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Development of analytical methods for determination of phosphorylated components of bacterial cell membranes
Mikulecká, Jana ; Čabala, Radomír (advisor) ; Feltl, Ladislav (referee)
Phospholipids are dominant components of bacterial cell membranes, where they create double layers. Bacteria differ in their phospholipid composition determination of which can help in identification of important groups of microorganisms. Phospholipid composition of bacteria is influenced by many environmental factors, therefore its variation can be observed within one bacterial stem also. Because of its simplicity, thin layer chromatography is usually applied to identification and determination of bacterial phospholipids. Disadvantage of this method are the high demands of time, carefulness and skills of the analytical personnel. The increasing interest in the phospholipid double-layer promotes the detailed investigation of their fatty acid composition because the more detailed analyses allows for more information yield about bacteria. Gas chromatography hyphenated with mass spectrometry seems to be the best choice for these purposes. Fatty acid identity and total fatty acid content in phospholipid molecules could be determined by this method. Additionally, number, position and isomerism of double bonds and presence of other functional groups on hydrocarbon chain could be determined. Whereas a suitable and...
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Determination of complexation constants of weak electrolyte analytes by Affinity capillary electrophroesis
Ansorge, Martin ; Šteflová, Jana (advisor) ; Ušelová, Kateřina (referee)
Capillary electrophoresis is not only excellent separation technique but it is often advantageously used for determination of physical-chemical parameters of compounds such as complexation constants of analyte and appropriate complexation agent. The most frequently used electrophoretic method for determination of complexation constants is affinity capillary electrophoresis. In the frame of this work the affinity capillary electrophoresis is used for determination of complexation constants of R and S enantiomers of flurbiprofen, particularly both its neutral and fully dissociated form with neutral β- cyclodextrin. For this purpose we utilized classical method introduced in literature, where complexation constants are determined at two pHs, where analyte is fully and partially dissociated, respectively. Simultaneously, the complexation constants were observed by the new method based on multidimensional nonlinear regression, where all data measured at different pHs and β-cyclodextrin concentrations were utilized for evaluation. Results show that classical method can be highly confusing, especially if unsuitable pH is chosen for determination of stability constants of neutral form of analyte. Multidimensional regression method provides substantially more reliable data but with higher error of estimate....
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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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Characterization of low-molecular-mass synthetic markers of isoelectric points by capillary zone electrophoresis and capillary isoelectric focusing
Brandejsová, Martina ; Kašička, Václav (advisor) ; Kubíčková, Anna (referee)
High-performance electromigration separation methods, capillary zone electrophoresis (CZE) and capillary isoelectric focusing (CIEF), have been applied to physico-chemical characterization of new synthetic low-molecular mass markers of isoelectric points. Amphoteric compounds on the basis of aminomethylnitrophenols, their derivatives and other structurally related substances were analyzed by CZE in a series of background electrolytes in a wide pH range, 1.86 - 11.18. From the measured pH dependencies of effective electrophoretic mobilities of analytes (beforehand corrected to reference temperature of 25 řC), their isoelectric points (pI) were determined. In addition, using the non-linear regression analysis of the above dependencies, acid-base dissociation constants (pKa) of ionogenic groups of selected analytes were calculated. Subsequently, the analytes with sharply defined isoelectric points were analyzed by CIEF. CIEF confirmed applicability of these compounds as markers of isoelectric points for calibration of pH gradient in CIEF in the determination of pI of amphoteric compounds, especially peptides and proteins. The determined pKa values of ionogenic groups in particular compounds will be utilized in the development of new pI markers with desired pI values.
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Portable and Miniaturized Separation Techniques Applicable for Food and Biotechnology Analysis
Dvořák, Miloš ; Pospíchal, Jan (referee) ; Bednář,, Petr (referee) ; Rittich, Bohuslav (advisor)
Capillary electrophoresis was used for determination of 6 fractions of caseins. Those fractions were measured in 144 samples of cow’s milk originated from the feeding experiment focused on explanation the influence of the feeding onto casein productions. In this work were separated 6 fraction of caseins first time with total resolution of the peaks. Capillary electrophoresis was applied for determination of short-chain organic acids during fermentation of wine must. It was compared the fermentation of must fermented by different yeast. The difference of profile short-chain organic acids during fermentation were not statistically significant. The once difference was in the utilisation of the malic acid and production of the lactic acid. A portable miniaturized system for medium pressure liquid chromatography was developed. The components were tested and system was used for the isocratic and gradient elution of various analytes (food dyes, parabens). New line of electroluminescent diodes (LEDs) for deep-UV areas of wavelength based on a different materials substrate was characterised. The new line was compared with old line LEDs. The new line LEDs was incorporated in deep-UV absorbance detectors. Detectors were characterised and tested for a detection various analytes in modes flow injection analysis and chromatography separation. First time was characterised this new line of the LEDs and the origin of the parasitic emission band produced by deep-UV LEDs light sources was explained. This origin is given by disturbances of a materials substrates. This work is a contribution for an advance of low-cost and portable systems and detection devices in the field of analytical chemistry.
