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Applications of monolithic columns in capillary liquid chromatography
Holdšvendová, Petra ; Coufal, Pavel (advisor) ; Pacáková, Věra (referee) ; Cvačka, Josef (referee)
INTRODUCTION From the nineties of the twent5r's century [l], when appeared the first references about the new type ofthe stationary phases designated for HPLC called monoliths, these became to be the object of interest of several scientific groups [2-4]. Their uncontested advantage in comparison with classical columns is their high stability in pH range from 2 to 12 where there is no possibility to use the classical columns. other advantages are their easy and cheap preparation and the absence of frits on the ends of the separating medium because the monolith is covalently bound to the inner side of the capillary [5]. On the other hand, their disadvantage remains to be worse in reproducibility of the stationary phase preparation than in case of classical columns. This PhD Thesis is focused on monoliths based on organic polymers, mainly the methacrylate monoliths. For the first time, this ýpe of monoliths was shown by Peters et al. [6] and since then there have been made several monolithic phases based on different separating features 17_101. Methacrylate monoliths are convenient for separation of the low molecular substances L7l as well as the high molecular substances like the proteins, oligonucleotides or saccharides n0-121.
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Refractometric study of very low density lipoprotein (LDL)
Šinglárová, Simona ; Pacáková, Věra (advisor) ; Staňková, Barbora (referee)
Knowledge of the chemical composition of lipoproteins has a broad meaning. In determining lipoprotein particle size fractions is necessary to preliminary separation by ultracentrifugation in density solutions. Usually the determination is performed by electrophoretic or gel filtration with a suitable detection, which distorts the results. A better solution offers a new detector, called Light Scattering Detector, which detects the bend of laser beam on molecules or particles in solution. To calculate the required particle size dependence on the concentration of the refractive index dn / dc. Concentration is determined gravimetrically and refractive indices by refractometric method. Dependence suits polynomial function. If it correlates with the chemical composition of VLDL lipoprotein can be determined by studying a larger number of samples.
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Use of the modern separation techniques for the analysis of insect pheromones
Žáček, Petr ; Jelínek, Ivan (advisor) ; Pacáková, Věra (referee) ; Cvačka, Josef (referee)
(EN) Communication mediated by chemical compounds (semiochemicals) is the most important way of information transfer in insects, especially in the social species. Gas chromatographic methods (one-dimensional, two-dimensional comprehensive, and preparative arrangement) coupled with mass spectrometric and/or electroantennographic detector were used for volatile or semivolatile semiochemicals analysis in various insect species. In this Thesis, biosynthesis of the bumblebee male sex pheromone in species Bombus terrestris, B. lucorum, and B. lapidarius was studied using putative biosynthetic precursors (sodium acetate, fatty acids) labeled with 2 H and 14 C. For the purpose of labeled volatile metabolites analysis, a method of separation and detection of isotopically labeled compounds in two-dimensional comprehensive gas chromatography was studied (2 H, 13 C). An "inverse isotope effects" was confirmed for compounds labeled with both isotopes in all examined types of columns. Concerning the biological samples, analysis of in vitro incubated tissues with labeled sodium acetate showed that pheromone components are synthesized de novo in the labial gland in B. terrestris and B. lucorum. Nevertheless, experiments of in vivo incubation of deuterated fatty acids confirmed biotransformation of the precursors...
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Determination of proteinogenic amino acids by high-performance separation techniques
Hodek, Ondřej ; Křížek, Tomáš (advisor) ; Pacáková, Věra (referee) ; Tůma, Petr (referee)
(EN) Proteinogenic amino acids are key components of living organisms. Thus, the latest metabolomics research has focused on developing fast and sensitive methods for the determination of amino acids. In this context, this thesis contains two studies describing development of high-performance separation techniques for the quantification of amino acids. In the first study, a capillary electrophoresis method was developed for the determination of free amino acids in tobacco plants, particularly focusing on optimizing the extraction of amino acids from solid plant materials. The extraction procedure was optimized using design of experiments (DoE) to obtain the highest possible extraction yield of amino acids. Factors such as volume and concentration of the extraction solvent (hydrochloric acid) were assessed as the most significant. Subsequently, the optimal values of these factors were determined using response surface methodology (RSM). Lastly, proteinogenic amino acids were quantified using capillary electrophoresis with contactless conductivity detection and calibration with internal standard, which improved the precision of the method. The second study aimed at developing a supercritical fluid chromatography method for the determination of free proteinogenic amino acids in human plasma. The most...
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