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Isolation and characterisation of biologically active substances
Kettnerová, Eliška ; Nesměrák, Karel (advisor) ; Musilová, Adéla (referee)
This Bachelor thesis aims at isolation and partial identification of biologically active substances which are produced by actinomycetes and can be potentially applied in medicine. Cultivation broths of actinomycetes containing their metabolites were purified and pre-concentrated by solid phase extraction. Then, the bioassay of the extracts by Kirby-Bauer test using the sensitive strain Kocuria rhizophila was performed. Biologically active metabolites were analyzed and isolated by ultra- performance liquid chromatography with photo diode array detector. Isolated substances were assayed by mass spectrometry, which yielded relative molecular mass values of the unknown compounds. The values were compared with relative molecular masses of compounds listed in a chemical database, which involves natural products including antibiotics. We revealed that the unknown biologically active substances do not refer to any already discovered compound present in the database suggesting that the unknown compounds may be novel. More mass spectrometry and nuclear resonance experiments have to be carried out in order to elucidate their structure. Key words: actinomycetes, antibiotics, SPE, UPLC, HPLC Subject heading: analysis of secondary metabolites, bioassay test, isolation of biologically active compounds,...
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Determination of sibiromycin by UPLC method.
Fadrhonsová, Ivana ; Cajthaml, Tomáš (referee) ; Bosáková, Zuzana (advisor)
Sibiromycin is produced by actinomycetes of Streptosporangium sibiricum and structurally belongs to family of pyrrolo-1,4-benzodiazepines. Sibiromycin is characterized by antibacterial and especially antitumor activity but due to its proved cardiotoxicity it cannot be used. The following research of possible therapeutic sibiromycin utilization is concerned on new nontoxic derivatives produced by genetic manipulated strains of S. sibiricum. For this purpose a new routine chromatographic UPLC-UV method with analyte preconcentration was developed and partially validated. A fermentation broth was extracted by solid phase extraction (SPE) on OASIS MCX columns with the sorbent based on a cation-exchange. Eluted extract was evaporated, reconstituted in methanol and loaded onto UPLC BEH C18 column and analyzed under gradient mode with mobile phases of methanol (A) and trifluoroacetic acid (B). Detection limit of the method was determined as 40 ng/ml, the recovery was 74.75 % and its reproducibility expressed as RSD was 5.18 %. The method was applied to comparison of sibiromycin production by S. sibiricum on 13 different fermentation media. It was found, that a composition of fermentation media influences not only the sibiromycin production but also a synthesis of sibiromycin nature derivatives, which were...
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Fytoestrogeny v potravinářství
Došková, Markéta
Phytoestroegens are biologically active compounds naturally occuring in plant materials. This group consists mainly of polyphenols characterized by structural similarities with female hormone 17-beta-estradiol. Plants produce phytoestrogens as their secundary metabolities, primarily due to the reaction as a response to various stress. Agents with estrogen effect contained in foodstuffs bring very positive impact on our health. On the other hand, some of the estrogen compounds may be harmful to the health depending on the concentration and the complex state of the consumer, as well as mycoestrogens, xenoestrogens and antiestrogens. Also for this reason is necessary to detect and quantify them in the sample of food. The major problem encountered in the studies of their effect is large amount of a variety of other substances in biological material, and according to their complicated structure was important to establish a reliable method, that was used also in the practical part of my experiment. It is an UPLC-MS/MS -- combination of Ultra High Performance Liquid Chromatography and Tandem Mass Spectrometry preceded by complex sample preparation which involves also SPE purification ("solid phase extraction").
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Determination of NSAIDs in wastewater
Krůzová, Sabina ; Mravcová, Ludmila (referee) ; Vávrová, Milada (advisor)
The thesis focuses on non-steroidal anti-inflammatory drugs in wastewater. It characterizes drugs and defines specific methods of determination naproxen and ibuprofen in wastewater. Besides their characteristics there are pre-analytics and final analytic methods described here which are used for determination of drugs in wastewater. To analyse we used wastewater from Veterinary and Pharmaceutical University in Brno. Samples were filtered and concentrated by solid phase extraction (SPE) and after that they were analysed by UHPLC with diode array detector. Thanks to these practices and methods, there were found, that concentration of naproxen in wastewater from VFU Brno was below the limit of quantification and ibuprofen wasn´t detected in most of cases.
