|
|
DEVELOPMENT AND OPTIMIZATION OF PACKING CHROMATOGRAPHIC COLUMNS
Černá, Kateřina ; Kozlík, Petr (advisor) ; Kubíčková, Anna (referee)
The aim of this thesis was to develop and optimize the packing of a new series of chromatographic columns called Astra with subsequent analytical measurements to determine the quality of optimized packing processes, the quality of the columns and to investigate the behavior of stationary phases. The packing processes for the following columns were developed and optimized - Astra C18 HE, AQ and DM 5 µm (250/150/100×4,6 mm, 150/100×3,0 mm), Astra C18-HE 3 µm (250/150/100×4,6 mm) , 150/100/50×3,0 mm, 150/100/75/50×2,1 mm), Astra AQ 3 µm (150/100×4,6 mm, 150/100/50x3,0 mm, 150/100/75/50×2,1 mm), Astra DM 3µm (150/100×4,6 mm, 150/100x3,0 mm, 150/100/50×2,1 mm) and for zwitterionic columns 3µm (150×4,6 mm, 100×2,1 mm). During the development of the packing processes, the following packing parameters were optimized. Composition of the suspension solution (slurry) - cyclohexanol:chloroform (1:10) for SF C18, AQ, DM and glycerin:methanol (1:3) for zwitterionic SF; concentration of the packing suspension; column pre-filling method - pre-filling with packing liquid (methanol); PID parameters, the values P = 3000, I = 100 and D = 100 were chosen; width of the pre-column, which must be equal to or greater than the inside diameter of the column; packing pressure and time; the column hardware type was chosen...
|
|
|
Analysis of historical pharmaceutical preparation containing chicory from the 18th century
Lener, Tomáš ; Nesměrák, Karel (advisor) ; Kubíčková, Anna (referee)
A historical pharmaceutical preparation - chicory extract - from the 18th century whose authenticity was investigated was qualitatively analyzed using HPLC with UV/VIS and MS2 detection. A current dry chicory root was used as a source of selected chicory chemotaxonomic markers and an indicator of the success of extraction with different solvents. The authenticity of the historical extract was proved by a water solubility test. A VIS spectrometric determination of inulin was used and the inuline content was determined to be 52.4% in the current dry chicory root and 7.45% in the historical extract. The detection wavelength 254 and 320 nm was selected from measured UV/VIS spectra of selected standards and the literature. The authenticity of the historical chicory extract was proven by tandem mass spectrometry, and, moreover, arteincultone - a new chicory chemotaxonomic marker - was identified. Key words: HPLC-MS, HPLC-UV/VIS, UV/VIS Spectrometry, Chicory, Historical extract, Chicory root, Arteincultone
|
|
|
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...
|
|
|
The study of the electrochemical and chemical oxidative degradation of salicylic and acetylsalicylic acid
Tonnerová, Barbora ; Kubíčková, Anna (advisor) ; Křížek, Tomáš (referee)
The aim of this Thesis was to study the degradation of salicylic acid by the electrochemical oxidation. The electrochemical oxidation of salicylic acid was compared to the electrochemical oxidation of acetylsalicylic acid. Two electrochemical cells were tested - batch cell and thin- layer flow-cell. The newly developed and validated method of ultra-high performance liquid chromatography has been used to analyse the degradation products, salicylic acid and acetylsalicylic acid. The optimal analysis was made by the Kinetex column C18 (2,1 x 100 mm; 1,7 µm) and mobile phase with two components - acetonitrile and water with addition of 0,1% formic acid. These two components of the mobile phase were mixed by the gradient program from 10 % to 60 % (v/v) acetonitrile. The mobile phase flow was set to 0,3 ml min-1 and the volume of injection was 2 µl. The detection was performed by photodiode array detector at the wavelength 240 nm. Total time of the analysis was 11 minutes. The electrochemical degradation has been studied by an anodic oxidation in a flow cell with a boron-doped diamond (BDD) anode and a stainless-steel cathode. The samples of salicylic acid and acetylsalicylic acid was dissolved in the ammonium acetate, concentration 200 mmol dm-3 at pH=4,0. The 20 % oxidation rate of salicylic acid was...
