The minimal refining method described in this study made it possible to neutralize crude canola oil using some weaker alkali such as Ca(OH)2, MgO and Na2siO3 as an alternative for NaOH. After citric acid degumming, more than 98% of phosphorous content was removed from crude oil. The free fatty acid content after minimal neutralization with calcium hydroxide decreased from 0.50 to 0.03%. Other quality parameters such as peroxide value, anisidine value and chlorophyll content were within commercially acceptable levels. The use of Trisyl silica and Magnesol R60 made it possible to remove the hot water washing step and to decrease the amount of remaining soap to less than 10 ppm. There was no significant change in chemical characteristics of canola oil after wet and dry bleaching. During traditional neutralization, total tocopherol loss was 19.6% while minimal refining with Ca(OH)2, MgO and Na2siO3 resulted in 7.0, 2.6 and 0.9 % reduction in total tocopherols. Traditional refining removed 23.6% of total free sterols, although after minimal refining free sterols content did not change significantly (p<0.05). Both traditional and minimal refining resulted in almost complete removal of polyphenols from canola oil. Total phytosterols and tocopherols in two cold press canola oils were 7700, 8400 mg/kg and 370, 350 mg/kg, respectively. Total phytosterols and tocopherols contents in solvent extracted canola oil were 9500, 500 mg/kg, respectively. The minimal refining method described in this study was a new practical approach to remove undesirable components from crude canola oil confirmed with commercial refining standards as well as preserving more healthy minor components. / This research project is supported by National Services and Engineering Research Council of Canada (NSERC).

No description available.

Calpains are calcium-dependent, intracellular, multi-domain cysteine proteases involved in many physiological functions regulated by calcium signaling, including cell motility. How calpains are activated in the cell is still unknown because the resting intracellular concentration of Ca2+ is orders of magnitude lower than that needed for half-maximal activation of the enzyme in vitro. Several stratagems by which calpains might overcome this Ca2+ concentration differential have been proposed. It is possible that post-translational modifications like phosphorylation, or accessory proteins that bind to calpain, might facilitate the enzyme’s activation at lower than optimal Ca2+ concentrations. Autoproteolysis (autolysis) and subunit dissociation are two other proposed activation mechanisms that could release constraints on the calpain core by breaking the link between the anchor helix and the small subunit to allow the active site to form. By measuring the rate of autolysis at different sites in calpain, it was demonstrated that while the anchor helix is one of the first targets to be cut, several other potentially inactivating autolysis sites, particularly in Domain III, can also be cleaved within the first minute. Thus autolytic activation would go hand in hand with inactivation. By fractionating and identifying calpain 2 autolysis fragments, I show that the small subunit does not dissociate away from the large subunit, but is proteolyzed to a 40-45 k heterodimer of the penta-EF-hand Domains IV and VI. It is likely that this autolysis-generated heterodimer has previously been misidentified as the small subunit domain VI homodimer that would be produced by subunit dissociation. A calpastatin affinity column was constructed and used to capture recombinant calpain 2 from bacterial cell lysate. This affinity column provides a tool to screen for and capture calpain complexed to potential binding partners in the presence of Ca2+. Here I propose a model for calpain 2 activation in vitro that does not involve autolysis, subunit dissociation, or calpain activators. / Thesis (Master, Biochemistry) -- Queen's University, 2010-10-25 16:03:52.364

With the rise of capillary HPLC systems, open tubular liquid chromatography (OTLC) has been garnering more attention due to the possible fundamental advantages of open tubular systems over conventional packed or monolithic systems. Performance has yet to reach its potential due in part to a variety of technical challenges, resulting in the need for very small injection volumes and sensitive detection. In this work, we have shown that with modern HPLC sample introduction and detection systems, along with careful fabrication of polymer stationary phases, that reverse phase open tubular liquid chromatography may be within reach. We have shown that, with small diameter (i.d. 30m) open tubular columns, complex multi-component mixtures (EPA 610, in-house drug mixture) can be separated. We have also shown that these columns are robust and can function over a wide range of flow rates (200-1000 nl/min), and may be useful for general reverse phase separation in the future. However, currently, more stationary phase development and procedure refinement is needed. Microstructured fibres (MSFs), a relatively new class of optical fibre which confine light within fibres through a refractive index change caused by the use of parallel air channels running throughout the length of the fibre, are explored as a new support material for open tubular liquid chromatography. The fine channel structures of MSFs enable reasonable sample volumes to be used compared to conventional open tubular systems, while offering a similar plug-like flow profile through the fibre. With current sample introduction and flow technologies, we have shown that the potential advantages of MSF columns is great even when simple C18 stationary phases are used; this was able to separate a four PAH mixture. However, a distribution in channel sizes caused by current manufacturing standards and a limited ability to evenly deposit polymer stationary phases in the fibres has kept MSF columns from reaching their full potential. / Thesis (Master, Chemistry) -- Queen's University, 2011-09-14 11:52:41.23

