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Separation of peptides on a newly designed cyclofructan-based stationary phase, characterization of separation system
Šímová, Veronika ; Kalíková, Květa (advisor) ; Janečková, Lucie (referee)
Hydrophilic interaction liquid chromatography is in the present time very often used for separation of polar compounds which are complicated separated in reverse phase HPLC or they are not sufficiently retained. Various stationary phases suitable for HILIC are commercially available and new stationary phases are still developing. In this work bare silica and isopropyl cyclofructan modified silica stationary phases were compared. Chromatographic behavior of sets of four pentapeptides and five nonapeptides was tested in detail under hydrophilic interaction liquid chromatography. Additionally, mobile phase composition changes the types of interaction and their strengths were studied using linear free energy relationship method. Obtained results denoted that the main role in the retention mechanism on the isopropyl cyclofructan stationary phases play hydrogen bond acidity and dipolarity/polarizibility, while dispersion interactions are preferred in the mobile phase. On the other hand, bare silica stationary phase provides only one interaction type - hydrogen bond acidity. Key words: peptides, cyclofructan, LFER, HILIC
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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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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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Odour nuisance assessment in the CR
Vančurová, Andrea ; Hůnová, Iva (advisor) ; Kalíková, Květa (referee)
The aim of this work is to monitor the issue of odorous substances in the Czech Republic. In most cases, the main source of information are studies prepared at the request of the municipal or city authorities. Individual areas affected with this problem, those that I focused on in this work, were selected according to numbers of inducements and complaints from local residents. The introduction of the theoretical part deals with the anatomy of the olfactory system and the reaction of brain perceptions on odour stimulus. Furthermore, I describe the possibilities of measuring odours. Due to low concentrations of odours the least used method is gas chromatography; another option is olfactometry, which is based on the principle of mixing pre-determined concentration of an odorant with neutral gas. In the studies that are the main source of this work, I used the method of questionnaires that are consisted of evaluations of the smell from the locals. Moreover, I also described the procedure of filing in complaints about odours in the neighborhood of residence and its support in the law. The core of the practical part are the complaints of people that were submitted to the Czech Environmental Inspectorate for the years 2006 to 2009. Includes both specific businesses that are repeat complaints and fines of CZK 100...
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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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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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Optimization of HPLC method for separation of tetracyclines
Kučerová, Gabriela ; Kalíková, Květa (advisor) ; Janečková, Lucie (referee)
The aim of this work is to develop and to optimize HPLC method for separation of a set of four tetracycline antibiotics - tetracycline, chlortetracycline, oxytetracycline, and doxycycline. Four different reversed octadecyl-silica stationary phases in various mobile phase compositions were examined in isocratic elution. The baseline resolution of all the analytes was obtained by using two columns - Astec C18 and Atlantis C18 I. The optimized separation system consisted of Atlantis C18 I. column, ACN/0.1% formic acid 15/85 (v/v) as the mobile phase and flow rate of 2 ml/min. The baseline resolution was achieved and the analysis time did not exceed 27.5 minutes.
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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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HPLC separation of chlorophenoxypropionic acid derivatives
Horová, Kateřina ; Kalíková, Květa (advisor) ; Šlechtová, Tereza (referee)
This Bachelor's thesis focuses on the separation of chlorophenoxypropionic acids and their derivatives by a high-performance liquid chromatography. The separations were performed in a reversed phase mode. These compounds are employed as herbicides, i.e. chemical substances to control unwanted plants, to kill weeds. The herbicides studied for the purposes of this thesis were 2-(4-chloro-2-methylphenoxy)propionic acid, 2-(2,4- dichlorophenoxy)propionic acid, 2-(2-chlorophenoxy)propionic acid, 2-(3- chlorophenoxy)propionic acid and 2-(4-chlorophenoxy)propionic acid. The study aimed at finding and optimizing chromatographic conditions for simultaneous separation of all five herbicides mentioned above. The analysis employed two types of columns, that is ZORBAX SB-C8 column and ZORBAX Eclipse XDB-C18 column whose level of polarity differ. As mobile phases, there were used acetonitrile/water, acetonitrile/formic acid, pH 2.1 and methanol/formic acid, pH 2.1 in various volume ratios. ZORBAX SB-C8 column, mobile phase methanol/formic acid (concentration of 0.365 mol/l, pH 2.1) 40/60 (v/v), temperature of 25řC, and flow rate of 1 ml/min delivered the most convenient separation conditions. Given these isocratic conditions, all the herbicides were baseline separated within 80 minutes. Keywords: HPLC,...
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