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Chiral separation of biologically active compounds by chromatography
Landl, David ; Kalíková, Květa (advisor) ; Kubíčková, Anna (referee)
- 4 - Abstract in English This diploma thesis is focused on the evaluation and comparison of the enantioselective potential of two columns CHIRAL ART Amylose-SA and CDShell-RSP using a set of 29 chiral drugs in high performance liquid chromatography. The separations of enantiomers were performed in three modes: reversed-phase, normal-phase, and polar- organic mode. The CHIRAL ART Amylose-SA column was tested in normal-phase mode, the CDShell-RSP column in reversed-phase and polar-organic modes. The CHIRAL ART Amylose-SA column contains amylose tris(3,5- dimethylphenylcarbamate) immobilized on 3 µm porous silica gel particles. The CDShell-RSP column contains a chiral selector hydroxypropyl-β-cyclodextrin, which is covalently bonded on 2.7 µm superficially porous particles. In the normal-phase mode, mobile phases composed of hexane and propane-2-ol were used. Furthermore, the effect of various additives (triethylamine, diethylamine, trifluoroacetic acid, and the mixture of diethylamine and trifluoroacetic acid) in the mobile phase on the enantioseparation of chiral drugs was tested. The most universal additive was a mixture of diethylamine and trifluoroacetic acid. A total of 22 chiral drugs were enantioseparated on the CHIRAL ART Amylose-SA column, 10 of them were baseline separated. Mobile phases for...
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Characterization of novel stationary phase in hydrophilic interaction liquid chromatography
Kadlecová, Zuzana ; Kalíková, Květa (advisor) ; Kozlík, Petr (referee)
The aim of this diploma thesis is detailed characterization of new diol-based column, i.e. Torus DIOL designed for supercritical fluid chromatography, in hydrophilic interaction liquid chromatography. This stationary phase contains diol ligands bonded on BEH ("bridged ethylene hybrid") particles. The BEH sorbent is more stable at high pH, pressure and temperature than conventional silica sorbents. Five fluorinated pesticides were successfully separated on Torus DIOL column. Retention mechanism and effect of different parameters were tested by analysis of small polar compounds (nucleobases, uridine and its derivatives, acidic, neutral and basic compounds). Both partitioning of analytes between an aqueous layer deposited on the surface of the stationary phase and the mobile phase and adsorption of analytes on the stationary phase participate in the retention mechanism. Effect of the mobile phase aqueous part pH was investigated with the following buffering solutions: formic acid (pH = 2.1), 10mM ammonium acetate (pH = 4.7 and pH = 9.5). Obtained results showed that aqueous part pH significantly affects retention of acidic compounds according to their pKA values. Retention of all tested analytes on Torus DIOL column slightly increased with increasing ionic strength of mobile phase. The retention...
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Preparative diastereomeric separation of silybin and its sulfates by RP-HPLC
Kolářová, Petra ; Tesařová, Eva (advisor) ; Kalíková, Květa (referee)
Silymarin is standardized extract isolated from fruits of Milk Thistle plant (Silybum marianum). The principal component of silymarin is silybin. This flavonolignan is mainly responsible for the medicinal effects of Milk Thistle fruits: antioxidant, hepatoprotective, anticancer and chemoprotective activities. Natural silybin exists as an equimolar mixture of diastereomers A and B whose preparative separation is very hard. It was shown that the biological activity of silybin A and B are different. Silybin in the blood conjugates mainly to sulfates. The structure or biological activity of the sulfates is not yet known. The aim of this work is to develop practically applicable method for preparative separation of diastereomers of silybin A and B, and sulfates, which are considered as one of the major metabolites of silybin. The preparative method for separation of silybin A and B in the mobile phase consisting of 50% MeOH on the chromatographic column Labio C18 25x250 mm, was developed. In addition, preparative method for separation of mixture of products accured from the sulphation of silybin-23-acetate in the mobile phase consisting of MeOH/H2O 60/40 (v/v) with addition of 10 ml/l HCOOH, was optimized.
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Study of interactions participating in the separation mechanism of chromatographic separation systems
Kalíková, Květa
The Thesis combines a theoretical part focused on better understanding of separation processes and interaction mechanisms with experimental applications for specific analytical purposes. HPLC is one of the preferently used separation techniques. As various interaction types can participate in the retention mechanism of analytes, the choice of the optimal separation system is often ambiguous. The selection of suitable separation conditions is very often made empirically. Such approach is rather time consuming. Therefore, it could be advantageous to utilize some physicochemical characteristics of the separation system. Linear free energy relationship (LFER) is one of the comprehensive semiempirical methods that allows characterization and comparison of stationary phases/separation systems and yield to better understanding of the intermolecular interactions, which play role in the separation processes. The optimization of separation processes can be disturbed or complicated by phenomena called system peaks. In the case of detectable system zone the resultant chromatogram includes more peaks than is the number of analytes in the sample. A serious problem that can arise if an analyte interacts with the system zones is resonance. This phenomenon can disturb completely the separation results and make...
