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Analysis of phospholipids by comprehensive gas chromatography
Šilhavecká, Simona ; Čabala, Radomír (advisor) ; Jelínek, Ivan (referee)
Phospholipids are an important group of polar lipids constituting the main component of cell membranes. Their proportion may vary depending on many factors of the surrounding environment in which the cell is located. Determination of membrane phospholipids is essential in many scientific, industrial and economic sectors. The aim of this work has been to develop a separation method for determination of membrane phospholipids by which it was possible to analyze phosphorylated parts of phospholipids and fatty acids from one sample. Comprehensive gas chromatography with mass detection (GC×GC-MS) was chosen for the assay. This method allows the separation of the entire sample on two serially connected different columns, among which is the interface called modulator. The preparation of the sample includes a cleavage of the phospholipid molecule by the enzyme phospholipase C, which released the phosphorylated polar headgroups. These polar parts had to be derivatized before analysis. The principle of the chosen derivatization consisted in the use of two different silylation agents (hexamethyldisilazane and N,O-Bis(trimethylsilyl) trifluoracetamide) in two steps. Conditions were selected for efficient separation of the silylderivatives of phosphorylated headgroups using GC×GC-MS using a cryogenic...
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Preparation of New Polymeric Fibers for Headspace GC Analyzes
Blechová, Karolína ; Čabala, Radomír (advisor) ; Štícha, Martin (referee)
The topic of this thesis was the preparation of fibers with new polymeric cyclophosphazene phase for a headspace microextraction on a solid phase followed by a separation on a gas chromatograph. The surface of platinum wires was covered by a layer of poly (cyclo) phosphazene- benzoquinone using electrochemical polymerization. The characteristic of the polymer layer was subsequently verified by the infrared spectroscopy. The new polymeric material was used as a stationary phase a for solid-phase headspace microextraction. Two different model solutions, a mixture of hydrocarbons in pentane and alcohols in water, were selected to test the selectivity of the film. It has been found that the films are extracting better hydrocarbons than alcohols. The extraction of the alcohol mixture sample was optimized by the experimental design, which makes it possible to determine the factors and their combinations, which have the greatest influence on the investigated response. A Minitab 16 program evaluated the final optima for individual alcohols as well as for the sum of the absolute areas of the whole mixture. A separation of model analytes was performed using a gas chromatograph with flame ionization and mass detector. Keywords electrochemical polymerization, poly (cyclo) phosphazene-benzoquinone film, solid...
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Enantioselective separation of certain analytes using supercritical fluid chromatography and high performance liquid chromatography
Martínková, Monika ; Tesařová, Eva (advisor) ; Čabala, Radomír (referee)
(EN) Cellulose tris-(3,5-dimethylphenylcarbamate) chiral stationary phase was used for separation of selected 24 analytes. Enantioseparations were realized using two systems, high performance liquid chromatography and supercritical fluid chromatography. Effect of mobile phase composition was studied. Five different aditives (isopropylamine, diethylamine, triethylamine, trifluoroacetic acid, isopropylamine combined with trifluoroacetic acid) and their influence on enantioseparation were tested. Influence of two different modifiers (methanol, propan-2-ol) combined with all aditives was also tested in supercritical fluid chromatography system. The aim of this work was to find optimized composition of mobile phase which was suitable for separation of the analytes studied and to compare separation potential among all mobile phases and also between used separations systems. The supercritical fluid chromatography was shown to yield better results, i.e. better resolution in shorter analysis time. However examples of analytes better resolved under optimized conditions in high performance liquid chromatography system have also been found. Keywords (EN) Chirality, enantiomers, enantioselective separation, chiral stationary phase, high performance liquid chromatography, supercritical fluid chromatography.
