC-ESI-MS, as well as their galloylated derivatives. Compared to the off-line SFE-LC approach, much less sample was required for extraction in the on-line method, since all the extracted components could be transferred to the LC column. Also, no extract processing/concentration step was needed in the on-line method. However, in the on-line mode, some polar compounds were lost (1) during the collection step (e.g. lower trapping efficiency on a single solid SFE trap when a high percentage modifier was used) and (2) during the water rinsing step (e.g. less retention of polar compounds on C18 trap). Therefore, this hyphenated technique is less desirable for the analysis of highly polar compounds. / Ph. D.

Pseudo-molecular ion formation is an artifact common to most analyses performed by electrospray ionization mass spectrometry. These species are non-covalent complexes formed between an analyte of interest and any other components (such as mobile phase, additives, and impurities) present in the ionized sample band. Published literature addresses pseudo-molecular ion formation in routine analyses as well as in complicated molecular recognition processes. The majority of these works are directed towards the formation of complexes in the positive ionization mode. Consequently, investigation of pseudo-molecular ion formation in the negative ionization mode is a logical extension of work in this area. Experiments presented here detail the work performed on elucidation of factors controlling ionization efficiency of aromatic acid pseudo-molecular ions by electrospray ionization in the negative ionization mode. Sets of tested acidic analytes, including ibuprofen derivatives and benzoic acid derivatives, were analyzed in the presence of various solution systems by flow injection analysis to determine the effect of pH, concentration, injection volume, and instrumental parameters on dominant ion forms observed in the mass spectra. These ion forms correspond to a deprotonated molecular ion ([M-H]-), a hydrogen-bound dimer ion ([2M-H]-), and a sodium-bridged dimer ion ([2M-2H+Na]-). Report of the latter ion form is unique to this work. Response of these ion forms were found to vary greatly with changing solution parameters, particularly in the presence of common LC-MS modifiers, such as triethylamine, acetic acid, formic acid, and ammonium formate. Results point to the formation of the sodium-bridged dimer ion during gas-phase processes following the release of ions from disintegrated droplets. Ab initio theoretical calculations and correlations with calculated solution phenomena (such as pKa and log P) were used to elucidate structural arrangements and dominant factors controlling pseudo-molecular ion formation by aromatic acids in the negative ionization mode. / Ph. D.

Electrochemical CO2 reduction reaction (CO2R) is a promising route to help reduce greenhouse gas emissions and reach carbon dioxide net zero emissions to combat global warming. Currently, in order to investigate catalytically produced products from CO2R offline methods such as gas chromatography (GC) and nuclear magnetic resonance (NMR) are used. These offline methods have a time resolution on the minutes to hours scale which leads to uncertainty of evaluating how products are produced from CO2R, such as knowing if a product is produced from electrochemical means or chemical conversion, and if a product is being produced in a linear rate of production or a different rate. This is where the ability to have real-time analysis of the products generated from CO2R is desirable, as it can more definitively answer many of these questions. Yet few analytical techniques have been developed in detail so far to achieve real-time analysis. Herein, we show the use of selected-ion flow-tube mass spectrometry (SIFT-MS) that quantitatively measures in realtime an array of 10 C1, C2, and C3 products from CO2R such as ethanol, ethylene or methane. The custom-developed SIFT-MS selected ion mode scan measures the concentration of gas and liquid-phase products of CO2R at the same time and is compatible with any electrolyzer cell. We demonstrate that the SIFT-MS technique can reliably and accurately determine product concentration in real-time through the evaluation of Cu foil and its comparison to traditional techniques. Considering the narrow range of developed and deployed techniques for real-time quantitative product analysis for CO2R, this study on SIFT-MS is a critical tool for future research in accelerating and optimizing catalyst design for electrochemical CO2R applications. / Thesis / Master of Applied Science (MASc) / The electrochemical reduction of carbon dioxide can be used within electrolyzer devices to help mitigate greenhouse gas emissions to combat global warming. The process is when carbon dioxide is extracted from sources such as industrial plants and undergoes electrochemical reduction to be converted into 16 or more products that can be then sold within the market for profit. The common analysis methods currently used to analyze how much of each product is produced from an electrolyzer device does not reveal all the information needed to best design electrolyzer devices. This has led way to new analysis methods that are being explored that can find all the information needed for product analysis that leads to optimal electrolyzer design. This work investigated uses a special type of mass spectrometry that will allow for the full information to be found on the products from electrochemical carbon dioxide reduction leading to enhanced electrolyzer designs.

