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Use of electrophoretic methods in the analysis of pharmaceutically important compounds
Šafra, Jiří ; Pospíšilová, Marie (advisor) ; Jokl, Vladimír (referee) ; Ušelová, Kateřina (referee)
The thesis deals with the use of electrophoretic methods in the analysis of pharmaceutically important compounds. In the theoretical part, the principles of basic techniques (like CZE, MEKC, ITP etc.) as well as on-line preconcentration methods (stacking, sweeping, ITP-CZE, tITP) are described. The first part of the experimental work deals with the use of on-line sample preconcentration techniques (ITP-CZE and LVSS with polarity switching) for the analysis of the complex matrices of plant origin. 1. The on-line ITP-CZE was used for the separation and quantification of phenolic acids derived from benzoic and cinnamic acids in methanolic extract of Epilobium parviflorum. BGE-S-BGE electrolyte system was used for the separation. The leading electrolyte in the ITP pre-separation step was 0.01M HCl, 0.02M imidazol, 0.2% HEC with pH 7.2; the terminating electrolyte was 0.01M HEPES with pH 8.2. The background electrolyte in the electrophoretic step contained 25mM MES, 50mM TRIS, 30mM boric acid, 10mM α-cyclodextrine, 0.2% HEC of pH 8.3. A single analysis took 25 min. No special sample pretreatment was required. 2. The next work focused on the application of a stacking-CZE method used for the separation and determination of eight phenolic acids in an extract of Epilobium parviflorum. Large-volume sample stacking...
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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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Determination of complexation constants by partial Filling method and Flow Induced Dispersion Analysis and their comparison with values determined by Affinity Capillary Electrophoresis
Ansorge, Martin ; Ušelová, Kateřina (advisor) ; Křížek, Tomáš (referee)
Partial Filling method (PF) and Flow Induced Dispersion Analysis (FIDA) were used for the determination of stability constants of model system of four profens (R-Flurbiprofen, S-Ibuprofen, S-Ketoprofen and S-Naproxen) complexing with β-cyclodextrin. When using PF method, only a part of capillary is filled with the selector and thus the analyte must migrate through the zone of selector first and then through the neat BGE (it has different mobilities in both zones). Dependency of the differences in migation time on the length of the selector zone is then studied. When FIDA is used, the analyte in pushed through the capillary by external pressure and dependency of the rate of peak dispersion of the analyte on the concentration of selector in capillary is observed. Moreover, PF experiments were theoretically studied by Simul 5 Complex computing programme. Values of stability constants obtained by both methods were compared with values obtained by frequently used Affinity Capillary electrophoresis (ACE) method. The comparison of stability constants values determined by PF method and FIDA shows that both investigated methods grants results comparable with those obtained by the ACE.
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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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