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Hexahistidine labeling of oligosaccharides
Partyka, Jan ; Křenková, Jana ; Foret, František
We have presented another approach for labeling of oligosaccharides suitable for\ncapillary zone electrophoresis coupled with mass spectrometric detection.\nOligosaccharides labeled by peptides with hexahistidine sequence have faster\nseparation resulting in two or three times shorter time in comparison to oligosaccharides\nwith singly charged labels.
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Use of Capillary Zone Electrophoresis for Determination of Selected Analgetics in Water
Čapka, Lukáš ; Opatřilová, Radka (referee) ; Márová, Ivana (referee) ; Vávrová, Milada (advisor)
From viewpoint of environmental analysis in the whole world became popular in the latest years the question of drugs’ breakthrough to the component of environment. These contami-nants belong to the biological active compounds, with different physical-chemical and biolog-ical properties and evince great tendency to bioaccumulation. They penetrate to the environ-ment because of their increasing of usage and wrong techniques of liquidation. The most often use drugs are preparations against pain – analgetics, and from this category there are non-steroidal anti-inflammatory drugs. The frequent usage of this compounds relate to their easy accessibility. From this large group of compounds was chosen for monitoring: diclofenac, ibuprofen, ketoprofen, salicylic acid, naproxen and acetaminophen; because they include in favorite preparations. The monitoring matrix was the wastewater from two waste water treat-ment plants (WWTP). The sampling was performed in inflow and outflow because the com-paring of concentration of selected contaminants and discovering of efficiency of removing the polutants reliance on treatment technology. For extraction of selected contaminants was used solid phase extraction (SPE) and for determination was used capillary zone electrophore-sis (CZE) with diode array detection (DAD). There was identified and quantified all of se-lected analgetics in inflow and so in outflow of WWTP. That means, this polutants infuse into surface water and then into other components of environment.
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Determination of surfactants by capillary zone electrophoresis.
Tůmová, Klára ; Zemanová, Jana (referee) ; Řezáčová, Veronika (advisor)
Surfactans are synthetically made surface-active ingredients contained in washing and cleaning products. They decrease the surface tension and remove dirt. Surfactans can be divided into four basic classes: anionic, cationic, non-inonic and ampholitic. The most commonly used are the anionic ones. Thanks to the massive use they penetrate into waste water and can disturb the environment. This thesis is focused on the optimization methods and the determination of three anionic surfactants by capillary zone electrophoresis.
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Determination of caseins by capillary electrophoresis
Mičíková, Ivana ; Zemanová, Jana (referee) ; Vespalcová, Milena (advisor)
The diploma thesis, Determination of the caseins by the capillary electrophoresis, is dealing with a creating of the analytical method for a determination of the caseins in cow‘s milk. The capillary electrophoresis was used as a separative method. The developed procedure was optimalized and verified on standards of the caseins and the real samples. The theoretical part gives an information about the compound of cow's milk, caseins, their usage in the food industry and the non-food technologies. There are mentioned some further nutritional factors, whose application in the cattle nutrition can affect the milk composition. The experimental part describes the procedure of the caseins isolation from milk, the used separative system and the determination of the caseins in the lyophilisated samples of milk. The separation was performed in two untreated fused-silica capillaries. Both of them had the same internal diameter: 50 m. The total length of the first capillary was 96 cm (71 cm of effective length) and of the second used capillary 125 cm (effective length 100 cm) The phosphate buffer (10 mM, pH 2,5) served as a basic electrolyte. The diploma thesis has arisen on the basis of the cooperation of the Institute of Food Science and Biotechnology, the Faculty of Chemistry, Brno University of Technology and the Research Institute for Cattle Breeding, Ltd., Department of Animal Nutrition Physiology in Pohořelice.
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