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Determination of non-esterified fatty acids in plasma
Lacinová, Petra ; Pacáková, Věra (advisor) ; Staňková, Barbora (referee)
The determination of non-esterified fatty acids (NEFA) in plasma is possible by many routine biochemical methods. For more detailed metabolic studies it is required to analyse the fatty acid (FA) profile. The NEFA profile determination is usually performed by the capillary gas chromatography (GC), but the sample preparation is not uniform. NEFA may be isolated by the extraction process or by the preparative thin-layer chromatography (TLC). The aim of this study was to compare these two separation procedures. The samples of pooled plasma from volunteer donors were analyzed by capillary GC after previous separation procedure by the TLC and the liquid-liquid extraction. The results were compared by the t-test for both the absolute concentration of individual FA provided by the internal standard (margaric acid) method and the relative abundance (Rel%). The reproducibility of the results was significantly better for the liquid-liquid extraction method than the TLC. The relative standard deviations (RSD) of the FA groups (unsaturated, monosaturated and polysaturated) were from 4.3 to 11.3 % vs. 8.6 to 33.8 % for the relative abundance and from 16.5 to 25.5% vs. 15.4 to 47.4% for the absolute concentration. For each FA, which were represented by more than 0.1 Rel %, RSD ranged from 4.5 to 47.8% vs. 9.5 to...
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Polymethacrylate monolithic columns in capillary liquid chromatography
Grafnetter, Jan ; Coufal, Pavel (advisor) ; Pacáková, Věra (referee) ; Ventura, Karel (referee) ; Sýkora, David (referee)
3 Conclusions This Thesis gives chromatographic and structural characteristics of the butyl methacrylate monolithic columns prepared for the capillary liquid chromatography. Results of this Thesis clearly demonstrate the possibility of preparation of capillary monolithic columns with a good repeatability of the principal chromatographic parameters. A completely new technique for preparation of the monolithic columns for use in the on-column detection mode was introduced. Different photometric detection modes were applied to monolithic capillary columns in CLC. The advantages and disadvantages of the on-column detection mode in comparison to the additional detection capillary were studied and described. It was shown that the best separation and the most sensitive quantification in the short analysis time could be reached by using the monolithic columns with detection window placed directry on the separation capillary, just after the stationary phase or within the monolith. The attempts leading to preparation of chiral monolithic stationary phases, which would allow enantioseparation of selected chiral analýes' were not totally successful. No time was left to find the suitable chromatographic conditions for enantioseparations. \ I
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Pharmaceutical application of high performance ion chromatography in analysis of organic anions and cations
Čujová, Sabína ; Pacáková, Věra (advisor) ; Čabala, Radomír (referee)
The thesis is focused on application of ion chromatography in pharmaceutical analyses of organic ions. Ion chromatography is increasingly used in the field of pharmaceutical analysis. This includes the analysis of impurities and metabolites. In the first part of this thesis, ion chromatography is compared with common separation techniques used in pharmacy, such as gas chromatography and high performance liquid chromatography. In the second part development and validation of methods of ion chromatography for purity evaluation and quality control of active pharmaceutical substances Rivastigmine hemitartrate and Pramipexole hydrochloride were carried out. Key words: ion chromatography, reversed-phase chromatography, ion-pair chromatography, ion-exclusion chromatography, ion-exchange chromatography, GC, HPLC
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Determination of pepsinogens using IgY and IgG
Kulhavá, Lucie ; Pacáková, Věra (advisor) ; Tichá, Marie (referee)
A decreased concentration of pepsinogen A in serum was found to be a marker of gastric cancer, similarly as a low ratio of pepsinogen A to pepsinogen C. In the present study we have compared properties of immunoglobulin fraction isolated from the egg yolks after immunization of laying hens with pepsinogen isolated from porcine gastric mucosa with those of present in rabbit antiserum obtained after the animal immunization with the same antigen. The characteristics of chicken antibodies against porcine pepsinogen and the comparison with rabbit antibodies raised against the same antigen was carried out using the following methods: ELISA, affinity chromatography on immobilzed antigen and bovine serum albumin, SDS and native electrophoresis and MALDI-TOF MS/MS. While the rabbit specific antibodies interacted with the used antigen and only slightly with bovine serum albumin and there was a diference between pre-immune IgG and specific IgG, in the case of chicken antibodies IgY it did not work. No diference was observed between ELISA tests performed with pre-immune serum and the serum after immunization with porcine pepsinogen and a high interaction of IgY with bovine serum albumin in pre-immune serum and specific IgY after the immunization were detected. Similar results were obtained in experiments with...
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Analysis of biologically active substances by modern separation methods
Bierhanzl, Václav ; Čabala, Radomír (advisor) ; Pacáková, Věra (referee) ; Sýkora, David (referee)
The thesis is dedicated to the phospholipids and their polar headgroups analysis by gas chromatography, capillary electrophoresis and mass spectrometry. Phospholipids are the most important polar lipids and they are classified into phospholipid classes according to their phosphorylated groups. Phospholipids can be found in cell membranes and the changes in their ratio are monitored to research the impact of external conditions on cells. Actually thin layer chromatography is still used for phospholipid class ratio analyses. It is not suitable for microbiological research due to its time demandingness. The presented compendium of papers engaged in phospholipid classification is targeted on Bacillus subtilis strain, which produces potential antibiotics with detergent effect - surfactin. Published methods can be used for research of optimal conditions for producing microbe cultivation. Because non-polar parts of the phospholipid molecule (fatty acids) can affect the analysis methods on spliced polar headgroups have to be designed. Capillary electrophoresis and gas chromatography methods were developed and the latter one was further optimized for simultaneous analysis with fatty acids. Additional part deals with an alternative approach which consists in direct injection on mass spectrometer of intact...
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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...
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