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Determination of glutathion in plant samples using capillary electrophoresis
Skála, Michael ; Křížek, Tomáš (advisor) ; Kubíčková, Anna (referee)
This work is based on a previously published method for determination of both glutathione forms by capillary zone electrophoresis equipped with UV spectrometric detector. Theoretical part described biological and biochemical functions of glutathione as a molecule. It mentioned historically used methods for glutathione determination, their accuracy, precision and reliability and focused on electrophoretic methods, especially capillary zone electrophoresis. Basic principles of capillary zone electrophoresis were described, followed by an overview of approaches to treatment of different types of samples, with special focus on plant samples. At the end, method this work was based on was described. The experimental part describes the development and optimization of this method. Firstly, optimum chemical composition and ionic strength of background electrolyte were determined - two electrolytes were compared, with NaCl and without NaCl - no significant differences were observed, so NaCl was excluded from BGE. Next, optimum concentration of hexadimethrine bromide was determined. The effect of HDB concentration on electroosmotic flow was investigated. Calibration curve for different concentrations of analytes in water was constructed and repeatability, LOD and LOQ of the method were determined....
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Modification of inner capillary surface for electrophoretic separations of liposomes
Píplová, Renata ; Křížek, Tomáš (advisor) ; Čokrtová, Kateřina (referee)
Electroosmotic flow is one of the main factors that affect the resolution of separations in capillary electrophoresis and its modification is often necessary. One way of modification of the electroosmotic flow is the use of dynamic or permanent coating of the inner capillary surface. Such modifications during liposome separation can also reduce the tendency of liposomes to adsorb to the capillary surface. The aim of this work was to find a stable coating that will sufficiently eliminate the electroosmotic flow and to test its effect on the separation of liposomes. At first, the dynamic coatings of the capillary with Pluronic F-127, polyvinylpyrrolidone K30, polydiallyldimethylammonium chloride, polyethylene glycol and polyvinyl alcohol were tested using four different methods. Individual polymers and methods differed in the rate of elimination of the electroosmotic flow as well as in the stability of the capillary coatings. Dynamic coatings with Pluronic F-127, polyvinylpyrrolidone K30 and polydiallyldimethylammonium chloride were selected for further liposome separation testing. Fluorescently labeled liposomes were separated using capillary electrophoresis with laser-induced fluorescence detection and the mobilities of liposomes separated using capillaries with different coatings were determined....
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Comparison of detection techniques for analysis of saccharides by capillary electrophoresis
Vlčková, Nikol ; Křížek, Tomáš (advisor) ; Kozlík, Petr (referee)
The aim of this work was to compare detection techniques of capillary electrophoresis to determine saccharides. The techniques were contactless conductivity detection, direct and indirect UV detection and fluorescence detection. Determined saccharides were glucose, fructose and sucrose, lactose was employed as internal standard. For conductivity detection the capillary has internal diameter of 20 µm. Voltage applied to capillary was 15 kV and temperature was 25 řC. 40mM NaOH was used as BGE. The calibration dependence was measured between 0,005 - 0,5 mg/ml. Repeteabilityof peak areas and migration times was measured in two concentration levels of 0,02 and 0,2 mg/ml. Detection and quantification limits were between 1,0 - 5,8 µg/ml. RSD values for repeatability of peak areas and migration times were between 0,01- 8,64 %. Difference in recovery from real concentration of analytes was between 7 - 22 %. For other detection methods the capillary had inner diameter of 50 µm. Voltage applied to capillary for indirect UV detection was 28 kV and temperature was 30 řC. Wavelength for detection was 207 nm and 50mM glycylgylycine with pH value of 12,8 was used as BGE. The calibration dependence was measured between 0,1-1 mg/ml. Repeteability of peak areas and migration times was measured in two concentration...
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Application of capillary electrophoresis for dried blood spots analysis
Ryšavá, Lenka ; Tůma,, Petr (referee) ; Petr,, Jan (referee) ; Kubáň, Pavel (advisor)
Blood samples are normally analyzed in the form of plasma or serum. As an alternative for blood sampling and storage, dry blood spots (DBS) have been increasingly used in recent years. In the actual dissertation thesis, DBS are processed and analyzed by capillary electrophoresis (CE), which has been very rarely used for DBS analysis in the past. However, CE offers several features, given by the recent development of this technique, not available for standard analytical methods. Direct coupling of microextraction techniques to CE and simultaneous determination of analytes present in complex matrices leads to minimization or elimination of deficiencies in DBS analysis, for example the effect of sample matrix, hematocrit effect, and inhomogeneous distribution of analytes in DBS. In the simplest possible arrangement, sample treatment, injection, and analysis of DBS samples are achieved exclusively by a single commercial CE instrument. Application of newly developed fully soluble materials for DBS sampling offers better extraction efficiency and more accurate quantitation. The developed concepts include new methods for efficient treatment of DBS samples and their direct analysis without the need for operator intervention and they provide sufficient selectivity and sensitivity for the determination of important analytes not only in DBSs but also in other complex samples.
