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Use of capillary electrophoresis with contactless conductivity detection for the analysis of proteins
Vatrasová, Kateřina ; Polášek, Miroslav (advisor) ; Jáč, Pavel (referee)
Charles University in Prague, Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Bc. Kateřina Vatrasová Supervisor: doc. RNDr. Miroslav Polášek, CSc. Consultant: PharmDr. Klára Petrů, Ph.D. Title of Diploma Thesis: Use of capillary electrophoresis with contactless conductivity detection for the analysis of proteins Nowadays there exist many methods for the determination of proteins. The aim of this work was to develop, optimize and characterize method for the analysis of a model mixture of proteins (α-lactalbumin, β-lactoglobulin, carboanhydrase, lysozyme and ribonuclease) by capillary electrophoresis with conductivity detection and to compare it with conventional capillary electrophoresis with UV detection. The influence of the capillary inner wall coating on the adsorption of proteins, the composition and concentration of convenient background electrolyte, and the voltage and temperature were examined as experimental conditions possibly affecting the separation. A 50-cm fused-silica capillary (effective length 35 cm for conductivity detector and 41.5 cm for UV detector) with internal diameter 50 μm was used for the analysis. The UV detection wavelength was 210 nm. The static coating of the inner capillary wall realized by successive multiple soaking with...
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Separace chirálních pesticidů pomocí cyklodextrinů kapilární elektroforézou
Denková, Barbora ; Jáč, Pavel (advisor) ; Polášek, Miroslav (referee)
In this diploma thesis, a preliminary study on the applicability of EKC-PF (with CD as chiral selector) to the separation of chiral pesticides was performed. In introduction, theoretical informations are presented concerning electrophoresis instrumentation and theory as well as different electrophoresis modes. Then, enantiomeric separation is introduced through cyclodextrins and possibilities of partial filling mode to finally terminate with a definition of chiral pesticides and some information about their enantioseparation. Concretely, in the experimental part, the evaluation of enantioresolution of several chiral pesticides (trichlorphon, cyproconazole, hexaconazole, imazalil, myclobutanil, penconazole, tebuconazole, propiconazole, and benalaxyl) using CM-β-CD as chiral selector was performed. First of all, a univariate optimization of variables (CM-β-CD injection time, voltage, temperature, pH, and buffer nature and concentration) was carried out for benalaxyl. Finally a screening, involving the effect of the CM-β-CD injection time, was performed for the other pesticides to check the potential of this CD as chiral selector.
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Study of α-bromophenylacetic acid suitability as a model analyte for chiral separations using capillary electrophoresis as a separation technique II
Bubáková, Zuzana ; Jáč, Pavel (advisor) ; Lochman, Lukáš (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Zuzana Bubáková Supervisor: PharmDr. Pavel Jáč, Ph.D. Title of diploma thesis: Study of α-bromophenylacetic acid suitability as a model analyte for chiral separations using capillary electrophoresis as a separation technique II Rizvi and Shamsi (Rizvi S.A.A., Shamsi S. A. Anal. Chem. 2006, 78(19), 7061-7069) reported the employment of α-bromophenylacetic acid (BPAA) dissolved in water/methanol mixture (1:1, v/v) as a model analyte for chiral separations in capillary electrophoresis (CE). However, the stability of BPAA in aqueous methanol is questionable (Kováčová G. 2018. Diploma thesis. Charles University, Faculty of Pharmacy). The aim of this work was to determine the reaction order of the decomposition of BPAA (c = 0.47 mM) in 50% aqueous methanol. The reaction kinetic study was performed by capillary electrophoresis in 50 μm (i.d.) poly(vinyl alcohol)-coated capillary (30 cm/24.5 cm) with UV detection. The BPAA and products of nucleophilic substitution (mandelic acid, α-methoxyphenylacetic acid, and Br-) were separated in 60 mM formate buffer (pH 3.0) at -30 kV; the detection was 200 nm. The first order reaction kinetics was confirmed by linear and non-linear regression, yielding the rate...
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Preparation of a method for detection of the extent of chelation of calcium and magnesium ions
Konečný, Lukáš ; Mladěnka, Přemysl (advisor) ; Jáč, Pavel (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmacology and Toxicology Candidate: Lukáš Konečný Supervisor: prof. Přemysl Mladěnka, Pharm.D., Ph.D. Title of Thesis: Preparation of a method for detection of the extent of chelation of calcium and magnesium ions Calcium and magnesium are biogenic elements belonging to the second group of the periodic table. Calcium is the fifth most abundant element in the human body. In addition to its important role in bone homeostasis, calcium is involved in other processes such as blood clotting, muscle contraction and regulation of nerve excitability Magnesium is the cofactor of many enzymatic reactions essential for a row of physiological functions. The aim of this work was to propose a novel screening method for determination of the degree of calcium and magnesium chelation. In the first step the UV-vis spectra of the selected indicator o-cresolphthalein complexone and its complexes with calcium and magnesium were measured. This was followed by verification of the linearity and the determination of the sensitivity of the method. Concurrently the stability of the indicator and its complexes with both ions was measured. In the last step, the suitability of the method was verified on a standard chelator ethylenediaminetetraacetic acid...
