|
|
Optimization of 5-fluorouracil determination by high-performance liquid chromatography
Durychová, Eva ; Křížek, Tomáš (advisor) ; Kozlík, Petr (referee)
This thesis is dedicated to the development of a method for the determination of 5-fluorouracil by high-performance liquid chromatography, which could be used for determination of its encapsulation efficiency by liposomes. First, separation of 1 mM standard of 5-fluorouracil was tested on several types of columns. Among tested columns belonged a C18 column, a C18 column with positive surface modification, a phenyl-hexyl column and several fluoride columns together with two HILIC columns. A mixture containing 10 mM CH3COONH4, pH = 4,5 and MeOH in the ratio of 98/2 was initially used as the mobile phase. The results showed, that 5-fluorouracil elutes on all columns too close to the dead time, where different impurities often elute. To avoid possible distortion of 5-fluorouracil signal by impurities potentially occurring in the real sample, several adjustments of chromatographic conditions were tested. The most effective solution was addition of 5 mM ion-pairing agent (namely tetrabutylammonium chloride hydrate) to the mobile phase, together with adjusting pH to 8,0 to support the ionization of the analyte. In the combination with mobile phase modified in this way, a phenyl-hexyl column with retention time 4,36 minutes reached the best result, therefore it was selected for the final determination of...
|
|
|
Electrophoretic determination of oxidized and reduced form of glutathion in plant material
Durychová, Eva ; Křížek, Tomáš (advisor) ; Kubíčková, Anna (referee)
This work is based on a previously published method for the determination of twenty proteinogenic amino acids by capillary electrophoresis with contactless conductivity detection and deals with possibility of its use for the determination of oxidized (GSSG) and reduced (GSH) forms of glutathione with amino acids. The theoretical part firstly deals with brief introduction of the properties and importance of glutathione and its protection against autooxidation, especially by alkylation with N-ethylmaleimide (NEM), which binds to GSH to form its alkylated form (GSH-NEM). Then, a brief summary of the available methods for determination of glutathione is given, with a focus on the already developed capillary zone electrophoresis methods. Finally, it briefly presents the electrophoretic method for the determination of twenty proteinogenic amino acids on which this work is based. In the experimental part, the integration of the determination of both forms of glutathione into the method for the determination of proteinogenic amino acids is presented. First, a simplified, less time consuming method was tested in shorter 50.0 cm capillary without hydroxyethylcellulose, where both forms of glutathione were separated at baseline within 10 minutes. Relative standard deviations of peak areas (GSH 2.1%; GSSG...
|
guest ::