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.

The performance of a Hewlett Packard Particle Beam LC/MS interface is evaluated using EPA appendix VIII and IX compounds. The behavior of these priority pollutants in the interface could determine its feasibility as a future EPA certified technique. The evaluation process consists of studies to determine minimum detectable quantities (MDQ), linear response, and band broadening contributions. The MDQ's of the analytes in electron impact and chemical ionization modes are extrapolated from experimental signal to noise data. The linearity study involves ten compounds analyzed at five different concentrations. The response factors (RF) are calculated and discussed. The study concerning the band broadening contributions of the interface involves four independent variables (helium flow, desolvation chamber temperature, source temperature, and 96 methanol) and their effects on peak width. A Box–Behnken experimental design is used and described. Response surfaces are generated from the best fit equation describing the data. / Master of Science

The main objective of this research was to identify the compounds responsible for persistent odors in biosolids during long-term storage using olfactometry measurements and to determine their generation pattern with regard to time of appearance and decline using gas chromatography-mass spectrometry (GC-MS). Another objective of this study was to investigate the effect of various digestion and dewatering techniques on odors and determine if there is a correlation between the peak concentration and time of appearance of short-tem organic sulfur odors and persistent odors. Headspace analysis was used to quantify short-term odor-causing organic sulfur compounds and persistent odors from compounds such as indole, skatole, butyric acid and p-cresol for an incubation period up to 150 days. A unique odor generation pattern was observed for each of the compounds analyzed for all the dewatered cakes tested in this study. Dewatered cake samples were also analyzed to determine their detection threshold by a trained odor panel and the results were consistent with the general pattern of odor generation observed in this study. Positive correlations were observed between the peak concentration of organic sulfur and persistent odor compounds whereas little or no relationship was observed between their times of appearance. The type of sludge used in digestion (primary sludge, WAS and mix) was found to affect the production of odor-causing compounds significantly. Primary sludge produces the highest odors followed by mix. WAS was found to produce biosolids with a low odor concentration. Positive correlation was observed between odor concentration and digestion SRT. Significant reduction in odor concentration was observed when the SRT was increased from 12-days to 25-days. At 45-day SRT, further reduction in odors was not very significant. Moreover, the results from this study indicate that methanogens play an important role in the degradation of both organic sulfur and persistent odors. Although the highest odors during biosolids incubation came from sulfur compounds, the persistent odors must be managed as part of a comprehensive sludge management approach. / Master of Science

Non-targeted metabolite profiling by liquid chromatography-mass spectrometry (LC-MS) was used to determine the metabolite responses of Arabidopsis roots to auxin or ethylene. Crosstalk between these hormones regulates many important physiological processes in plants, including the initiation of lateral root formation and the response to gravity. These occur in part through alterations in the levels of flavonoids, specialized plant metabolites that have been shown to act as negative regulators of auxin transport. However, much remains to be learned about auxin and ethylene responses at the level of the metabolome. LC-MS analysis showed that a number of ions changed in response to both hormones in seedling roots. Although classes of specialized metabolites such as flavonols and glucosinolates change in abundance in response to both auxin and ethylene, there was little overlap with regard to the specific metabolites affected. These data will be integrated with information from transcriptomic and proteomic experiments to develop framework models that connect phytohormones and specialized metabolism with specific physiological processes. Previous studies by imaging techniques have shown that flavonols increase in response to both auxin and ethylene in the root elongation zone, but LC-MS showed that flavonols decreased in abundance in response to these hormones. Therefore a method was developed for targeted metabolite profiling of flavonols in individual root tips by flow injection electrospray mass spectrometry. This method uncovered spatial differences in metabolic profiles that were masked in analyses of whole roots or seedlings, and verified that flavonols increase in response to these hormones in root tips. / Master of Science

