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Mobilization of electroosmotic flow markers in capillary electrophoresis
Martínková, Eva ; Křížek, Tomáš (advisor) ; Sobotníková, Jana (referee)
Capillary zone electrophoresis is a separation method used to separate charged substances in electric field. Substances migrate to detector due to their own electrophoretic mobility and electoosmotic flow in capillary. Separation is possible because of electroforetic mobility of substances, which is directly proportional to the charge and undirectly proportional to the hydrated radius of ion. This means that neutral molecules form just one neutral zone, which is detected together. Well absorbing neutral substances are thus used as markers of average (mean) EOF. It was found, that some of the markers have dispositions to be mobilized with respect to the EOF. This mobilization is caused by interactions of free electron pair on atoms of nitrogen or oxygen of marker with components of background buffer. The aim of the study was to measure mobilization of some commonly used markers with different cations of acetate buffer and thus find "ideal" marker, whose mobility is almost the same as EOF and find combinations of marker+buffer, which are better to be avoided. Eleven markers in combination with six cations of background electrolyte were studied. We have found, that relative mobilities of some of chosen markers come up to five percent of limit mobilities of small inorganic cations. An appropriate...
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Quantitative Structure-Retention Relationships of Salicylthioamide Derivatives
Kalužíková, Aneta ; Nesměrák, Karel (advisor) ; Sobotníková, Jana (referee)
The aim of the thesis is to study the relationship between the structure of N-benzylsalicylthioamide derivatives and their retention behavior in high-pressure liquid chromatography. Furthermore, the influence of various substituents on electron absorption spectra in ultraviolet spectral range was studied. All studied derivatives have two absorption maxima in ultraviolet spectral range at 260 nm and 293 nm. The substitution of an auxochrome on the acyl ring leads to bathrochromic shift and hyperchromic shift, especially of the first absorption maxima. The presence of an auxochrome on the amide ring leads to hyperchromic shift as well. HPLC reversed-phase chromatography, using XDB-C18 ZORBAX column and mobile phase with various composition of acetonitrile, was used to obtain retention times of the derivatives. Using this data, the correlation equations between retention factor (mobile phase with no acetonitrile), resp. slope of the dependence of the logarithm of retention factor on the volume fracture of acetonitrile in mobile phase, and partition coefficient octanol-water, resp. hydrophobic substituent constants, were derived. Key words: QSAR, HPLC, spectrometry, benzylsalicylthioamides
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Various methods of preparation of capillary columns packed with Sephadex for gel chromatography
Tolasz, Jakub ; Coufal, Pavel (advisor) ; Sobotníková, Jana (referee)
In this diploma thesis, capillary columns with an inner diameter of 530 microns filled with gel particles were prepared. These columns are intended for gel chromatography. Three various methods of filling were used for their preparation. The first method was based on filling of wet particles in suspension with the organic solvent, followed by drying and swelling of particles with water into the form of gel. The second method used filling of wet particles in the suspension form with the aqueous phase. The last method started with suction of dry gel particles by air and continued with their subsequent swelling with water into the form of the gel. The prepared columns have been compared analysing hyaluronic acid and thiourea. The basic parameters specified for the gel column chromatography were determined by using phenylalanine. The pressure characteristics depending on the flow rate of mobile phase were used to compare the quality of packing of the columns prepared by various methods.
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Determination of Organic Acids in Beer by HPLC method
Kukleva, Ekaterina ; Bosáková, Zuzana (advisor) ; Sobotníková, Jana (referee)
Kukleva Ekaterina Abstract Optimization and validation of HPLC method with RI or UV detection system for determination of six major organic acids in beer was made. Beer is very complicated matrix, therefore, the samples needs pretreatment using SPE. For solid phase extraction Algient Bond-Elute SAX (SAX) and Supelco ENVI-Carb (CB) cartridges were used and compared. Differences between those two cartridges is in their principle of pretreatment. While on SAX cartridge dissociated organic acid ions will be retained and interfere substances not, on CB cartridge interfering carbohydrates will be retained instead of organic acid ions, which will get through sorbent. Recoveries for organic acids on SAX and CB cartridges were80 - 110 % and 20 - 50 %, respectively. For determination of six organic acids, by name citric, malic, succinic, lactic, acetic and oxalic, Eurokat H (Knauer) HPLC column was used with 0.01 N sulfuric acid as a mobile phase. Time of analysis was 25 minutes per sample. Organic acid ions were detected using Refractive Index (RI) and Ulraviolet (UV) detectors. Peak resolution was 1,2 - 1,8 and 1,1 - 1,6 for RI and UV detection, respectively.
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Preparation and testing of capillary packed column sephadex G-10
Tolasz, Jakub ; Sobotníková, Jana (referee) ; Coufal, Pavel (advisor)
Sephadex is a common sorbent in biochemistry for separation of molecules of a wide range of molecular masses. Sephadex is also used for purification or desalination of isolated proteins. Up to now, the gel chromatography has been performed in wide separation columns. Therefore, the isolation techniques for proteins had to be very effective to isolate enough proteins as a sufficient amount of the sample. The preparation and application of a capillary separation column packed with sephadex could decrease the needed sample amount and enable work with hardly isolable proteins obtained only in small quantities. In this bachelor project, the capillary column was packed with sephadex using the slurry packing procedure. The principle of monolithic columns was used to create a gel in the column. The column was packed in organic solvent firs, and then hydrated when flushing with water to generate the gel.
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Modern reverse stationary phases based on silicagel, zirconium dioxide and organic monoliths: application to analysis of biologically active compounds
Soukupová, Klára ; Sobotníková, Jana (advisor) ; Pacáková, Věra (referee) ; Cvačka, Josef (referee)
Charles University in Prague Faculty of Science Department of Analytical Chemistry Modern Reversed Stationary Phases Based on Silicagel, Zirconium Dioxide and Organic Monoliths; Their Used in Separation of Biologically Active Compounds Synopsis of PhD. Thesis Prague 2008 Klára Soukupová The PhD. Thesis was carried out at the Department of Analytical Chemistry, Faculty of Science, Charles University in Prague, in the period of 2003-2008. Author: Mgr. Klára Soukupová Supervisors: RNDr. Jana Suchánková-Sobotníková, Ph.D Doc. RNDr. Eva Tesařová, CSc. Prague 2008 1 Table of contents 1. Introduction 2 2. Objectives of the Thesis 3 3. Results and Discussion 4 4. Conclusions 12 5. References 13 Appendix 1: List of Publications, Lectures and Poster presentations 14 Appendix 2: Curriculum Vitae 16 2 1. Introduction High performance liquid chromatography (HPLC) is modern instrumental method used mainly in analytical chemistry. Most used separation mode is reversed phase liquid chromatography (RP HPLC). This method is very useful because of high separation efficiency, reliability and wide choice of stationary phases with different selectivity. Most used stationary phases in RP HPLC are bonded phases, which have many advantages: good availability, wide application range, fast establishment of equilibrium and ability to...
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