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Determination of enantiomers of amino acids by HPLC
Michalčíková, Regina ; Tesařová, Eva (advisor) ; Janečková, Lucie (referee)
This Bachelor's thesis deals with determination of amino acids enantiomers, namely alanine in the experimental part, by high performance liquid chromatography using chiral stationary phases based on teicoplanin. The theoretical part is focused on summarizing the fundamental issue of chirality and chiral amino acids in foods, the characteristics of high-performance liquid chromatography, as one of the most appropriate methods to enantioseparation amino acids, and description of the methods of derivatization, which are used in determination of enantiomers of amino acids. The experimental part deals with the separation of alanine enantiomers, with the choice of the best separation system for the separation and determination of D-and L-alanine in fruit juice.
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Chromatograhic characterization of chiral and achiral separation systems
Kučerová, Gabriela ; Tesařová, Eva (advisor) ; Čabala, Radomír (referee) ; Ševčík, Juraj (referee)
Dissertation thesis is a 5-publications' collection concerning characterization and application potential of cyclodextrins, polysaccharides and macrocyclic antibiotics based chiral stationary phases. The effects of stationary phase and mobile phase are studied. This approach ensures the complex insight into separation systems studied. Systems with different nature of chiral selector were studied by HPLC. Namely, macrocyclic antibiotics and derivatized polysaccharides were used for experiments. Former ones provided better results for enantioseparation of non-coded amino acids than latter ones. Dynamic coating procedure was used for preparation of a new chiral stationary phase. Characterization of new cationic cyclodextrin based chiral stationary phase was performed. Linear free energy relationship method was used for characterization of two different separation systems, i.e. newly prepared stationary phase and commercially available stationary phase. Based on results obtained, newly prepared stationary phase showed better results for separation of different achiral groups of analysts. New stationary phase prepared by dynamic coating was compared with chromatographic system, in which the chiral selector was used as a mobile phase additive. The chiral selector used for the two different approaches was...
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Use of Chiral Separations for the Determination of Enzyme Enantioselectivity
Břicháč, Jiří ; Zima, Jiří (advisor) ; Tesařová, Eva (referee) ; Flieger, Miroslav (referee)
Conclusion Enantioselective enzymes pray important rore in metaboric pathways of chirar compounds. Enantioselective metabolism of lipid peroxidation product HNE was studied using measurement of enzyme kinetics. Difference in ability to detoxiý individua| HNE enantiomers by rat brain mitochondriar ALDHs was found. Based on kinetic parameters of purified recombinant enz),Ťnes' it was concluded, that rat ALDH5A enantioselective|y oxidized (R)-HNE' whereas rat ALDH2 was not enantioserective. Moreover, direct and indirect RP-HPLC methods for the separation of HNEA enantiomers were deveroped and validated. The indirect method involved pre-corumn derivatization with a chiral amino agent ANPAD' and subsequent separation ofdiastereomers on a Spherisorb oDS2 column. The direct separation of HNEA enantiomers was performed using the porysacharide-based CSP, Chiralpak AD-RH. Enantioserective enzymes are useful biocatalyzators a'owing cheap preparation of optically pure chemicals. screening of epoxide hydrolases produced by various microorganisms was performed and enantioselective enzymes were found using chiral GC on cyclodextrin-based CSps. In summary, this thesis describes development and optimization of the analytical methods for determination of various chiral compounds implicated in enzyme enantioselectiviý. All...
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Study on stability of triazine based herbicides in water
Petrlíková, Kateřina ; Tesařová, Eva (advisor) ; Janečková, Lucie (referee)
The first aim of this thesis was to find a suitable analytical method for monitoring of stability of four s-triazine herbicides in water samples. Two columns - stationary phases C18, namely products of Waters and Astec were tested in HPLC system. The composition of ACN/H2O mobile phase was changed in the range from 90/10 to 60/40 (v/v). Baseline separation of prometone, propazine and prometryne was achieved on the Astec C18 column with mobile phase ACN/H2O 70/30 (v/v). This separation system was used for the study of stability. The stability of s-triazines was monitored in the period of six weeks. The samples of atrazine, prometone, propazine and prometryne water from water tap. All samples were detected at the wavelength of 221nm. No significant changes were found in the contents of s-triazines in the samples were measured in samples of water - water from Vltava, acidified water from Vltava, water from water tap and acidified water from water tap. All samples were detected at the wavelength of 221nm. No significant changes were found in the contents of s-triazines in the samples.
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Optimization and validation of analytical method for determination of selected drugs
Geryk, Radim ; Tesařová, Eva (advisor) ; Janečková, Lucie (referee)
The aim of this work was finding suitable conditions for separation of enantiomers of citalopram and citadiol, optimization of chromatographic system and validation of the analytical method for determination of selected enantiomers in drugs. Drugs are often chiral compounds. In many cases, only enantiomer has required pharmacological effects. Advanced chemical technologies associated with the synthesis, separation and analysis of the individual enantiomers caused an increase of the number of new chiral drugs in the form of single pure enantiomers, the chiral switch. Citalopram is one of the widely used antidepressants of selective serotonin reuptake inhibitors (SSRI). Citalopram is used for treatment of depression, panic anxienty or obsessive compulsive disorder of pathological laughing and crying. The pharmacological activity is associated with the S-citalopram, while R-enantiomer is essentially inactive and even counteracts the activity of escitalopram. Citadiol is a chiral syntetic precursor of citalopram. This thesis was focused on monitoring enantioseparation "behaviour" of selected enantiomers. HPLC method with chiral stationary phases based on macrocyclic antibiotics, cyclofructans and cellulose was applied for enantioseparation of the above-mentioned compounds. The optimized chromatographic...
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HPLC determination of silybin diastereoisomers in plasma of laboratory animals
Kolářová, Petra ; Tesařová, Eva (advisor) ; Bednář, Petr (referee)
Silybin is the main component of silymarin, a standardized extract obtained from the seeds of milk thistle (Silybum marianum). Flavonolignan silybin has antioxidant, hepatoprotective, chemoprotective and antitumor activities. Natural silybin occurs as an approximately equimolar mixture of two diastereoisomers - silybin A and silybin B. Analytical separation of these diastereoisomers is possible but preparative separation is complicated. The biological activity of the silybin A is different from the silybin B. Silybin diastereoisomers are mainly conjugated to glucuronides and sulfates in organism. A mixture of both silybin diasteroisomers is used in the majority of reported biological, chemical and pharmacokinetic studies. For the first time, optically pure silybin A and silybin B were used for pharmacokinetic study in this thesis. The object of this work was determination of the concentration of free and total silybin in rats plasma in relation to time. Theoretical introduction describes the current state of the problem of chemistry, pharmacology and pharmacokinetics of silybin diastereoisomers. Second part is focused on the selection of appropriate analytical column, chromatographic method and suitable procedure for preparation of biological samples for the determination of the silybin...
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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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