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Analysis of biologically active compounds using high performance separation methods
Adamusová, Hana ; Bosáková, Zuzana (advisor) ; Jelínek, Ivan (referee) ; Tůma, Petr (referee)
In the first part of this doctoral thesis, a new analytical HPLC-MS/MS method for monitoring of concentration changes of 17β-estradiol (βE2) during in vitro mouse sperm capacitation was developed. Capacitation was performed for three initial concentrations of βE2 (200, 20 and 2 μg/L). For all the concentrations a similar trend for the total unbound βE2 was observed. In general, the βE2 concentration decreased to reach its minimum and then increased again. The position of the minimum differed for the individual tested βE2 concentrations. Experimentally obtained results were subjected to the kinetic analysis. The curves fitted through the experimentally determined points displayed an autocatalytic character. For the agreement between the curves obtained by fitting through the experimental points and the theoretical calculated curves, it is necessary to assume that the first step is adsorption of βE2 onto the surface of the sperm controlled by Langmuir isotherm. The kinetic study was also used to study the effects of fluorides and aluminium fluoride complexes on the capacitation of mouse sperm. The experimental points were in very good agreement with the shape of the theoretical curves and this fact verifies the mechanism of the mouse sperm capacitation kinetics. In the second part of this work, two...
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Effect of background electrolyte anions on markers of electroosmotic flow in capillary electrophoresis
Čokrtová, Kateřina ; Křížek, Tomáš (advisor) ; Kubíčková, Anna (referee)
Mobility of the electroosmotic flow is an important quantity in capillary electrophoresis because its value is needed to determine the effective electrophoretic mobility of analytes. The effective electrophoretic mobility is used for electrophoretic determination of physico-chemical constants, such as dissociation constants or stability constants, but it is also important in analytical chemistry because identification of analytes is based on the value of the effective mobility. The most common way to measure electroosmotic flow is to add a neutral substance to the sample. However, the neutral substance can gain electrophoretic mobility due to interactions with the components of the background electrolyte. This may cause an inaccurate determination of the velocity of the electroosmotic flow. The aim of this work was to measure the mobility of several common and less frequently used markers in the background electrolyte containing different anions and to find suitable marker - anion combinations in order to avoid major measurement errors. Relative mobility of eight markers in the pure background electrolyte and in the background electrolyte containing a salt of the studied anion - sodium chloride, sodium perchlorate or sodium sulphate - was measured and related mobility of thiourea. Acetate buffer...
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Study of interaction between protamine and heparin and its applicability in capillary electrophoresis
Martínková, Eva ; Křížek, Tomáš (advisor) ; Kubíčková, Anna (referee)
Heparin is an acid mixture of glycosaminoglycans with high negative charge density which naturally occurs in human body. Due to its ability to bind antithrombin III and thus accelerate inhibition of thrombin it has anticoagulant effect. This is abundantly used in clinical practice for operations, in case of embolia or heart-attacks. Protamine is a mixture of small basic peptides, which is used in clinical practice as a heparin antidote. The interaction between heparin and protamine is electrostatic and is also used for determination of heparin in human plasma or blood using affinity methods. In my study it was found that if protamine and heparin are mixed in one vial, a complex is formed. Its resulting charge depends on concentration ratio of protamine and heparin. On the other hand, in case the protamine is injected as a sample and heparin is added to background electrolyte as a protein-binding ligand, it is possible to determine heparin from decreasing protamine peak area. Because of the complexity of protamine-heparin interaction, tetraarginine was used as structurally close model of protamine to increase repeatability of measurements. The method for determination of heparin was optimalised. It uses 20 mM or 60 mM ortho-phosphoric acid as background electrolyte, 1 mg/mL solution of tetraarginine...
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Study of vortioxetin complexation by native cyclodextrins and its determination using capillary electrophoresis
Počtová, Žofie ; Křížek, Tomáš (advisor) ; Sobotníková, Jana (referee)
In this master thesis, the formation of supramolecular complexes between vortioxetine active substance and cyclodextrins (α, β, γ) is examined and the stability constants of these complexes are determined. The stoichiometric ratios of cyclodextrin and vortioxetine 1:1 and 2:1 are considered. The results suggest that the 1:1 ratio of cyclodextrin and vortioxetine is more accurate for each of used cyclodextrins and that the strongest interaction is between vortioxetine and β-cyclodextrin. Further, a method for vortioxetine assay in a tablet is developed by using non-aqueous capillary electrophoresis technique. The precision, accuracy, and robustness are validated and the values of detection and quantification limits are determined. This method can be used as a verification method for the results obtained by HPLC as the separation principle is different in capillary electrophoresis. Also, the stability of vortioxetine as a pure powder and as a mixture with excipients is examined under the conditions of elevated temperature and humidity.