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The use of solid phase extraction for fractionation of lipids in cheese matrix
Scholzová, Kristýna ; Vespalcová, Milena (referee) ; Vítová, Eva (advisor)
This bachelor thesis deals with Solid Phase Extraction, as a method for isolation of free fatty acids from cheese matrix to be subsequently determined using gas chromatography with FID detection. The theoretical part provides information about milk fat composition and related reactions being a source of flavour compounds, free fatty acids including. The main objective of the theoretical research is to summarize alternatives to determining fatty acids in cheese, focusing on current advancement and use of Solid Phase Extraction for this purpose. The practical part discovers proper technique and conditions for fractionation of lipids by Solid Phase Extraction, using aminopropyl bonded phase columns. Agilent SampliQ Amino columns were utilized to separate lipid mixture and different lipid classes were eluted using suitable organic solvents. Composition and purity of lipid extracts were verified combining thin layer chromatography and gas chromatography. Before being determined by gas chromatography, free fatty acids were converted into methylesters, based on ČSN EN ISO 12966-2. Besides discussing proper experimental conditions, the assesment also comes with solving optimal volumes of sample and eluting solvents to apply. The results of this thesis are considered to be useful base for subsequent experimental optimization and following integration of validated method into process of determining free fatty acids.
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Preconcentration of Trace Analytes on Modified Sorbents and their Determination on Waters
Holubová, Zuzana ; Čelechovská, Olga (referee) ; Holoubek,, Ivan (referee) ; Otruba,, Vítězslav (referee) ; Sommer, Lumír (advisor)
The work has been focused on the preconcentration techniques for determination of 9Be, 51V, 59Co, 60Ni, 89Y, 111Cd, 208Pb, 232Th and 238U on the modified sorbents, all measurements were performed on ICP-MS. The instrument setup was optimized. The solution of internal standard (200 µg•l-1) was used during all measurements. The internal standard were chosen as follows: 6Li for 9Be, 45Sc (51V), 72Ge (59Co, 60Ni, 89Y), 103Rh (111Cd), 209Bi (206Pb, 207Pb, 208Pb, 232Th and 238U). Another parameters such as influence of mineral acids (HCl, HNO3), surfactant (Septonex®, Zephyramin, Ajatin, Brij 35 and Sodium dodecyl sulphate) and organic reagents (Ammonium pyrrolidinedithiocarbamate, 8-Hydroxyquinoline-5-sulphonic acid, 1,2-Dihydroxyanthraquinone-3-sulphonic acid and 4-(2-Pyridylazo)resorcinol) and the effect of some matrix components on intensity of instrument signal determination was investigated. For preconcentration polar Silicagel and modified nonpolar Silicagels (Silicagel-C18, C8 nad Phenyl) were used. Another experimantal part was dedicated to preconcentration on nonpolar Amberlite XAD-16 and Strata SDB-L and intermediately polar sorbent Amberlite XAD-7. Recoveries of sorption process were observed in the presence of all testing types of surfactants in certified concentration 5•10-4 mol•l-1. Surfactants were always applied in the conditioning step. The suitable combinations of surfactant and organic reagent were tested for increasing of recoveries of analytes. Testing organic reagents were added in five times mass excess againts concentration of analytes, the concentration of the organic reagents was 900 µg•l-1. The organic reagent was always added to the solution for preconcentration before this solution was led on the sorbent. The composition and a necessary volume of elution mixture for quantitative elution were tested, too. Only one sorbent was chosen from each group of sorbents with similar properties. The chosen sorbent showed the best recoveries in majority of target analytes. The highest recoveries from silica-group was reached for Silicagel-C18, from nonpolar Amberlite was chosen Strata SDB-L and intermediately polar Amberlite XAD-7. The influence of some matrix components (anionts and cations) on recoveries of analytes was investigated for chosen sorbent. The optimized process was applied on real samples of nature water and industry water. In experimental part all analytes are marked as specific izotopes, which were measured by ICP-MS. Of course, all izotopes of analytes undergo preconcentration techniques.
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Preconcentration Techniques for Determination of Uranium Using the Modified Sorbents.