Combining cesium (Cs) bombardment with positive secondary molecular ion detection (MCs+) can extend the analysis capability of Secondary Ion Mass Spectrometry (SIMS) from the dilute limit (<1%) to matrix elements. The MCs+ technique has had great success in quantifying the sample composition of III-V semiconductors as well as dopants and/or impurities; however, it has been less effective at reducing the matrix effect for IV compounds, particularly Si-containing compounds, due to Cs overloading at the surface during the analysis from the Cs primary ion beam. The Cs overloading issue is attributable to the mobility and relocation of the implanted Cs to the surface; this effect happens almost instantaneously. Once the surface is overloaded with Cs, the excess Cs begins to reneutralize the ionization Cs and, as a result, the MCs+ technique is ineffective at reducing the matrix effect. This research provides new insights for improving the MCs+ technique and elucidating the Cs mobility. A combination of multiple experimental techniques and theoretical modeling was implemented to assess the Cs retention, up-take, and distribution differences between group III-V and IV materials. Early experiments revealed a temperature-dependent component of the Cs mobility, prompting an investigation of this phenomenon. Therefore, we designed, built, and installed a variable temperature stage for our SIMS with temperatures ranging from -150 to 300 C. This enabled us to study the temperature-dependent component of the Cs mobility and the effect it has on the secondary ion emission processes. Additionally, a method was devised to quantify the amount of neutralization and ionization due to the relocated Cs. The results allow for a more thorough understanding of the material dependence on the Cs+-sample interaction and the temperature component of the Cs mobility. / Ph. D.

<p dir="ltr">Lignocellulosic biomass is a significant sustainable carbon source that may facilitate the reduction of the dependency on fossil fuel and meeting of the ever-rising need for energy and chemicals. The main components in biomass include cellulose, hemicellulose, and lignin.Fast pyrolysis, rapid heating in the absence of oxygen, has been demonstrated to be a promising technique for converting biomass into valuable small chemicals, such as drop-in fuel.</p><p dir="ltr">Lignin is the most abundant aromatic biopolymer in nature. The proportional content of the phenylpropanoid monomeric units (4-hydroxyphenyl (H), guaiacyl (G), and syringyl (S)) in lignin is usually determined using a tedious and slow method (taking two days per sample) involving derivatization followed by reductive cleavage (DFRC) combined with GC/MS or NMR analysis. Therefore, a fast mass spectrometric method for the determination of the monomer content was developed. This method is based on fast pyrolysis of a lignin sample inside the ion source area of a linear quadrupole ion trap mass spectrometer. The evaporated pyrolysis products are promptly deprotonated via negative-ion mode atmospheric pressure chemical ionization ((-)APCI) and analyzed by the mass spectrometer to determine the monomer content. The results obtained for the wild-type and six genetic variants of poplar were consistent with those obtained by the DFRC method. However, the mass spectrometry method requires only a small amount of sample (50 μg) and the use of only small amounts of three benign chemicals, methanol, water, and ammonium hydroxide - as opposed to DFRC, which requires substantially larger amounts of sample (10 mg or more) and large amounts of several hazardous chemicals. Furthermore, the mass spectrometry method is substantially faster (three minutes per sample), more precise, and data interpretation is more straightforward, as only nine ions measured by the mass spectrometer are considered.</p><p dir="ltr">Understanding the various factors affecting the fast pyrolysis product distribution of biomass is critical for achieving a fast pyrolysis process with high energy- and carbon-efficiencies. The presence of inorganic salts (sodium, potassium, calcium, etc.) within the lignocellulosic feed is known to impact the product distribution. Cellulose, the most abundant biopolymer, has a simple linear structure consisting of glucose anhydride (dehydrated glucose) monomers connected through glycosidic bonds. By studying the primary and secondary products, as well as char yields, obtained upon fast pyrolysis of undoped cellobiose samples (a model compound of cellulose) and those doped with inorganic salts, mechanistic insights were obtained on the influence of inorganic compounds on the product distributions by using fast pyrolysis/atmospheric pressure chemical ionization mass spectrometry (py/APCI MS) and fast pyrolysis/gas chromatography with flame ionization and low-resolution mass spectrometry detection (py-GC/FID/MS).</p><p dir="ltr">Polyurethane, a synthetic polymer, has many applications in various fields due to its versatile properties. One such application is the use as insulating material in submarines. However, the varying conditions of temperature, humidity, and salinity pose a significant challenge in predicting the lifetime of polyurethane due to aging and degradation. The degradation products of polyurethane foams were investigated by using electrospray ionization/mass spectrometry (ESI/MS). Adipic acid was found to be a degradation indicator that is associated with changes in the chemical composition and mechanical condition of polyurethane foams. An ESI/MS method was developed for monitoring adipic acid content, providing a potential tool for predicting the lifetime and performance of polyurethane foams. Both naturally aged and artificially aged foam samples were studied. The results show that humidity facilitates the degradation process of polyurethane foams. This quantitative method will greatly facilitate predicting the lifetime and performance of polyurethane foams, especially from the point of view of on-board maintenance examinations in submarines.</p>

