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Gradient chromatofocusing for separation of proteins
Bílek, Jan ; Kalíková, Květa (advisor) ; Riesová, Martina (referee)
This bachelor thesis is focused on separation of four model proteins - conalbumin, bovine serum albumin, beta-lactoglobulin A and beta-lactoglobulin B - by gradient chromatofocusing high-performance liquid chromatography with two commercial anion-exchange columns. The first column contained a strong anion exchanger (Agilent Bio SAX) and the second one contained a weak anion exchanger (Agilent Bio WAX). Three different mobile phase pH gradients were optimized, one-hour gradient and two half-an-hour gradients. The baseline separation of mixture containing conalbumin and beta-lactoglobulines A and B was achieved on both anion- exchange columns using each of the three optimized gradients and with flow rate 1 ml/min. Three bovine serum albumin isoforms were resolved in all tested systems using a strong anion exchanger as the stationary phase. The highest resolution of mixture containing conalbumin and beta-lactoglobulines A and B was obtained with a strong anion exchanger as the stationary phase in thirty minutes. The highest resolution of bovine serum albumin isoforms was achieved with a strong anion exchanger as the stationary phase in sixty minutes. Key words: gradient chromatofocusing, protein separation, high performance liquid chromatography
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System zones in capillary electrophoresis
Riesová, Martina ; Gaš, Bohuslav (advisor) ; Kašička, Václav (referee) ; Gebauer, Petr (referee)
Martina Riesová Abstract of Ph.D. Thesis Capillary electrophoresis is a method of choice in many analytical laboratories for its high separation efficiency, rapidity, low consumption of chemicals and therefore low costs. Inherent to each electrophoretic separation system are system peaks, which can significantly affect or confuse the electrophoretic results. In capillary zone electrophoresis, position of system zones can be predicted easily and reliably by means of prediction software based on a theoretical description of electromigration. However, the prediction of only position of a system zone may not be sufficient for identification of system peaks in obtained electropherograms. Therefore, an existing theoretical model was significantly extended and new version of PeakMaster software (PeakMaster 5.3) was introduced in the framework of this thesis. PeakMaster 5.3 enables to predict not only the positions of system zones, but also their shapes and polarity. Thus, PeakMaster 5.3 improves the prediction possibility of overlapping or interaction of system peaks with analyte peaks. Moreover, composition of the sample can be optimized in order to obtain convenient shapes and amplitudes of system peaks. The applicability of capillary zone electrophoresis can be extended by addition of a complexation agent into...
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Determination of limiting electrophoretic mobilities of charged cyclodextrins
Boublík, Milan ; Riesová, Martina (advisor) ; Křížek, Tomáš (referee)
In the frame of this work, dependences of electrophoretic mobilities of four defined multiple charged cyclodextrins on ionic strength of background electrolyte were measured using capillary zone electrophoresis. Dependencies of mobility on ionic strength do not follow Onsager law in the case of large and multiple charged species. Therefore, empirical fitting function was used to determine limiting mobilities of studied cyclodextrins. Obtained values of limiting mobilities as well as the resulting regression function are essential for correct determination of complexation constants of corresponding cyclodextrins with various analytes.
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Chiral HPLC separation of newly synthesized amino acids
Kučerová, Gabriela ; Kalíková, Květa (advisor) ; Riesová, Martina (referee)
The aim of this work was to develop and to optimize HPLC method for enantioseparation of newly synthesized derivatives of amino acids. The set of these analytes contained four N- blocked derivatives of D,L-Phenylalanine, three N-unblocked derivatives of D,L- Phenylalanine, , one methylated derivative of D,L-Tyrosine and D,L-Tyrosine. Two separation modes i.e. reversed phase and polar-organic modes and two columns i.e. Chirobiotic® T and Chirobiotic® T2 were used. Chiral stationary phases of these columns were composed of macrocyclic antibiotic teicoplanin coated on silica gel support. As mobile phases in revesed phase mode, methanol and acetate buffer were used with Chirobiotic® T column. The most suitable concentration were 20 mM and the most suitable pH value were 4.00. Under the above mentioned conditions eight analytes of ten were separated and the optimal conditions were found. Polar-organic mode and Chirobiotic® T column were suitable only for enantioseparation of three N-blocked derivatives of D,L- Phenylalanine. Mobile phases were composed of methanol with small additions of triethylamine and acetic acid. Chirobiotic® T2 column in reversed phase mode was not suitable for tested set of analytes. Only partial separation of D,L-Tyrosine derivative and one N-unblocked D,L-Phenylalanine...
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Determination of dissociation constants and limiting mobilities of some ampholytes
Maliňáková, Lucie ; Riesová, Martina (advisor) ; Beneš, Martin (referee)
Capillary zone electrophoresis is a simple and automated method which enables simultaneous determination of the dissociation constants and limiting ionic mobilities of the analyte from one series of experiments. The small consumption of the sample is main advantage of this method. The dependence of the effective mobilities of 7 selected ampholytes on pH in the range of 1.6 to 6.8 was measured. A set of simple buffers with constant ionic strength and satisfactory buffering capacity was designed using computer program PeakMaster. The ampholytes were detected by contactless conductivity detector. Dynamic capillary coating was used to minimize interactions of analytes with silanol groups on the inner wall of the capillary. Experimentally obtained effective mobilities were fitted with the suitable function. Actual mobilities and the mixed dissociation constants corresponding to the actual ionic strength were parameters of the regression function. Thermodynamic dissociation constants and limiting ionic mobility of ampholytes were calculated using the extended Debye-Hückel law with the linear member and the Onsager law. Key words: capillary zone electrophoresis, dissociation constant, limiting mobility, ampholytes
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Oscillating electrolytes in electrophoresis
Riesová, Martina
Oscillating electrophoretic systems are systems exhibiting oscillations in concentration profiles of all ions when electric current is passing through them. These systems were predicted from linear model of electromigration and the oscillations are a direct consequence of complex system eigenmobilities. We searched for ternary oscillating systems composed of two acids and one base. A tool we used was program PeakMaster, which was modified to enable easier and faster finding of systems with complex eigenmobilities and optimization of their composition. We found eight ternary systems with complex eigenmobility and inspected them experimentally. Five of them proved oscillating behavior. We also found one quaternary oscillating system. To compare theory and experiment we calculated in PeakMaster a theoretical position of the system zones with complex eigenmobilities. In experiments we recognized the system zones like a small gap in the pattern of oscillation. Position of this gap was in very good agreement with theoretical prediction by PeakMaster. We confirmed that the complete oscillating pattern travels in the capillary with velocity determined by real part of complex eigenmobility. Also we tested a robustness of oscillation in some systems and compared simulated the amplitude of oscillations with...
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Monitoring of water quality in protected landscape area streams - Křivoklátsko
Kesnerová, Lucie ; Benešová, Libuše (advisor) ; Riesová, Martina (referee)
This thesis deals with monitoring of surface water quality in CHKO Křivoklátsko. The main purpose of monitoring is to obtain information about a state of flow and development during the previous period and to determine the pollution stage according to relevant standards. The theoretical part describes the basic information about the locality where they come from water samples and basic characteristics of sets of parameters. This section also includes theoretical assesment of sediment. The experimental part shows the practical determinates of each parameter, with the calculation of the quantities present and physical indicators. This section is followed by evaluation of measurement results, which are arranged in tables, including readings of all required parameters. The paper concludes an assessment of the monitoring of quality and compare the current state compared with the previous reporting period at each sampling point. Key words: chemical composition of water, water quality, sediments
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