|
|
The use of a capillary electrophoresis and capillary electrophoresis on chip for clinical research
Pavlíček, Václav ; Tůma, Petr (advisor) ; Pacáková, Věra (referee) ; Dlouhý, Pavel (referee)
Present the dissertation thesis on the topic "The use of a capillary electrophoresis and electrophoresis on chip for clinical research" is divided into two separate parts. The first chapter summarizes the problems of development new method of very rapid determination of uric acid and creatinine using capillary electrophoresis in human urine and serum, when the entirely original solution has been developed for implementation of very fast electrophoretic separations in a commercial electrophoretic apparatus. The separations were performed in a laboratory made capillary formed by connecting a 9.7 cm long separation capillary with inner diameter (d) 25 µm with an auxiliary 22.9 cm long capillary with d 100 µm. The coupled capillary is characterized by the effective separation path 8.3 cm (short-end injection mode) and the high electric field intensity around 2.3 kV per cm. The coupled capillary was tested at several high-speed analysis; i) the determination of creatinine in human urine, background electrolyte - 20 mM citric acid/NaOH, pH 3.0; ii) the determination of uric acid in human urine, background electrolyte - 20 mM MES/NaOH, pH 6.0. Under these experimental conditions, migration times of 12.2 s for creatinine and 8.6 s for uric acid were achieved and the attained separation efficiency was...
|
|
|
SPE optimization for determination of perfluorinated organic acids
Ševčík, Václav ; Čabala, Radomír (advisor) ; Pacáková, Věra (referee)
This thesis is focused on optimization of preparation of aqueous environmental samples for determination of persistent pollutants - perfluorinated organic acids, which are potentially dangerous for living organisms. The goal of the thesis is optimization of SPE conditions for preconcentration of these compounds for their determination by GC-MS. Perfluorinated organic acids with carbon chain lenghts of C6-C12 were selected as analytes. Effect of sample pH value, effect of type and elution solvent volume, addition of indifferent salt and ion pair reagent have been studied during the optimization. The results show that the extraction efficiency depends on analyte carbon chain length and reaches values from 75 to 110 % for C6 - C8 and 55 to 95 % for C9 - C12 acids. The overall increase in extraction efficiency was more pronounced for acids with shorter chain length (for C6 up to ten times), whereas for long chain acids the improvement was only moderate.
|
|
|
Immobilization of protease V8 on magnetic particles for application to proteolytic cleavage of pepsin A
Čepa, Adam ; Pacáková, Věra (advisor) ; Tichá, Marie (referee)
This thesis is part of a long-standing research in the field of diagnosis of the stomach diseases, which is based on the gastric enzyme pepsin A mapping. It was found that a phosphorylation in the primary structure of this enzyme may serve as a marker of incipient stage of carcinogenesis. This thesis is focused on the immobilization of protease V8 isolated from microorganism Stafylococcus aureus to magnetic agarose beads. Protease V8 is a promising candidate for producing peptide maps of pepsin A. The influence of pH, temperature and reaction time on the enzyme to activity has been studied and the optimal conditions for hydrolytic catalysis of formation of peptide fragments of pepsin A.
|
|
|
Study of influence of 17β-estradiol and 17α-ethynylestradiol on capacitation of mouse sperm in vitro.
Adamusová, Hana ; Bosáková, Zuzana (advisor) ; Pacáková, Věra (referee)
Environmental estrogens (natural and synthetic) belong to a group of contaminants called endocrine disrupting chemicals (EDCs). These compounds interfere with a function of endocrine system. They can bind to natural estrogen receptors or block synthesis of endogenous hormones. 17β-Estradiol (BE2) is a natural endocrine disruptor produced more by women than men. Also a synthetic 17α-ethynylestradiol (EE2) belongs to EDCs. It is used as a main part of birth control pills. Together with other endocrine disruptors EE2 is probably responsible for feminization of male animals living in effluents of waste water treatment plants. The aim of this thesis was to develop a method for determining free 17β-estradiol and 17α-ethynylestradiol available to capacitating sperm. In order to determine a status of BE2 during mouse sperm capacitation in vitro, a high performance liquid chromatography (HPLC) method with tandem mass spectrometry (MS/MS) detection was used. A reversed-phase separation mode using a SunFire C18 column with a simple mobile phase composed of acetonitrile and water at the ratio 40/60 (v/v) containing 0,1% formic acid in both components was applied. Under the optimized separation conditions, calibration curves for 17β-estradiol and 17α-ethynylestradiol were measured in the concentration range of 1-250...
|
|
|
Characterization of poly(1,4-diethynylbenzene) by IGC method
Petrášová, Sabina ; Sedláček, Jan (advisor) ; Pacáková, Věra (referee)
Poly(1,4-diethynylbenzene) ( -conjugated polymer) was prepared as an insoluble polymer network via chain coordination polymerization of 1,4-diethynylbezene catalyzed with [Rh(NBD)acac] complex. Thermodynamic properties and acid-base characteristics of the prepared poly(1,4-diethynylbenzene) were studied by means of Inverse Gas Chromatography (IGC) in the temperature range 80-100 řC. Retention data of selected testing substances were used to determine the Gibbs energy of sorption, the sorption enthalpy and their acid-base and disperse parts as well as the disperse contribution to the surface energy and parameters of KA, KD, ANHPS and DNHPS quantifying the acid-base character of the studied polymer. The results showed that poly(1,4-diethynylbenzene) interacted more efficiently with Lewis bases than with Lewis acids. The values of experimental sorption enthalpy were used for the determination of the parameters KA and KD. Values of these parameters classify poly(1,4-diethynylbenzene) as the material with a slightly acid character. This conclusion is further supported by the results of H. P. Schreiber method based on the application of ANHPS and DNHPS parameters for the evaluation of the acid-base properties of the material. The infrared spectroscopy proved that poly(1,4-diethynylbenzene) contained...
|
|
|
Development and Application of High Performance Separation Methods in Connective Tissue Analysis
Braun, Martin ; Štulík, Karel (advisor) ; Pacáková, Věra (referee) ; Samcová, Eva (referee)
24 Annotation: This work is concerned with the development and application of high performance separation methods suitable for determination of clinically considerable fragments of collagen and elastin as the most important proteins of connective tissues. For their analyses, various chromatographic and electrophoretic methods have been used, including high performance liquid chromatography (HPLC), capillary zone electrophoresis (CZE) and micellar electrokinetic capillary chromatography (MEKC). This work has aimed at optimization of the developed and modified analytical separation procedures and methods and their utilization for determination of clinically important markers of connective tissue degradation. The key validation parameters of the analytical methods used in this work were evaluated too, which enabled their comparison with other approaches. One of the most important results of this work is thus high sensitivity, precision, repeatability and robustness of the methods which was found in a number of biological sample types. Successful was also very good HPLC separation of the analyzed cross-links in both collagen and elastin tissues. High performance methods are described which have been suggested and optimized for separation and quantitative determination of pentosidine, pyridinolines and...
|
|
| |
|
| |
|
| |
|
| |
guest ::