Metaproteomics, as a proteomic approach to analyse environmental samples, is a new and expanding field of research. The field promises new ways of determining the status of the organisms present in a sample, and could provide additional information compared to metagenomics. Being a novel field of research, robust methods and protocols have not yet been established. In this thesis, we examine several methods for a reliable extraction of protein from soil and periphyton samples. The extraction should preferably be fast, compatible with downstream analysis by mass spectrometry and extract proteins in proportion to their presence in the original sample. A variety of methods and buffers were used to extract proteins from soil and periphyton samples. Concentration determinations showed that all of these methods extracted enough protein for further analysis. For purification and digestion of the samples, several methods were used. The purified samples were analysed on three different mass spectrometers, with the Orbitrap Velos Pro delivering the best results. The results were matched against four genomic and metagenomic databases for identification of proteins, of which the UniProt/SwissProt database gave the best result. A maximum of 52 proteins were identified from periphyton samples when searching against UniProt/SwissProt with strict settings, of which the majority were highly conserved proteins. The main limitation for this type of work is currently the lack of proper metagenomic databases.

Selenium has been shown to have multiple biochemical effects ranging from nutrient deficiency at low levels to toxicity at high levels. This duality of concern has led to a demand for increased numbers of highly accurate and precise determinations of selenium in biological materials. A convenient procedure was developed for determining selenoamino acids by HPLC-THG-AAS, based on the derivatization of these analytes with Sanger's reagent. Selenomethionine, selenocystine and selenocysteine (after blocking the free selenol group with phenylmercurio cation) were converted to their N-2,4-dinitrophenyl derivatives, and separated on a Nucleosil 5-NO$ sb2$ column with methanolic mobile phase containing acetic acid and triethylamine. Furthermore, an improved HPLC-AAS interface design was modified and optimized for the detection of selenium in HPLC column eluate. The new design was (i) compatible with aqueous mobile phases containing volatile buffers and (ii) provided equivalent molar response to analytes containing Se($-$II), Se(+IV) and Se(+VI). A method for simultaneously determination of selenate, selenite, selenocystine, selenomethionine and selenoethionine was developed by using the HPLC-AAS system with aqueous acetic acid containing ammonium acetate as eluate solution on the cyanopropyl column. The equivalent low ng limits of detection (1-2 ng as Se) for different oxidation states of selenium analytes were obtained using several different mobile phases and/or columns. A phenol extraction procedure for selenate, selenite, selenocystine, selenomethionine and selenoethionine was evaluated for the determination of these selenium analytes in natural waters and wheat samples. The current HPLC-AAS system provides an inexpensive alternative to conventional techniques for the determination of selenium analytes in environmental samples.

Standards of Amadori rearrangement products (ARP) were synthesized for the purpose of developing analytical techniques and performing mechanistic studies related to their thermal decomposition. Several synthetic strategies were explored. An HPLC analytical system with a diode array detector was coupled to a fluorometer and an electrochemical detector, in order to detect simultaneously and on-line, a wide variety of degradation products of ARPs and to follow their kinetics. The potential of such a system to analyze complex Maillard mixtures was demonstrated. The kinetics of the reaction of glucose with morpholine (a Strecker inactive analogue of proline was used in order to simplify the kinetics) to produce Amadori morpholine was studied under experimental conditions that minimize side reactions and maximize Amadori product formation. At specific time intervals, the samples were analyzed for the presence of reactants and Amadori product by the multidetector HPLC system. Color and fluorescence were also measured. The data obtained were used to calculate the rate constants for the formation and degradation of Amadori product. A mechanistic model that statistically fitted the kinetic data was proposed. To further understand the details of the decomposition mechanism of Amadori proline, different mass spectrometric experiment were performed. High resolution, linked-field scan and neutral loss experiments have indicated that 1-((2$ sp prime$-carboxyl)pyrrolidinyl)-1-deoxy- scD-fructose (Proline Amadori product) followed two main pathways of fragmentation under electron impact conditions; one initiated by the ring oxygen and the other by the amino acid nitrogen, producing two well stabilized fragment ions; oxonium and imminium ions. In addition, ortho-elimination reactions initiated by O or N-centered radical sites were shown to produce the most intense peaks and diagnostically important ions for the identification of Amadori products. However this approach can only pro