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Analytical approaches to study group interactions of azole pesticides with important biological active compounds
Kovač, Ishak ; Jaklová Dytrtová, Jana (advisor) ; Száková, Jiřina (referee) ; Navrátil, Tomáš (referee)
This dissertation presents a developed capillary electrophoresis method for analysing residues of triazole fungicides (TAF) from real samples ("Sweet cherry" tomato - Solanum lycopersicum L) and shows its potential application in environmental analysis. The developed method was validated in-house and characterised by a relatively short analysis time and low cost with sufficient LOD and LOQ. As an additional part of this dissertation, which rose from the tomato plants' treatment with TAF, we studied the impact of triazole fungicides on plant development and their impact on biologically active compounds. Determination of TAF residues from tomato peel carried out with capillary electrophoresis. Capillary electrophoresis is a versatile technique with minute amounts of sample required, with high efficiency in separating small molecules, such as selected TAF. The developed method, cyclodextrin-modified micellar electrokinetic chromatography, accomplished a baseline separation of the TAF mixture from tomato peel. The optimal conditions for separation were in the background electrolyte comprised of phosphoric acid and Tris (100 mM concentration, respectively), 20% v/v methanol, with apparent p[H+ ] 4.8. The constituents of the mixed micellar pseudo-stationary phase were ionic detergent sodium dodecyl...
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Analysis and characterization of liposomes using capillary electrophoresis
Orgoníková, Andrea ; Křížek, Tomáš (advisor) ; Coufal, Pavel (referee)
4 Abstract This diploma thesis deals with the analysis of three different types of liposomes using capillary electrophoresis with UV-VIS and laser induced fluorescence detection and the use of liposomes as a pseudostationary phase for analyte separations by liposome electrokinetic chromatography method. In experiments with DSPC-DSPG-PEG2000-DMPE liposomes encapsulating 5-fluoruracil, the peak of liposomes with encapsulated 5-fluoruracil and the peak of free 5-fluoruracil were successfully identified in the electropherograms. Both peaks showed the same absorption spectrum in the UV region, thus confirming their identity. It was proved that capillary electrophoresis with UV-VIS detection is useful for the separation and detection of free and encapsulated drug, which is necessary to determine the efficiency of encapsulation. By monitoring the change of effective analyte mobility after the addition of liposomes to the background electrolyte the applicability of the investigated liposomes in liposome electrokinetic chromatography was evaluated. A change in mobility was observed for negatively charged 5-fluoruracil and positively charged tryptamine and p-toluidine. The absolute value of the effective mobility of negatively charged 5-fluoruracil decreased by 18.2 % due to interactions with liposomes and effective...
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Electrophoretic determination of oxidized and reduced form of glutathion in plant material
Durychová, Eva ; Křížek, Tomáš (advisor) ; Kubíčková, Anna (referee)
This work is based on a previously published method for the determination of twenty proteinogenic amino acids by capillary electrophoresis with contactless conductivity detection and deals with possibility of its use for the determination of oxidized (GSSG) and reduced (GSH) forms of glutathione with amino acids. The theoretical part firstly deals with brief introduction of the properties and importance of glutathione and its protection against autooxidation, especially by alkylation with N-ethylmaleimide (NEM), which binds to GSH to form its alkylated form (GSH-NEM). Then, a brief summary of the available methods for determination of glutathione is given, with a focus on the already developed capillary zone electrophoresis methods. Finally, it briefly presents the electrophoretic method for the determination of twenty proteinogenic amino acids on which this work is based. In the experimental part, the integration of the determination of both forms of glutathione into the method for the determination of proteinogenic amino acids is presented. First, a simplified, less time consuming method was tested in shorter 50.0 cm capillary without hydroxyethylcellulose, where both forms of glutathione were separated at baseline within 10 minutes. Relative standard deviations of peak areas (GSH 2.1%; GSSG...
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