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Development of electrophoretic method for determination of sulphate in blood plasma
Hnízdilová, Lucie ; Křížek, Tomáš (advisor) ; Hraníček, Jakub (referee)
Cílem této bakalářské práce bylo vyvinout metodu pro stanovení síranů v krevní plazmě pomocí kapilární zónové elektroforézy. Byla použita křemenná kapilára o vnitřním průměru 50 μm a efektivní délce 41,5 cm. Plazmatické proteiny byly sráženy acetonitrilem. Jako optimální základní elektrolyt byla určena 0,4M kyselina mravenčí s 15mM chromanem draselným. Separační napětí bylo 25 kV. Vzorek byl dávkován napětím -5 kV po dobu 40 s. K vyhodnocení byla využita nepřímá UV detekce při 260 nm. Vzhledem ke složité matrici je vhodné provést kvantifikaci metodou standardního přídavku. Po vztažení ploch píků na vnitřní standard (500μM chlorečnany) byla relativní směrodatná odchylka 5,97 %. Výhodou této metody oproti dříve používaným srážecím metodám je krátká doba analýzy a jednodušší úprava vzorku. Klíčová slova kapilární elektroforéza, anorganické ionty, sírany, krevní plazma
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Changes in the background electrolyte caused by electrolysis in capillary zone electrophoresis
Novotný, Tomáš ; Gaš, Bohuslav (advisor) ; Křížek, Tomáš (referee)
4 Abstract Electrode reactions are one of the inevitable side effects which occur during standard separation measurement on capillary zone electrophoresis. Electrolytic products arising from the electrolysis of water, which is the main procces occuring at the electrodes in the majority of cases, can cause changes in background electrolyte located in vials and inside the separation capillary. The precision and reproducibility of the separation experiments is decreased due to the degradation of background electrolyte in capillary zone electrophoresis. Changes in the background electrolyte are highly dependent on the composition of the buffer itself: some electrolytes are changed more than the others within the same configuration of the experiment. A correct choice of the buffer with the right manipulation of the background electrolyte leads to higher precision and reproducibility of the separation measurements. Keywords: Capillary zone electrophoresis, electrolysis, background electrolyte, contactless conductivity detection
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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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Utilization of statistical approach in development of HPLC methods
Vymyslický, Filip ; Křížek, Tomáš (advisor) ; Kozlík, Petr (referee)
The aim of this diploma thesis was to develop a systematic procedure for the development of HPLC methods using the design of experiments. The system was developed based on the development of three HPLC methods for the determination of the purity of active substances using the design of experiments approach. The HPLC method for determining the purity of esomeprazole was selected to develop the systematic process for the development of HPLC methods by statistical approach. Experimental space was explored to find more suitable separation conditions. The second method used to develop the systematic procedure was the method for determining the purity of bisoprolol. This method was converted to a column of smaller size and the composition of the aqueous part of mobile phase was modified compared with the original pharmacopoeial method. Experimental space was explored to find more suitable separation conditions using the design of experiments. Last the method for determining the purity of risperidone was chosen. The composition of the aqueous part of mobile phase was changed in contrast to the original pharmacopoeial method. Experimental space was explored to find more suitable separation conditions using the design of experiments. For all studied HPLC methods, the values of the monitored chromatographic...
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Determination of low-molecular-mass heparin using affinity capillary electrophoresis
Molnárová, Katarína ; Křížek, Tomáš (advisor) ; Sobotníková, Jana (referee)
Unfractionated heparin, which is a widely used anticoagulant, is frequently replaced with low-molecular-mass species. They are used due to their more predictable anticoagulant effect with less bleeding complications and also they have prolonged anticoagulant effect. For monitoring of low-molecular-mass heparin levels, anti-factor Xa assay is used, which has some significant drawbacks. This work is dedicated to determination of low-molecular-mass heparin, namely Fraxiparine, using affinity capillary electrophoresis. Heparin is a polysaccharide which does not exhibit a significant UV absorption; therefore, its indirect detection method was used. Fraxiparine forms a stable complex with protamine. Protamine is an arginine-rich, positively charged peptide which is used to suppress heparin anticoagulant effect. Because protamine has a complex, not precisely defined structure, it was replaced by well-defined tetraarginine. The method uses phosphoric acid of 9 mmol L-1 concentration with addition of 0.1% (w/v) hydroxyethylcellulose as the background electrolyte. The samples are injected hydrodynamically into the capillary by a pressure of 5 kPa. First, the zone of Fraxiparine was injected, followed by the zone of tetraarginine (5 s). After that, 30 kV voltage was applied for 30 s. During this time the...
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Separation of liquid crystal forming substances using non-aqueous capillary electrokinetic chromatography
Čokrtová, Kateřina ; Křížek, Tomáš (advisor) ; Kubíčková, Anna (referee)
Liquid crystals are widely used in electronics, medicine and other fields. Analytical separations are important in the development of new liquid crystals to control the purity of synthesized substances. The sample analysis is important for detection of impurities formed during synthesis and for determination of chiral purity of the substance. Liquid crystal-forming substances cannot be separated by capillary zone electrophoresis due to the absence of readily ionizable groups. Electrokinetic chromatography is a method in which a suitable surfactant is added to the background electrolyte. The uncharged substances then interact with the electrically charged surfactant to obtain an effective charge. Separation can occur if they interact differently with the added surfactant. Another problem complicating the analysis is the very low solubility of analytes in water. Separations in this work were therefore carried out under non-aqueous conditions in acetonitrile. However, under these conditions a poor repeatability of the migration times of the substances was observed. Therefore, capillaries with differently coated inner walls were used in subsequent measurements. Surface modification should improve the repeatability of migration times. Several types of capillary coating have been tested. Dynamic coating...
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Effect of separation electrolyte ions on baseline stability in capillary electrophoresis
Kolečkářová, Tereza ; Křížek, Tomáš (advisor) ; Kubíčková, Anna (referee)
Unstable electropherogram baseline of potassium buffer, found while studying electrophoretic mobilities of electroosmotic flow markers in different separation buffers [1], makes it harder for higher concentrations of buffers to evaluate the results. This work investigates, whether the above described phenomenon is confined to potassium ions, and if it is not, what conditions influence it. The measure of instability of signal baseline is taken as its standard deviation and it is compared for potassium, sodium, ammonium and lithium buffers. For potassium and sodium buffers influence of high voltage on the system is studied using pressure to mobilize solution inside the capillary. It is proven that the instability for potassium buffer is increased with high voltage. The instability of the signal is then compared for different voltage levels. The results show to the influence of Joule heating. The stability of signal baseline for all studied cations was found to be decreasing with increasing ionic strength and conductivity. This work concludes, that this phenomenon does not confine only to potassium ions. Even though the potassium buffer does not have the highest conductivity, signal instability was the highest however. The degree of instability of signal baseline for potassium buffer depends not only...
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