The need for selective and inexpensive detectors in liquid chromatography is of considerable interest in the determination of sulfur compounds. Of the available-selective sulfur methodologies, flame photometric detector coupled to gas chromatography is the most widely used. It has proven to be a sensitive and selective method for detection of heat stable and volatile sulfur compounds. Fundamentally, this technique is not applicable to high boiling and/or thermolabile sulfur compounds. More recently, hyphenated flame photometric detector has been utilized, with limited success, to monitor sulfur species in liquid chromatography. However, existing HPLC-FPD methodologies have never been applied to real samples, due to the low population of S 2, the emitting species, and the quenching effects of the other species present in the flame. / In this work, two total consumption high-performance liquid chromatography flame photometric (HPLC-FPD) interfaces compatible with either methanolic or aqueous mobile phases are described and optimized for monitoring low volatile and thermally fragile sulfur compounds in biological samples. Each interface was fuelled either by methanol or by hydrogen. The all quartz interfaces enclosed four consecutive thermal processes: (a) thermovaporization of the HPLC effluent; (b) pyrolysis of the organic matrix (including sulfur species) in a kinetic H2/O2 flame; (c) conversion of the oxidized sulfur compounds to H2S in a reducing post-combustion stage fuelled by hydrogen; and (d) transport of the generated hydrides towards a hydrogen radical rich surrounding of an inverted hydrogen-oxygen diffusion flame. Chemiluminescence induced in the last step was integrated as a narrow beam in a light-guide positioned remotely from the analytical cool flame and oriented towards a photomultiplier unit. Radioisotopic assays demonstrated that sulfur (as H235SO4) was transferred quantitatively to the analytical flame. Indirect evidence suggested that sulfur was hydrogenated in the post-combustion step via a thermochemical hydride generation process to mediate the formation of S2. The linearity of calibration graphs (0.9950 < r < 0.9986), where r is the correlation coefficient) and unprecedented HPLC-FPD limits of detection for sulfur compounds (1.5 etag/s for 2-methylthiophene, 2.25 etag/s for carbon disulfide, and 4.5 etag/s for ethanesulfonic acid) allowed for the speciation of sulfur species in garlic extracts. Alternatively, modification of the methanol fuelled interface to a hydrogen fuelled reactor allowed detection of thiosulfinates and high molecular weight sulfur compounds in horse kidney and garlic extracts, respectively.

The design and construction of an electrochemical detector with two working electrodes located on the opposite walls of a thin-layer cell and its use as a detector for High Pressure Liquid Chromatography (HPLC) in the analysis of catecholamines in human cerebrospinal fluid are described. The location of the electrodes in this manner permits an electrochemically reversible or quasireversible couple to be electrolized more than once as it passes through the detector. If one electrode is held at a potential where oxidation takes place and the second electrode at a potential where reduction of this oxidized form back to the starting material occurs, then the current produced increases proportionately to the number of conversions that take place. A comparison of this cell in the dual-working-electrode and single-working-electrode mode shows an improvement in the signal-to-noise ratio by a factor of six. This HPLC system with electrochemical detection has been used for the first time to detect norephinephrine (141 pg/mL) and dopamine (262 pg/mL) in human cerebrospinal fluid.

Detecting the presence of insects at low densities can avoid total deterioration of stored grains because corrective actions can be implemented early. Tribolium castaneum (Herbst) and Cryptolestes ferrugineus (Stephens) are the major insect pests of the Canadian grain handling industry. Identification of the volatile organic compounds released by insects can be used to detect insects in stored grains. An attempt was made to identify the volatile organic compounds released by T. castaneum and C. ferrugineus by headspace analysis. The volatiles in the head space of vials with insects, insects and wheat flour, and insects and wheat, were analyzed using a GC-MS coupled with an automatic headspace sampler. Wheat with fifteen percent moisture content was used in this study along with two different insect densities. Feasibility of the automatic headspace sampler in headspace analysis was found to be positive. The sampler can do sample conditioning, absorption, trap cleaning and desorption of the volatiles into the GC-MS and speed up the process. The samples extracted at 20 strokes with 1000 µL per stroke, and desorbed at 250°C gave a clear peak of compounds. The amount of volatiles produced by T. castaneum adults varied based on insect densities, the concentration of Methyl-1, 4-benzoquinone; Ethyl-1, 4-benzoquinone; and 1-Tridecene released by ten adult insects were 355, 390 and 530 µg/L compared to 300,310 and 210 µg/L of Methyl-1, 4-benzoquinone; Ethyl-1, 4-benzoquinone; and 1-Tridecene produced by five adult insects. Extreme high and low temperature leading to death produced very high amounts of volatiles compared to insects kept at 35°C. The larvae of the T. castaneum insects did not produce any volatiles at ambient condition as well as at extreme cold and warm conditions. The C. ferrugineus adults did not produced any detectable amount of volatiles even at the higher insect density after up to 3 days. The results of the combination of T. castaneum and C. ferrugineus insects gave the same volatile organic compounds as produced by T. castaneum insects alone. The 1-Tridecene produced by T. castaneum was not reported previously in other studies.