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Detailed characterization of macrocyclic glycopeptide-based chiral stationary phases in SFC
Folprechtová, Denisa ; Kalíková, Květa (advisor) ; Křížek, Tomáš (referee) ; Kohout, Michal (referee)
The choice of a suitable chiral stationary phase is a key factor for successful enantioseparation in chromatographic methods. Macrocyclic glycopeptides are chiral selectors that, due to their unique structure, allow to interact with the analyte by various types of interactions simultaneously, which contributes to their complex retention and enantiodiscrimination mechanisms. Macrocyclic glycopeptides are widely used chiral stationary phases in liquid chromatography, but they have not been thoroughly tested in sub/supercritical fluid chromatography, which currently rank among important separation techniques in terms of chiral analyses. Therefore, the aim of this thesis is a detailed characterization of the enantiodiscrimination and retention mechanisms of macrocyclic glycopeptide-based chiral stationary phases packed with core-shell particles, namely TeicoShell, VancoShell, and NicoShell columns in sub/supercritical fluid chromatography. First, the effect of the mobile phase composition, especially the type and amount of organic modifiers and additives, on individual chromatographic parameters, i.e., retention, resolution of enantiomers, and peak shapes was studied in detail using selected sets of structurally different biologically active chiral compounds and materials. Based on the obtained...
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Study of retention and separation of peptides in HPLC
Boudová, Hana ; Kalíková, Květa (advisor) ; Kubíčková, Anna (referee)
This diploma thesis deals with the study of retention and separation of structurally different peptides and digest products of cytochrome c using reverse phase and mixed modes of liquid chromatography. As the model peptides, five unmodified dipeptides, nine dipeptides containing a protective benzoyl group on the N-terminal amino acid and eight therapeutic peptides containing five to ten amino acids were selected. The chromatographic behaviour of the model peptides was tested on three different columns: XBridge C18, XSelect CSH C18 and Atlantis Premier BEH C18 AX under different conditions. The influence of the pH of the aqueous part of the mobile phase, i.e. 2,1; 3,0; 4,7 and 6,8, and the amount of acetonitrile in the mobile phase on retention and separation of model peptides was tested. The effect of the concentration of ammonium acetate buffer, pH = 6,8 in the mobile phase on the retention and peak symmetry in the concentration range of 10-50 mmol L-1 was assessed. The chromatographic conditions for the baseline separation of 20 different model peptides were optimized. The conditions were as follows: XSelect CSH C18 column, mobile phase composed of acetonitrile/10mM ammonium acetate, pH = 6,8 (v/v) under gradient elution. The suitable chromatographic conditions for the analysis and identification...
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Determination of L-tryptophan in food supplements in high performace liquid chromatography
Pešková, Pavlína ; Kalíková, Květa (advisor) ; Kubíčková, Anna (referee)
This bachelor's thesis deals with optimalization of chromatographic conditions for determination of tryptophan by HPLC method with UV detection. The amount of tryptophan has been determined in two food supplements. To find suitable conditions for the determination of tryptophan, the influence of the composition of the mobile phase on analyte retention and peak symmetry was tested in two different chromatographic modes - reversed-phase mode using the Atlantis dC18 column and mixed-mode using the Atlantis PREMIER BEH C18 AX column. Specifically, the effect of the type and amount of organic modifier (acetonitrile/methanol), the effect of the pH of the ammonium acetate buffer (4,7;5,9;7,0) and its concentration (5mM/10mM) in the mobile phase was tested. Based on the obtained data, the optimal chromatographic conditions were selected (in terms of analysis time and peak symmetry). In the optimized conditions selected validation parameters such as repeatability, reproducibility, limits of detection and quantification were determined. The optimized conditions were as follows: mixed-mode Atlantis PREMIER BEH C18 AX column, mobile phase acetonitrile/5mM ammonium acetate, pH 7,0 10/90 (v/v), separation temperature 25 řC, flow rate 1 ml/min and UV detection at 254 nm. Under these conditions, the tryptophan...