Moos, Martin ; Řehůřková, Irena (referee) ; Kanický, Viktor (referee) ; Kráčmar, Stanislav (referee) ; Sommer, Lumír (advisor)
The work has been focused on the preconcentration techniques for determination of uranium on the modified sorbent, with the final use of ICP-OES and ICP-MS. First ICP-OES at a wavelength of 385.958 nm was used for determination of uranium. Parameters were optimized and the effect of mineral acids (HCl, HNO3), tensides (Septonex, Ajatin) and organic reagents like (4-(2-pyridylazo)resorcinol, ammonium pyrrolidinedithiocarbamate, 8-hydroxyquinoline-5-sulphonic acid and 1,2-dihydroxyanthraquinone-3-sulphonic acid) was observed. For ICP-MS an isotope 238U was chosen. As an internal standard 209Bi (200 ?g•l-1) was used. Modified silica gel was used (Silicagel tethered with C18, C8 alkyls and phenyl) and two types of Amberlite XAD 4 and XAD 16 for the preconcentration of uranium (VI). Commercially produced Amberlite was modified as follows: Drying at 100 °C (24 hours), milled, minced sorbent was selected and fractionated to particles size ranging from 0.32 to 0.63 ?m and finally activated in methanol. The deionised water and Septonex (5•10-3 mol•l-1) at pH 8 were used for Amberlite conditioning. The optimal sorption of uranium (VI) proceeded at pH 8 in the presence of 4-(2-pyridylazo)resorcinol or ammonium pyrrolidinedithiocarbamate with Amberlite XAD-16 moreover with 1,2-dihydroxyanthraquinone-3-sulphonic acid. All organic reagents have mass concentration five time larger than uranium . For the elution of uranium the mixture of 1 mol•l-1 HNO3 with acetone (ratio of 1:1) was used. Acetone was evaporated and the final analysis performed using ICP-OES. The preconcentration of uranium (VI) using Silicagel-C18 conditioning was performed with ethanol, deionised water and Zephyraminu (5•10-4 mol•l-1) at pH 8. For the Silica-C8 and Silicagel-Phenyl the same conditioning procedure without zephyramin was used. Silica-C18 had the highest sorption efficiency with 1,2-dihydroxyanthraquinone-3-sulphonic acid, Silicagel-C8 with ammonium pyrrolidinedithiocarbamate and Silicagel-Phenyl with 8-hydroxyquinoline-5-sulfonic acid, all organic reagents have mass concentration five time larger than uranium concentration. As an optimal elution mixture acetone and ethanol (ratio of 1:1) in the presence of 1 mol•l-1 HCl was evaluated. Acetone and ethanol was evaporated and the final analysis was performed using ICP-MS. Silicagel-C18 was evaluate as the most effective in the presence of zephyramin and 1,2-dihydroxyanthraquinone-3-sulphonic acid. Sorption was also tested in the presence of 20 ?g•l-1 microelements (Be, V, Co, Ni, Y, Pb, Th, Cd) and in the presence of a defined concentrations of K, Na, Ca, Mg, Al and Fe. Direct analysis of uranium was carried out on samples of water from the river Ploucnice and Turonian aquifers containing uranium, 3,5 and 19,3 ?g•l-1. Different type of water required preconcentration of uranium (VI) on Silica-C18 in the presence of 1,2-dihydroxyanthraquinone-3-sulphonic acid and zephyramin. The analysis results gave statistically satisfactory results, which were confirmed using standard addition of 20 ?g•l-1.
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The possibilities of assessment of free fatty acids
Hornáková, Miroslava ; Vespalcová, Milena (referee) ; Vítová, Eva (advisor)
This thesis deals with the determination of free fatty acids in natural and processed cheese. In the theoretical part the possibilities of extraction, fractionation and determination of lipid fractions, characterization of fatty acids and various methods of their determination are described, including the determination of free fatty acids. In the experimental part the selected method for determination of free fatty acids was optimized and partly validated; this method was then applied to samples of processed cheese analogues and natural Gouda type cheese. For extraction of lipids from the sample the method according to ČSN 0107 was selected, solid phase extraction (SPE) was used for separation of free fatty acids. The method according to ČSN EN ISO 5509, using methanol solution of potassium hydroxide, was applied for esterification, fatty acids methyl esters were determined by gas chromatography with FID detection.
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Determination of platinum in Brno's agglomeration
Dvořák, Rostislav ; Řezáčová, Veronika (referee) ; Komendová, Renata (advisor)
This thesis is focused on the determination of platinum in samples of road dust and soil by atomic absorption spectrometry. The theoretical part describes the basic physical and chemical properties of platinum, platinum function in automotive converters and its behavior in the environment. In the experimental section summarizes the methods used to decompositions samples, concentration of the analyte and the main results of the measurements.
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