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Testing of potential capability of chiral ionic liquids with long alkyl chain in capillary electrophoresis chiral separations II
Müllerová, Kateřina ; Jáč, Pavel (advisor) ; Lochman, Lukáš (referee)
Department: Department of Analytical Chemistry Candidate: Kateřina Műllerová Supervisor: PharmDr. Pavel Jáč, Ph.D. Title of the diploma thesis: Testing of potential capability of chiral ionic liquids with long alkyl chain in capillary electrophoresis chiral separations II Chiral ionic liquid (−)-N-dodecyl-N-methylephedrinium bromide (DMEB) was tested as a selector for chiral separations of selected drugs such as chiral quinolones, ketoprofen, and flurbiprofen by capillary electrophoresis (CE). The effect of several parameters on enantioseparation was examined: (i) type and pH of the separation buffer, (ii) type and amount of organic modifier, and (iii) the concentration of DMEB. When using this chiral selector, only the separation of ofloxacin enantiomers was observed, while the enantiorecognition of other chiral model analytes was not successful under the conditions tested. As a next step, a CE method for the assay of levofloxacin was developed to demonstrate the potential of DMEB as a chiral selector in quality control of single-isomer drugs. The best separation was achieved with 20 mM tris buffer of pH 8.5 containing 100 mM DMEB and 20 % (v/v) of acetonitrile as the background electrolyte. The separation took place in 50 µm id fused silica capillary (80.5 cm / 72 cm) at −30 kV with UV detection...
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Development of CE-C4D method for the analysis of saccharides in honey
Šebestová, Gabriela ; Jáč, Pavel (advisor) ; Šatínský, Dalibor (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Bc. Gabriela Šebestová Supervisor: PharmDr. Pavel Jáč, Ph.D. Title of the diploma thesis: Development of CE-C4D method for the analysis of saccharides in honey Honey is a commonly used natural mixture which quality must be monitored due to possible mishandling or adulteration. This diploma thesis is focused on the development of a capillary electrophoresis method with capacitively coupled contactless conductivity detection (CE-C4D) for the determination of carbohydrates in honey samples. Design of Experiments (DoE) was used to optimize the separation conditions. This approach allows to find suitable separation conditions by means of multivariate optimization. During the optimization, the impact of the following factors was investigated: the composition of the background electrolyte, the separation voltage and temperature during separation and the inner diameter of the separation capillary. Two methods were optimized: one with normal electroosmotic flow (EOF) and the other using reversed EOF after CTAB addition. The method in the normal EOF mode was used for validation and analysis of honey samples. A fused silica capillary with an inner diameter of 20 m, a total length of 48.7 cm and an effective...
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Peptide Mapping of Biopharmaceuticals Using CE
Kosolapov, Dmytro ; Jáč, Pavel (advisor) ; Khalikova, Maria (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Dmytro Kosolapov Supervisor: PharmDr. Pavel Jáč, PhD. Consultant: PharmDr. Juraj Lenčo, Ph.D. Title of graduation thesis: Peptide Mapping of Biopharmaceuticals Using CE This work focuses on optimization of capillary electrophoresis (CE) method for the peptide mapping. and enzymatic digestion of proteins using trypsin. Myoglobin from equine heart and a reference antibody were used as substrates. Two digestion approaches were compared: a conventional tryptic digestion in solution and digestion with a commercial SMART Digest Trypsin Kit that is based on immobilized trypsin. The digestion was monitored with RP-HPLC. After a short optimization of tryptic digestion, a number of experiments were carried out to determine the optimal conditions for peptide mapping by means of CE. All the measurements were carried out using a PVA-coated capillary of 50 µm internal diameter, effective length of 56 cm and total length of 64.5 cm. Samples were applied hydrodynamically at a pressure of 50 mbar, the separation voltage was 30 kV, the temperature of the capillary was set to 20řC. The analytes were detected with UV-VIS detector at 200 nm. The effect of BGE type and concentration (30, 60, 90, 120, 150 mM formic acid...
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The capabilities of capillary electrophoresis in the separation of boswellic acids
Poušková, Jitka ; Jáč, Pavel (advisor) ; Polášek, Miroslav (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Bc. Jitka Poušková Supervisor: PharmDr. Pavel Jáč, Ph.D. Title of the diploma thesis: The capabilities of capillary electrophoresis in the separation of boswellic acids The main aim of this work was testing of capabilities of capillary electrophoresis for the separation of boswellic acids in Boswellia serrata extract as well as in a mixture of standards. The effect of five different buffers and some additive (such as cyclodextrins, organic modifiers or surfactants) in the background electrolyte was tested. The most suitable components of the background electrolyte, were examined at different concentrations to achieve complete separation of boswellic acids. First experiments were carried out in a fused silica capillary with 50 μm internal diameter, 48.5 cm total length and 40 cm effective length, followed by the separations in a longer capillary with 83.5 cm total length and 75 cm effective length. The separation voltage was +30 kV. The UV detection was at 200 nm, 250 nm and 280 nm. The best results were achieved with the background electrolyte composed of 80 mM phosphate buffer at pH 7.0 with 10 % (v/v) of methanol and 80 mg/ml of 2-hydroxypropyl-β- cyclodextrin. With this electrolyte, Boswellia...
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