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Determination of tryptamine and tryptophan in biological material by capillary electrophoresis
Šimonová, Alice ; Křížek, Tomáš (advisor) ; Taraba, Lukáš (referee)
The aim of this bachelor thesis was the development of the method for the determination of tryptamine and tryptophan in biological material by capillary electrophoresis. Total length of silica capillary with inner diameter 75 m was 50.0 cm and effective length was 8.5 cm, injection of the sample was on the short end of the capillary. The composition of background electrolyte, injection method and on-line preconcentration techniques were optimized. Background electrolyte was acetic acid of 1,6 mol/l concentration, sample was injected hydrodynamically with pressure of 5 kPa for 5 s and driving voltage was -30 kV. The analytes were detected at wavelength of 220 nm and the inner standard aniline was detected at wavelength of 200 nm. Time of separation was only 2 minutes, which is the main advantage of this method. The limits of detection were 0.002 mmol/l for tryptamine and 0.001 mmol/l for tryptophan, the limits of quantification were 0.006 mmol/l for tryptamine and 0.005 mmol/l for tryptophan. Repeatability of peak areas and migration times of standards of 0,045 mmol/l concentration related to inner standard showed values of relative standard deviation lower than 5 %. The use of optimized method was tested on Nicotiana tabacum leaves and on cultivating medium of oomycete Pythium oligandrum. Key...
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Indirect determination of heparin by capillary electrophoresis
Filounová, Barbora ; Křížek, Tomáš (advisor) ; Kubíčková, Anna (referee)
Heparin is a mixture of sulfonated polysaccharides which is negatively charged. Heparin is a substance which is important in organism and fundamentally affects its physiology. Main attribute of heparin is anticoagulation - it prevents the complete blood coagulation. This anticoagulant effect balances the hemocoagulation by influencing the coagulation pathway. In some cases a pharmacological application of heparin is needed so the heparin is administrated as a injection of physiological solution of sodium or calcium heparine salt. Monitoring of level of the heparin in blood is problematic - methods used today are based on the measurement of a time required for blood clot formation. The result evaluation is done by comparing a sample with reference solution. These methods are relatively imprecise, can not be used in "on-line" setting and are highly influenced by general health condition of patient. In this work some principles of affinity capillary electrophoresis were adapted from another work - heparin was determined indirectly by monitoring of decrease of the peak area of protamine. Protamine is medically used antidote of heparin because they create a stable complex together. In this work protamine was replaced by well defined tetraarginine because the most frequent amino acid in protamine is...
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Preparation and characterization of polyaniline-coated stationary phases doped with silver
Pátereková, Viktória ; Křížek, Tomáš (advisor) ; Sobotníková, Jana (referee)
The aim of this study is a preparation of polyaniline stationary phases doped with silver for application in HPLC. Various polyaniline coated stationary phases differing in the addition of AgNO3 were polymerized. Some of them were subjected to Ag sedimentation, in others AgNO3 was added after polymerization. Stationary phases were investigated by scanning electron microscopy, atomic absorption spectroscopy, Raman spectroscopy, and Fourier transformation infrared spectroscopy. Columns filled with prepared stationary phases were compared with silver-free polyaniline column by separating a mixture consisting of caffeine, theobromine and theophylline in three chromatographic modes (NP-pure ACN, HILIC-98/2 (v/v) ACN/water and RP-20/80 (v/v) ACN/water) at a flow rate of 5 µL/min with UV detection at 265 nm and also by separating a mixture of 2'-aminoacetophenone, 3'-aminoacetophenone and 4'-aminoacetophenone in the same chromatographic modes, at a flow rate of 5 µL/min except from RP mode where a flow rate of 10 µL/min was used to accelerate separation. Polyaniline-coated columns doped with silver showed different selectivity in the RP mode of the mixture of caffeine, theobromine and theophylline when compared to polyaniline-coated columns without the addition of silver. Further, the columns were tested for...
