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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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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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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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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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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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Mobilization of electroosmotic flow markers in capillary electrophoresis
Martínková, Eva ; Křížek, Tomáš (advisor) ; Sobotníková, Jana (referee)
Capillary zone electrophoresis is a separation method used to separate charged substances in electric field. Substances migrate to detector due to their own electrophoretic mobility and electoosmotic flow in capillary. Separation is possible because of electroforetic mobility of substances, which is directly proportional to the charge and undirectly proportional to the hydrated radius of ion. This means that neutral molecules form just one neutral zone, which is detected together. Well absorbing neutral substances are thus used as markers of average (mean) EOF. It was found, that some of the markers have dispositions to be mobilized with respect to the EOF. This mobilization is caused by interactions of free electron pair on atoms of nitrogen or oxygen of marker with components of background buffer. The aim of the study was to measure mobilization of some commonly used markers with different cations of acetate buffer and thus find "ideal" marker, whose mobility is almost the same as EOF and find combinations of marker+buffer, which are better to be avoided. Eleven markers in combination with six cations of background electrolyte were studied. We have found, that relative mobilities of some of chosen markers come up to five percent of limit mobilities of small inorganic cations. An appropriate...
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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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Advanced Separation Techniques and Data Processing in Capillary Electrophoresis
Ansorge, Martin ; Ušelová, Kateřina (advisor) ; Ševčík, Juraj (referee) ; Masár, Marián (referee)
Techniques of capillary electrophoresis are essential for both the quantitative and qualitative analysis of a large variety of compounds. They find application in many areas of chemistry, from pharmacological industry and food processing, up to highly specialized biotechnological laboratories. Over the last hundred years, their mathematical model has been described with precision. Thus, current research mainly aims for the development of more advanced and more specialized applications. During the greatest boom of affinity capillary electrophoresis within the nineties, many authors would describe similar phenomena under different names. The first part of this work focuses on the consolidation and unification of the problematics and terminology of this method. It also discusses the phenomena which can affect electromigration and separation during electrophoretic processes. We will focus on a specific subset of affinity capillary electrophoresis, partial filling capillary electrophoresis, which, to our knowledge, has not been fully theoretically explored yet, and present a mathematical model allowing the determination of complexation constants and state some limitations of this approach. The second part of this thesis focuses on the development and application of computer softwares, which are meant...
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Development of the electrophoretic method for determination of phosphates and borates in cooling mixtures
Listakhava, Iryna ; Křížek, Tomáš (advisor) ; Kubíčková, Anna (referee)
In this diploma thesis, a method of routine analysis of commercial coolants was developed, focused on the simultaneous determination of inorganic anions - phosphates and borates - as corrosion inhibitors in fresh mixtures and the markers of recycling quality. The method was developed for the instrumental technique of capillary zone electrophoresis with indirect detection at 254 nm. The following were optimized: background electrolyte (its composition, concentration, pH), measured samples (coolant dilution, suitable internal standard) and measurement conditions (capillary length, sample dosing). The influence of the sample matrix on the measurement results, the robustness of the calibration line (or the agreement of the response with the actual analyte concentration) and also the repeatability of the method were tested. Optimizations and tests resulted in a method based on a standard addition with the following parameters: background electrolyte of pH = 11.50 containing sodium chromate at 10 mmol l-1 concentration and CTAB at 1 mmol l-1 concentration; dilution of the coolant sample 20-50×, addition of internal standard MES at 0,1 mmol l-1 concentration, electrokinetic sample injection at -5 kV for 10 s, capillary length 50.0 cm, inner diameter 75 μm. The repeatability of the method expressed as a...
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