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Application of solvation mocel to retention description of selected compounds in liquid and gas chromatography
Jirkal, Štěpán ; Čabala, Radomír (advisor) ; Feltl, Ladislav (referee) ; Sýkora, David (referee)
(EN) The solvation model based on LSER was applied to study the retention behaviour of analytes in liquid and gas chromatography. In a first chapter, a retention description of 21 solutes was investigated by using the solvation model in a wide range of mobile phase composition methanol-water and acetonitrile-water. Generally, the retention of aromatic compounds was better described by the solvation model, compared to aliphatic compounds. Effect of the particular analytes used to formulate the LSER model on ability of retention description was studied. Different results of a retention estimation was achieved by using the regression set of compounds including aromatic solutes only or by contrast aliphatic solutes only. The solvation model developed on the basis of oxygen derivatives provided distinct results in comparison to model formulated with nitrogen derivatives only. The second chapter of this work, focused on gas chromatography, dealt with a description of retention of 152 isomers C5-C8 alkenes by the LSER model. The solvation descriptor L was obtained by using two estimation methods Havelec-Ševčík (HS) and Platts-Butina (PB), the descriptor E was calculated according to its definition. Two models for retention description of alkenes were constructed, the HS model and the PB model, derived...
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Study of peptide digestion kinetics by trypsin and chiral separations of biologically active compounds by HPLC
Šlechtová, Tereza ; Tesařová, Eva (advisor) ; Čabala, Radomír (referee) ; Mikšík, Ivan (referee)
This dissertation thesis composes of two parts; the first part focus on the characterization of trypsin, enzyme frequently used in proteomic research for the investigation and identification of protein sequences, and its peptide digestion kinetics. The second part is aimed to the enantioseparations of biologically active compounds. First part of this project focus on tryptic digestion of synthetic peptides and the development of HPLC method for the identification of synthetic peptides and their fragments. Using the in-solution digestion and HPLC method, relative kinetic constants were determined for problematic sequences. Amino acids responsible for the decrease in trypsin catalytic activity and their location towards the cleavage site were studied. Certain slight exopeptidase activity of trypsin was noted, especially at the end of peptide chain. Furthermore, three columns with immobilized trypsin used in HPLC were compared concerning their catalytic activity. The immobilization of enzymes on solid support is used to elevate the amount of enzyme present during digestion and to assure better repeatability and reproducibility of obtained results. Activity of a new trypsin column synthesized at the University of North Carolina at Chapel Hill was compared to two commercially available trypsin columns....
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Analysis of catalytic oxidation product mixture using gas chromatography method and controlled evaporation mixer
Haupt, Josef Michael ; Schwarzová, Karolina (advisor) ; Čabala, Radomír (referee)
The goal of this master's thesis was to develop a method for gas chromatography using the Controlled Evaporation Mixer (CEM) for gas mixture sampling. Gas mixture contained chemical substances formed during manufacturing of acrylic acid by two-step oxidation of propylene. The analyzed mixture composed of permanent gases, light hydrocarbons and compounds containing oxygen. During the method development several capillary columns with polar and nonpolar stationary phase were used. After selection of the most suitable column (SPB-1 Sulfur, Supelco, USA) the method was further optimized for the maximal possible separation efficiency. However, it was impossible to separate propane and propene on SPB-1 Sulfur column. These gases were possible to determine on TCD detector and separate on respective columns prior to the detector. For that purpose, the optimization of multiple-port valves switching inside the gas chromatograph was performed. The next step was calibration of gas chromatograph for a purpose of quantitative analysis of selected substances. The calibration solution containing solvent and an analyte was evaporated and diluted with nitrogen by CEM to obtain desired analyte concentration. Formaldehyde and propane had a very low response on detectors, acetaldehyde was undetectable in desired...