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Study of retention and separation of pesticides in different modes of liquid chromatography
Dérerová, Tímea ; Kalíková, Květa (advisor) ; Riesová, Martina (referee)
4 Abstract This bachelor thesis is focused on a separation of six pesticides in reversed-phase and mixed-mode high performance liquid chromatography using Atlantis dC18, XSelect CSH C18, XSelect CSH Fluro-Phenyl and Atlantis Premier BEH C18 AX columns. The same manufacturing parameters - column and carrier particle dimensions opened up the possibility of their direct comparison in terms of retentivity and selectivity. The stationary phases were categorized using Walters test, in terms of hydrofobicity, silanol activity and efficiency. The analytes of interest were pesticides with a different mechanism of action in the environment. Two of them contained an acidic functional group that caused their elution at system void volume if pure water was used as aqueous part of mobile phase. For suppression of dissociation of acidic pesticides, the buffers, i.e., 10mM ammonium acetate, pH 4.7 and 10mM ammonium formate, pH 3.0 were used instead of pure water. Tested columns exhibited different retention and selectivity of analytes. For separation conditions optimization, an experimental approach, including changes in mobile phase composition and pH, and separation temperature, was used. Retention time, resolution and peak shape were all dependent on these chromatographic parameters. The retention of tested pesticides...
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Optimization of separation and quantification of prospective products of big bang
Smitka, Jaroslav ; Tesařová, Eva (advisor) ; Kalíková, Květa (referee)
The thesis is a contribution to research aimed at modeling the conditions of the Big Bang and monitoring possible reaction products. As the starting compounds were selected 2- amino-1-butanol, 3-methoxy-1-propanol, 2-methyl-1-butanol, formamide, some of which were subsequently bombarded by PALS at J. Heyrovsky Institute of Physics, Academy of Sciences. Gas chromatography hyphenated with mass spectrometry and liquid chromatography with UV detection were used for analysis. In GC-MS, calibration of 3- methoxy-1-propanol and 2-methyl-1-butanol was carried out and LOD and LOQ were calculated. Comparison of 3-methoxy-1-propanol and bombarded 3-methoxy-1-propanol did not show the emergence of new products since 3-methoxy-1-propanol was in excess. The found compounds can be more likely identified as impurities present in a solvent or in the individual standards. The optimized HPLC system has managed to separate the three standards. Formamide, which is unsuitable for analysis by gas chromatography, due to its high polarity, however, eluted in all tested systems with system peak (the dead time marker). Nevertheless, the bombarded sample was analyzed for comparison with the original standard. Comparison, however, showed no reaction products in the bombarded sample. Key words: big bang laser sparks formamide...
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Separation and determination of possible products of enzymatic cleavage of 4-nitrophenyl-N,N'-diacetyl-β-D-chitobioside using capillary electrophoresis
Velvarská, Romana ; Křížek, Tomáš (advisor) ; Kalíková, Květa (referee)
This work deals with the development and optimization of conditions of a method that can be used to compare the activity of the enzyme β-N-acetylhexosaminidase in hydrolysis of a natural substrate and a chromogenic substrate, which is often used in the study of enzyme kinetics. As a substrate, 4-nitrophenyl-N,N'-diacetyl-β-D-chitobioside was selected for cleavage. This oligosaccharide contains bond, which the enzyme cleaves in the natural substrate, and the bond that occurs in the chromogenic substrate. To determine the products arising from enzymatic hydrolysis of 4-nitrophenyl-N,N'-diacetyl-β-D-chitobioside, capillary zone electrophoresis was used. First, it was necessary to find the optimal composition of the electrolyte, its pH and concentration. The optimal background electrolyte was a solution of sodium tetraborate at a concentration of 25 mmol/l and a pH of 10.25. Subsequently, repeatability, calibration curves and linearity, limit of detection and limit of quantification were investigated. Repeatability of migration times ranged up to 0.6%, the repeatability of peak areas between 2.5 and 6.3%. Limits of detection were ranging from 0.005 to 0.120 mmol/l. Finally, the optimized method was successfully used to monitor the actual enzyme cleavage.
