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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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Study of (bio)molecular interactions and enzymatic reactions by capillary electrophoretic methods
Růžička, Martin ; Hudeček, Jiří (advisor) ; Mikšík, Ivan (referee) ; Křížek, Tomáš (referee)
Non-covalent interactions participate in majority of processes in living organisms. The strength of interaction between (bio)molecules can be characterized by binding constants of respective complexes, which can be determined by variety of physico-chemical methods. From these methods, capillary electrophoresis features several advantages: (1) interactions takes place in aqueous solutions between free molecules without necessity of their immobilization, (2) short analysis time, (3) small consumption of analyzed compounds, and (4) easy automation of analyses. Within this dissertation thesis, methods of partial-filling affinity capillary electrophoresis (PF-ACE) were developed for study of interactions between double-strand DNA oligonucleotides and well characterized intercalator ethidium bromide as a model compound. Subsequently, binding constants of oligophenylene derivatives complexes with DNA oligonucleotides were determined. The PF-ACE method was optimized for study of enantioselective interactions between helquats and selected chiral acidic aromatic analytes. Several compounds whose enantiomers were separated using helquats as chiral selectors were identified. Capillary electrophoresis was applied as a separation analytical method for monitoring of peptide substrate cleavage by rhomboid...
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Chromatographic characterization of polyaniline-coated stationary phases
Taraba, Lukáš ; Křížek, Tomáš (advisor) ; Pacáková, Věra (referee) ; Srbek, Jan (referee)
(EN) This dissertation thesis is focused on physicochemical and chromatographic characterization of polyaniline-coated stationary phases. In the first part, surfaces of bare silica and octadecyl silica sorbents were modified by in-situ chemical polymerization of aniline hydrochloride and their subsequent systematic characterization was performed by using the linear solvation energy relationship approach in the HILIC mode of capillary LC. In addition, several common physicochemical techniques were used to characterize properties of these altered materials. The modified sorbents were then packed into capillary columns. The retention interactions taking place between solute and the separation system were evaluated on the basis of retention data of a number of various solutes. The results showed that polyaniline coating had a significant effect on the retention promoting interactions of both polyaniline-coated stationary phases. The assumed mixed-mode retention mechanism was proven for both the stationary phases. The second part dealt with investigation of the separation potential of polyaniline- coated silica stationary phase in different chromatographic modes. The retention factor curves of structurally similar solutes were constructed as a function of organic modifier portion in the mobile phase....
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Chromatographic characterization of an ion-exchange column
Seidlová, Bára ; Sobotníková, Jana (advisor) ; Křížek, Tomáš (referee)
The following evaluation procedure was selected for the charterized ion-exchange column Phenosphere™ SCX. First of all, a set of biologically interesting analytes was selected. This set contained several basic drugs, specifically five tricyclic antidepressants (TCA), an anesthetic called procaine and a substance with antimalaric and antiarrythmic effect - quinidine. For this set of basic substances were the separation conditions optimized on a characterized ion-exchange column Phenosphere™ SCX. The effect of the mobile phase composition on chromatographic behaviour of selected drugs was investigated, for instance the effect of used buffer and its concentration, buffer pH and the effect of addition of the organic modifier (in this case methanol). Under optimized chromatographic conditions for basic drugs analysis the ion-exchange column was characterized using LSER (Linear Solvation Energy Relationship). The chromatographic behaviour of the test substances with known molecular descriptors was found and then the multiple linear regression between retention factors of test substances and their molecular descriptors was done. The results of the multiple linear regression were discussed and then the interactions contributing to the analyte retention on the investigated cation-exchange column...
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Characterization of protamine influence on indirect electrophoretic determination of heparin
Petrák, Ondřej ; Křížek, Tomáš (advisor) ; Kubíčková, Anna (referee)
Cílem této bakalářské práce bylo studium interakcí heparinu a protaminu v prostředí kapilární zónové elektroforézy pomocí jejich vlivu na velikost plochy píku tetraargininu. Byla použita křemenná kapilára o vnitřním průměru 50 µm a celkové délce 50 cm. Efektivní délka kapiláry byla 41,5 cm a vzorek byl dávkován na dlouhý konec kapiláry. Byl optimalizován způsob dávkování jednotlivých zón, vhodné složení základního elektrolytu bylo již známo z předešlých experimentů, na které tato práce navazovala. Základní elektrolyt byl složen z 0,1 M roztoku kyseliny trihydrogenfosforečné a 0,1% roztoku hydroxyethylcelulózy. Vzorek byl dávkován hydrodynamicky tlakem 5,0 kPa po dobu 120 s. Následně byla stejným tlakem po dobu 90 s dávkována zóna základního elektrolytu. Poslední dávkovanou zónou byla zóna tetraargininu o koncentraci 1 mg/ml, která byla dávkována hydrodynamicky tlakem 5,0 kPa po dobu 3 s. Posledním krokem bylo vložení separačního napětí 30 kV po dobu 30 s. Tetraarginin byl detegován při λ = 200 nm. Byly změřeny vlivy samotného heparinu/ protaminu a jejich směsí v různých vzájemných poměrech. Bylo ověřeno, že se zvyšující se koncentrací heparinu dochází k poklesu plochy píku tetraargininu. Nově bylo zjištěno, jaký vliv na velikost tohoto účinku má přítomnost protaminu. Zde došlo k překvapivému...
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Development of an UHPLC method for stability study of venetoclax
Máchalová, Jitka ; Křížek, Tomáš (advisor) ; Kozlík, Petr (referee)
Tato práce se zabývá vývojem chromatografické metody pro stanovení venetoclaxu a kontrolu jeho čistoty pomocí techniky UHPLC. Venetoclax je nízkomolekulární inhi- bitor antiapoptického proteinu Bcl-2, který hraje důležitou roli při regulaci apoptózy a při vzniku hematologické malignace. Venetoclax se tedy používá pro léčbu chronické lymfocytární leukemie. Kvůli problematické rozpustnosti analytu byl jako rozpouště- dlo vzorku zvolen 10mM vodný roztok H3PO4 : ACN s 40% objemovým zastoupením acetonitrilu. Dále byla nalezena optimální kombinace stacionární a mobilní fáze. Optimalizace byla prováděna především s ohledem na faktor chvostování píku vene- toclaxu a jeho rozlišení od nečistoty eluující v jeho těsné blízkosti. V optimalizované metodě byla použita kolona Acquity UPLC BEH Phenyl (100 × 2,10 mm; 1,7 µm) a jako mobilní fáze směs pufru (10mM H3PO4/NH3 o pH 2,5) s přídavkem 0,5% trifluoroctové kyseliny a ACN. Součástí validace metody bylo testování přesnosti, opa- kovatelnosti, meze detekce a kvantifikace, linearity a lineárního dynamického rozsahu, robustnosti metody a testování stability vzorku. Opakovatelnost retenčního času byla 0,07 % a opakovatelnost plochy píku byla 0,45 %. Hodnota meze detekce metody byla 1,6 · 10−5 mg mm−1 a hodnota meze kvantifikace byla 5,4 · 10−5 mg mm−1 . Linearita...
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