|
|
|
Study of degradation behavior of abacavir using a diamond film electrode
Pražáková, Lucie ; Kubíčková, Anna (advisor) ; Kozlík, Petr (referee)
In this work the electrochemical degradation behavior of the drug abacavir is studied with the use of boron-doped diamond electrode. Oxidative degradation of the active substance abacavir, used to treat patients with HIV disease, was studied in a batch of three-electrode and two-electrode settings. In this work, the characteristics of five working boron-doped diamond electrodes, differing in a boron/carbon doping ratio of 500 ppm, 1000 ppm, 2000 ppm, 4000 ppm and 8000 ppm, were studied. The degradation in the three-electrode arrangement took place under potenciostatic electrolysis at potentials from 0,6 V to 2,0 V. The degradation in the two-electrode arrangement took place under conditions of potenciostatic electrolysis at higher potentials from 2,0 to 4,5 V. Optimal degradation, to the required values up to 25 %, was achieved at a potential of 3,5 V for an oxidation time of 10 minutes on a boron-doped diamond electrode with a boron/carbon doping ratio of 500 ppm and 1000 ppm. Separation and determination of abacavir and its degradation products were performed by ultra-high performance liquid chromatography with a mass detector. The degradation of abacavir produced two known degradation products, DP1 with relative retention times of 0,40 and DP2 with relative retention times of 0,45. The...
|
|
| |
|
| |
|
|
Characterization of new HILIC stationary phases
Vaclová, Jana ; Kozlík, Petr (advisor) ; Kubíčková, Anna (referee)
Hydrophilic interaction liquid chromatography is a frequently used separation method for analysis of polar compounds. It is an alternative method to reversed-phase chromatography, where these compounds show insufficient or very weak retention. A high number of stationary phases are currently available for HILIC and new ones are still being developed. The aim of this diploma thesis was to characterize and compare three relatively new HILIC columns containing an unmodified silica gel (HILIC-A), aminopropyl modified silica (HILIC-B) and polyhydroxyl chain modified silica (HILIC-N) as stationary phase. Based on the study of the effect of acetonitrile content in mobile phase on the retention of a model set of peptides, a multimodal retention mechanism was demonstrated. Analysis of 18 model analytes with different pKa values showed, that the composition of aqueous part of mobile phase (buffer), more specifically it's concentration and pH value, has a significant impact on retention of ionized analytes and peptides on the studied stationary phases. A significant contribution of ionic interactions to retention was observed on HILIC-B and HILIC-A columns. The retention of basic compounds on the HILIC-B column increased with increasing ionic strength, while it decreased on the HILIC-A column. Increasing the...
|
|
|
HPLC study of pharmacological coupling reactions
Krouská, Hana ; Kubíčková, Anna (advisor) ; Kozlík, Petr (referee)
In this work decomposition of phenylisothiocyanate in aqueous and methanolic solutions was studied. RP-HPLC with UV detection was chosen to monitor these reactions. A simple method of isocratic elution has been developed. Due to a long total analysis time, it was necessary to develop also gradient elution method. Using these methods degradation rate of phenylisothiocyanate in methanol and in an aqueous solutions were monitored. Time course of decomposition exhibits exponential shape that is typice for the first-order kinetics. The measurements also showed that disintegration of phenylisothiocyanate is slower in water than in methanol. In both cases it is very slow - decomposition of half of the original quantity of phenylisothiocyanate occurs within days.
|
|
|
HPLC study of chemical reactions
Krouská, Hana ; Kubíčková, Anna (advisor) ; Kozlík, Petr (referee)
In this work hydrolysis of phenylisothiocyanate and subsequent decomposition of diphenylthiourea were studied. RP-HPLC with UV detection was selected for monitoring of these reactions. The new method of isocratic elution was developed and mobile phase composition and flow rate were optimized. Hydrolysis of isothiocyanate was studied as a function of pH and reaction temperature. The subsequent decomposition of diphenylthiourea was carried out at 80 řC depending on the pH.
|
guest ::
RSS feed.