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Determination of niacin and its metabolites by HPLC-MS
Pultarová, Kamila ; Čabala, Radomír (advisor) ; Staňková, Barbora (referee)
Nicotinic acid (NA) is a hypolipidemic agent with pleiotropic effects on plasma lipoproteins. Its medicamentous form with delayed secretion leads to pyrimidine metabolites, which make increased risk of hepatotoxicity and should be monitored. The aim of the presented study was to introduce HPLC-MS method for the determination of NA, nicotinamide (NAM), nicotinuric acid (NUA), 1-methyl-nicotiamide (MNA), nicotinamid-N-oxide (NNO), 1-methyl-2-pyridon-5-carboxamide (2-Pyr) and 1-methyl- 4-pyridon-5-carboxamide (4-Pyr) in blood plasma useful for the monitoring of hypolipidemic therapy. We have compared calibration dependences of individual metabolites using two columns - Hypercarb (grapfite carbon) and Hypersil Silica (silicagel). Both columns revealed linear calibration dependences for all mentioned analytes except MNA in the concentration range 10-2000 ng/ml for chemical standards; calibration dependence in human blood plasma measured with the column Hypersil Silica was linear in the range 20-4000 ng/ml for all tested analytes. Correlation coefficient varied between the value 0,9400 and 0,9997. Analysis time was 15 min with the column Hypercarb and 27 min with Hypersil Silica. Biological samples were extracted using solid phase with sulphonyl group. Reproducibility of the results of biological samples...
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Analysis of reaction products of phthalaldehyde with selected amino acids
Křížová, Věra ; Čabala, Radomír (advisor) ; Cvačka, Josef (referee)
This thesis focuses on analysis of reaction products of phthalaldehyde with selected amino acids using the combination of high-performance liquid chromatography with mass spectrometry. Only one product with dihydroisoindole structure is formed in the case of simple amino acids (glycine, glycine ethyl ester, alanine, α-aminobutyric acid, valine, leucine and isoleucine). Reactions of phthalaldehyde with amino acids with two amino groups (lysine, asparagine, glutamine and arginine) yield different types of compounds. Main products are formed by the interaction of both of the amino groups with one molecule of phthalaldehyde. Apart from this type of product, these reactions also result in formation of the expected analogous structures of dihydroisoindoles. Moreover, the formation of products containing one molecule of amino acid and two molecules of phthalaldehyde is not excluded. The products were structurally analysed by the use of high-performance liquid chromatography and tandem mass spectrometry. Reaction products of phthalaldehyde with α-aminobutyric acid, valine, leucine, isoleucine and lysine were isolated and further analyses such as electron ionisation with double focusing sector analyser and nuclear magnetic resonance were performed. Pieces of information resulting from the analysis of...
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Analysis of products of cardanol metathesis by HPLC-MS/MS method
Flenerová, Zuzana ; Čabala, Radomír (advisor) ; Vrkoslav, Vladimír (referee)
Cardanols are more and more used in the industry of polymers, resins, coatings, friction materials, etc. At right conditions, these lipid-like chemicals can undergo metathesis reaction. The main aim of the diploma thesis is an analysis of products of cardanol metathesis by HPLC-MS/MS. Standard 3,3'-hexadec-8-en-1,16-diyldiphenol, one of the main products of cardanol metathesis, was prepared for quantitative HPLC analysis. The standard was prepared in two steps. The first step was cardanol ethenolysis to give 3-(non-8-enyl)phenol. The second step was self-metathesis of 3-(non-8- enyl)phenol to give 3,3'-hexadec-8-en-1,16-diyldiphenol. The standard was characterised by HPLC/MS, MS/MS, EI-MS, UV/VIS, IR, Raman a NMR spectroscopy. The second part of the thesis is dedicated to the study of a mechanism of double-bond localization method in long chain hydrocarbons by acetonitrile APCI-MS. The formation of adducts [M + 55]+ , which are the key substances of this method, was studied using simple alkene models by MS and MS/MS. At the end, the structure and the mechanism of the formation of adducts [M + 55]+ were suggested. Key words: Cardanol, olefin metathesis, HPLC-MS, double-bond localization, APCI, acetonitrile
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