Yes / Background. The spirochete bacterium Treponema pallidum ssp. pallidum is the etiological agent of syphilis, a chronic multistage disease. Little is known about the global T. pallidum proteome, therefore mass spectrometry studies are needed to bring insights into pathogenicity and protein expression profiles during infection. Methodology/Principal Findings. To better understand the T. pallidum proteome profile during infection, we studied T. pallidum ssp. pallidum DAL-1 strain bacteria isolated from rabbits using complementary mass spectrometry techniques, including multidimensional peptide separation and protein identification via matrix-assisted laser desorption ionization-time of flight (MALDI-TOF/TOF) and electrospray ionization (ESI-LTQ-Orbitrap) tandem mass spectrometry. A total of 6033 peptides were detected, corresponding to 557 unique T. pallidum proteins at a high level of confidence, representing 54% of the predicted proteome. A previous gel-based T. pallidum MS proteome study detected 58 of these proteins. One hundred fourteen of the detected proteins were previously annotated as hypothetical or uncharacterized proteins; this is the first account of 106 of these proteins at the protein level. Detected proteins were characterized according to their predicted biological function and localization; half were allocated into a wide range of functional categories. Proteins annotated as potential membrane proteins and proteins with unclear functional annotations were subjected to an additional bioinformatics pipeline analysis to facilitate further characterization. A total of 116 potential membrane proteins were identified, of which 16 have evidence supporting outer membrane localization. We found 8/12 proteins related to the paralogous tpr gene family: TprB, TprC/D, TprE, TprG, TprH, TprI and TprJ. Protein abundance was semi-quantified using label-free spectral counting methods. A low correlation (r = 0.26) was found between previous microarray signal data and protein abundance. Conclusions. This is the most comprehensive description of the global T. pallidum proteome to date. These data provide valuable insights into in vivo T. pallidum protein expression, paving the way for improved understanding of the pathogenicity of this enigmatic organism. / This work was supported by the grants from the Flanders Research Foundation, SOFI-B Grant to CRK, http://www.fwo.be/, a Public Health Service Grant from the National Institutes of Health to CEC, (grant # AI-051334), https://www.nih.gov/ and a grant from the Grant Agency of the Czech Republic to DS and MS (P302/12/0574, GP14-29596P), https:// gacr.cz/.