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Development of method for determining aminoacid loading in solid phase peptide synthesis
Mácha, Hynek ; Nesměrák, Karel (advisor) ; Křížek, Tomáš (referee)
A simple method has been developed to determine amino acid loading in solid phase peptide synthesis. The method is applicable for the most common type of synthesis, which use FMOC as protective group and piperidine as a deprotecting agent. Both products of deprotection reaction are separated by HPLC and determined using an UV detector; an internal standard is added. The method gives true values that have been verified by an independent method. The RSD is 1.52%. The method is more accurate than the published methods and allows the determination from the waste of synthesis. The employing of the internal standard eliminated the necessity of dilution accuracy or known volumes.
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Validation of HPLC method for determination of purity of sofosbuvir and its transfer to UPLC system
Vymyslický, Filip ; Křížek, Tomáš (advisor) ; Kubíčková, Anna (referee)
The aim of this bachelor thesis was validation of HPLC method for determination of purity of sofosbuvir and its transfer to UPLC system. The parameters included in the validation plan were: the system suitability test, robustness, accuracy, linearity, recovery, limit of detection, and limit of quantification. Robustness was tested by the design of experiments approach. Method was transferred from the HPLC system to the UPLC system. The transfer was performed by randomization test, which was evaluated by statistical methods, namely by pairwise T-test and correlation analysis.
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Effect of background electrolyte anions on markers of electroosmotic flow in capillary electrophoresis
Čokrtová, Kateřina ; Křížek, Tomáš (advisor) ; Kubíčková, Anna (referee)
Mobility of the electroosmotic flow is an important quantity in capillary electrophoresis because its value is needed to determine the effective electrophoretic mobility of analytes. The effective electrophoretic mobility is used for electrophoretic determination of physico-chemical constants, such as dissociation constants or stability constants, but it is also important in analytical chemistry because identification of analytes is based on the value of the effective mobility. The most common way to measure electroosmotic flow is to add a neutral substance to the sample. However, the neutral substance can gain electrophoretic mobility due to interactions with the components of the background electrolyte. This may cause an inaccurate determination of the velocity of the electroosmotic flow. The aim of this work was to measure the mobility of several common and less frequently used markers in the background electrolyte containing different anions and to find suitable marker - anion combinations in order to avoid major measurement errors. Relative mobility of eight markers in the pure background electrolyte and in the background electrolyte containing a salt of the studied anion - sodium chloride, sodium perchlorate or sodium sulphate - was measured and related mobility of thiourea. Acetate buffer...
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Optimization of electrophoretic determination of protamine and insulin
Molnárová, Katarína ; Křížek, Tomáš (advisor) ; Hraníček, Jakub (referee)
This work deals with optimization of a method for separation and detection of protamine and insulin using capillary zone electrophoresis. The composition of background electrolyte, the solution pH and the injection method were optimized. Citric acid in a concentration range of 80 to 240 mmol L-1 and chloroacetic acid ranging from 50 to 150 mmol L-1 were tested sequentially. The optimized method uses a fused silica capillary with inner diameter of 50 μm. The total length of capillary is 50.0 cm, effective length is 8.5 cm. The injection of the sample is performed on the short end of the capillary. The method uses chloroacetic acid of 100 mmol L-1 concentration as the background electrolyte. Driving voltage is 20 kV. Sample is injected hydrodynamically with a pressure of 5 kPa for 3 s. The analytes are detected spectrophotometrically at wavelength of 200 nm. The method allows for determination in case of protamines in concentration range between 4 μg mL-1 and 300 μg mL-1 and insulin from 5 μg mL-1 to 300 μg mL-1 . The limits of detection are 1 μg mL-1 for protamine and 2 μg mL-1 for insulin. Repeatability of migration times and peak areas tested at 30 μg mL-1 and 200 μg mL-1 concentration levels using hydrodynamic injection showed values of relative standard deviation lower than 6 % suggesting...
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