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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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Chiral separation of biologically active compounds by chromatography
Landl, David ; Kalíková, Květa (advisor) ; Kubíčková, Anna (referee)
- 4 - Abstract in English This diploma thesis is focused on the evaluation and comparison of the enantioselective potential of two columns CHIRAL ART Amylose-SA and CDShell-RSP using a set of 29 chiral drugs in high performance liquid chromatography. The separations of enantiomers were performed in three modes: reversed-phase, normal-phase, and polar- organic mode. The CHIRAL ART Amylose-SA column was tested in normal-phase mode, the CDShell-RSP column in reversed-phase and polar-organic modes. The CHIRAL ART Amylose-SA column contains amylose tris(3,5- dimethylphenylcarbamate) immobilized on 3 µm porous silica gel particles. The CDShell-RSP column contains a chiral selector hydroxypropyl-β-cyclodextrin, which is covalently bonded on 2.7 µm superficially porous particles. In the normal-phase mode, mobile phases composed of hexane and propane-2-ol were used. Furthermore, the effect of various additives (triethylamine, diethylamine, trifluoroacetic acid, and the mixture of diethylamine and trifluoroacetic acid) in the mobile phase on the enantioseparation of chiral drugs was tested. The most universal additive was a mixture of diethylamine and trifluoroacetic acid. A total of 22 chiral drugs were enantioseparated on the CHIRAL ART Amylose-SA column, 10 of them were baseline separated. Mobile phases for...
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Characterization of novel stationary phase in hydrophilic interaction liquid chromatography
Kadlecová, Zuzana ; Kalíková, Květa (advisor) ; Kozlík, Petr (referee)
The aim of this diploma thesis is detailed characterization of new diol-based column, i.e. Torus DIOL designed for supercritical fluid chromatography, in hydrophilic interaction liquid chromatography. This stationary phase contains diol ligands bonded on BEH ("bridged ethylene hybrid") particles. The BEH sorbent is more stable at high pH, pressure and temperature than conventional silica sorbents. Five fluorinated pesticides were successfully separated on Torus DIOL column. Retention mechanism and effect of different parameters were tested by analysis of small polar compounds (nucleobases, uridine and its derivatives, acidic, neutral and basic compounds). Both partitioning of analytes between an aqueous layer deposited on the surface of the stationary phase and the mobile phase and adsorption of analytes on the stationary phase participate in the retention mechanism. Effect of the mobile phase aqueous part pH was investigated with the following buffering solutions: formic acid (pH = 2.1), 10mM ammonium acetate (pH = 4.7 and pH = 9.5). Obtained results showed that aqueous part pH significantly affects retention of acidic compounds according to their pKA values. Retention of all tested analytes on Torus DIOL column slightly increased with increasing ionic strength of mobile phase. The retention...
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Enantioselective potential of sulfobutylether-β-cyclodextrin based chiral stationary phase
Folprechtová, Denisa ; Kalíková, Květa (advisor) ; Kubíčková, Anna (referee)
The aim of this diploma thesis was to prepare two new chiral stationary phases by dynamic coating of sulphobutylether-β-cyclodextrin (SBE-β-CD) with varying degrees of substitution onto strong anion-exchange stationary phases. The enantioselective potential and stability of the newly prepared chiral stationary phases were tested using a set of chiral analytes. The set contained structurally diverse analytes, i.e. benzodiazepines (oxazepam, lorazepam), phenothiazines (thioridazine, promethazine), β-blockers (labetalol, pindolol, propranolol, alprenolol), profens (carprofen, fenoprofen, flurbiprofen, indoprofen), flavanones (6-hydroxyflavanone, 7-hydroxyflavanone), DL-tryptophan and its derivatives (5-OH-DL-tryptophan, 5-F-DL-tryptophan, DL-tryptophan butylester and blocked aminoacid (t-Boc-DL-tryptophan)), dipeptides (glycyl-DL-phenylalanine, glycyl-DL-tryptophan) and Troger's base. Measurements were carried out in reversed-phase high-performance liquid chromatography. Mobile phases consisted of methanol/formic acid (pH 2.10) and methanol/10mmol l-1 ammonium acetate buffer (pH 4.00) in various volume ratios. The chiral stationary phase containing hexasubstituted SBE-β-CD was suitable for enantioseparation of eleven analytes. Four of them were baseline enantioresolved and seven partially. The chiral...
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Study of interactions participating in the separation mechanism of chromatographic separation systems
Kalíková, Květa ; Tesařová, Eva (advisor) ; Pacáková, Věra (referee) ; Ševčík, Juraj (referee)
Charles University in Prague Faculty of Science Department of Physical and Macromolecular Chemistry Study of interactions participating in the separation mechanism of chromatographic separation systems The PhD Thesis Report Květa Kalíková Prague 2009 The PhD thesis was carried out within the PhD study during years 2005-2009 at the Department Physical and Macromolecular Chemistry, Faculty of Science, Charles University in Prague. Supervisor: Doc. RNDr. Eva Tesařová, CSc. 2 Content 1. Introduction 4 2. Aims of the thesis 4 3. Results and discussion 5 3.1 Characterization of separation system using LFER model (publication I) 5 3.2 Occurrence and behavior of system peaks in RP HPLC with solely aqueous mobile phases (publication II) 6 3.3 Examples of optimized separation systems and their application 8 3.3.1 Achiral separation systems 8 3.3.1.1 HPLC separation of estrogens (publication III) 8 3.3.1.2 HPLC method for separation of 1,4-benzodiazepines (publication in progress) 9 3.3.2 Chiral separation systems (publication IV) 10 3.3.2.1 Optimization and validation of HPLC method for enantioseparation of (±)-cloprostenol (publication V) 11 4. Conclusions 12 5. References 13 6. List of publications 14 7. List of presentations 15 8. Curriculum Vitae 17 3 1. Introduction High performance liquid chromatography (HPLC)...
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