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Characterization and comparison of new reversed-phase HPLC columns
Putzová, Klára ; Kalíková, Květa (advisor) ; Kozlík, Petr (referee)
The aim of the bachelor thesis was to evaluate and compare interaction/separation properties of three chromatographic columns, namely XBridge C18, XSelect CSH C18 XSelect HSS C18, designed for reversed-phase high performance liquid chromatography. All the columns contain the same ligand, i.e. octadecyl but they differ in the stationary phase support. The columns were compared in terms of silanol activity, hydrophobicity, shape selectivity and efficiency. For this purpose, the simple chromatographic tests designed by Walters, Engelhardt and Galushko were used. Furthermore, the separation of five selected pesticides, namely acetochlor, fomesafen, isoproturon, metazachlor triflumuron, were optimized on tested columns in simple mobile phases composed of acetonitrile or methanol as organic modifiers and water. All the tested columns showed low silanol activity and medium to high hydrophobicity acording to the chromatographic tests used. The low silanol activity corelates with endcapping procedure used for all the columns. All pesticides were more retained in methanolic mobile phase due to the lower elution strength of methanol compared to acetonitrile in reversed-phase high performance liquid chromatography. The analysis time of separation varied for each column under the same chromatographic conditions. The...
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Optimization of HPLC separation of penicillin derivatives
Šímová, Veronika ; Kalíková, Květa (advisor) ; Tesařová, Eva (referee)
Penicillins are part of β-lactam antibiotics, which are wide spread in human and veterinary medicine. The aim of this bachelor work was to develop and to optimize HPLC method for separation of four derivatives of penicillin - amoxicillin, 4-hydroxyphenoxymethylpenicillin, benzylpenicillin and phenoxymethylpenicillin. The reserved phase HPLC with octodecyl stationary phase was used in this work. The baseline separation of all analytes was not obtained in isocratic elution. For this reason, the gradient elution was examined on the baseline resolution of all analytes was achieved and the analysis time did not exceed 22 minutes. The stability of methanolic solutions of penicillins was tested during eight days. Key words: HPLC, antibiotics, derivatives of penicillins, reverse mod, stability
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Chiral HPLC separation of newly synthesized amino acids
Kučerová, Gabriela ; Kalíková, Květa (advisor) ; Riesová, Martina (referee)
The aim of this work was to develop and to optimize HPLC method for enantioseparation of newly synthesized derivatives of amino acids. The set of these analytes contained four N- blocked derivatives of D,L-Phenylalanine, three N-unblocked derivatives of D,L- Phenylalanine, , one methylated derivative of D,L-Tyrosine and D,L-Tyrosine. Two separation modes i.e. reversed phase and polar-organic modes and two columns i.e. Chirobiotic® T and Chirobiotic® T2 were used. Chiral stationary phases of these columns were composed of macrocyclic antibiotic teicoplanin coated on silica gel support. As mobile phases in revesed phase mode, methanol and acetate buffer were used with Chirobiotic® T column. The most suitable concentration were 20 mM and the most suitable pH value were 4.00. Under the above mentioned conditions eight analytes of ten were separated and the optimal conditions were found. Polar-organic mode and Chirobiotic® T column were suitable only for enantioseparation of three N-blocked derivatives of D,L- Phenylalanine. Mobile phases were composed of methanol with small additions of triethylamine and acetic acid. Chirobiotic® T2 column in reversed phase mode was not suitable for tested set of analytes. Only partial separation of D,L-Tyrosine derivative and one N-unblocked D,L-Phenylalanine...
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