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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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Determination of electrophoretic mobility of alkaline cations in micellar systems
Müllerová, Ludmila ; Gaš, Bohuslav (advisor) ; Kašička, Václav (referee)
Micellar electrokinetic chromatography is a widely used analytical separation technique. To achieve separation, it uses interaction of analytes with charged micelles present in the background electrolyte (BGE). Not only the mobilities of analytes but also mobilities of small inorganic ions, usually contained in the BGE, are influenced by the presence of micelles. Description of interactions between inorganic ions and micelles is needed for better understanding electrophoretic separations in BGEs containing micelles. Determination of physico-chemical constants (interaction constants, mobilities of complexes) by capillary electrophoresis is based on accurate determination of effective mobilities. For this reason, accurate determination of mobility of electroosmotic flow (EOF) is also necessary but complicated in BGEs containing charged micelles, because the neutral EOF marker can be mobilized by interaction with the micelles. In this work a new two-detector method for determination of effective mobility in interacting BGEs was proposed. In this method the analyte is placed in the BGE containing charged micelles, while the marker zone is in the BGE without micelles and so the possible interaction is avoided. Using this method, dependence of sodium cation mobility on concentration of lithium...
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