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Chiral separation of diquats and determination of stability constants of their complexes with cyclodextrins by capillary electrophoresis
Bílek, Jan ; Kašička, Václav (advisor) ; Jelínek, Ivan (referee)
Capillary zone electrophoresis was used for chiral separation of eleven diquat derivatives. These N-heteroaromatic dications containing structural motif of 2,2'-bipyridine have recently been studied for their interesting electrochemical properties as well as for the axial chirality of their molecules. The combination of these properties could potencially lead to interesting applications in the future. For enantioseparation of diquats (DQ) commercially available randomly sulfated α-, β-, and γ-cyclodextrins with high degree of substitution were used. A succesfull chiral separation was achieved using all of the three sulfated cyclodextrins as chiral selectors (CS). Baseline enantioseparation was achieved for 82 %, 91 % respectively 100 % of the analyzed DQ in the presence of HS-α-CD, HS-β-CD, HS-γ-CD respectively. The highest separation efficiency and resolution were obtained in the backround electrolyte containing 22 mmol/L NaOH, 35 mmol/L H3PO4 (pH2,5) and 6 mmol/L HS-β-CD. Using three available nonracemic DQ an identification of the particular M- and P-enantiomers was done for the three corresponding DQ structures. Apparent stabillity constants of complexes of the DQ derivatives with above mentioned cyclodextrins as CS were determined by means of capillary affinity electrophoresis. The stability...
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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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Influence of the interacting constituent of the background electrolyte on electrophoretic separation
Müllerová, Ludmila ; Dubský, Pavel (advisor) ; Kašička, Václav (referee) ; Petr, Jan (referee)
Capillary electrophoresis is a widely used separation method of analytical chemistry. Addition of a selector into the background electrolyte extends its applicability to separation of enantiomers or of compounds of similar physicochemical properties. In analytical practice, mixtures of selectors are also commonly used - either prepared intentionally to achieve better separation or because commercially available selectors may be mixtures of compounds differing in the degree of substitution and substituent positions. Mathematical description of these systems, which are highly relevant in analytical practice, can simplify search for optimal separation conditions. Also, it provides a useful insight into the separation mechanism. In this work, a model of electromigration of an analyte interacting with a mixture of two selectors is proposed and experimentally verified. This model results from a more general description of systems with an arbitrary number of selectors. The model shows that a selector mixture can be treated as a single selector if the ratio of the respective selector concentrations is kept constant. When the mixture is prepared intentionally, this description predicts, how separation potential of the mixture changes with its composition. Thus it allows the optimal composition and total...
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Determination of enzyme activity of cellobiase using capillary electrophoresis
Velvarská, Romana ; Křížek, Tomáš (advisor) ; Kubíčková, Anna (referee)
This thesis deals with optimization of capillary electrophoresis conditions for the separation of unreacted substrate and products (cellobiose, glucose) generated by the enzymatic cleavage of cellotriose with enzyme cellobiase. The optimal separation electrolyte was shown to be a solution containing 30 mmol·dm-3 sodium hydroxide with addition of 1.0 mmol·dm-3 sodium phosphate and 1.0 mmol·dm-3 sodium phosphate dibasic (pH = 12.38). With this separation electrolyte calibration curves and repeatability were measured and the limits of detection and quantification were determined. The relative standard deviations of peak areas ranged up to 6.1 % and they did not exceed 0.2 % for the migration time. Limits of detection were determined in the range from 0.020 to 0.026 mmol·dm-3 and limits of quantification from 0.066 mmol·dm-3 to 0.085 mmol·dm-3 . Subsequently the enzyme reaction was monitored in offline setup. Furthermore, the offline procedure was automated and the relative standard deviation of cellodextrines after 30 minutes of hydrolysis was ranging up to 13.0 % of glucose and cellotriose and 3.0 % cellobiose using this automated method, which makes it suitable for semiquantitative and comparative measurements. The last part of the experiment was to carry out enzymatic reaction in online setup....
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