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Optimization of microextraction technique for analysis of water by chromatographic methods
Otrubová, Lucie ; Čabala, Radomír (advisor) ; Sobotníková, Jana (referee)
New liquid phase microextraction (LPME) has been optimized. Plastic adapter with tapered tip was used as extraction device. Concentration of analytes was determined by GC-MS. Face centered design was used to optimize LPME and the results were evaluated by Minitab 16 programme. Volume of sample, volume of extraction solvent, stirring rate, extraction time, ionic strength and time after microextraction were optimized. Response of system was defined as the sum of the peak relative areas. Toluene, tetrachlorethylene, ethylbenzene, xylenes, mesitylene and naphthalene were the studied analytes. Methylhexadecanoate was used as an internal standard. Optimal system conditions were as follows: 20 mL sample volume, 300 μL extraction solvent volume, 20 minutes, stirring speed 700 rpm, no addition of salt, and time after microextraction 3.5 minutes. Determination coefficient was 0.9700 and the lack-of-fit was insignificant which indicated good agreement of the model with the experimental data. The preconcentration factor ranged from 26 (tetrachlorethylene) to 39 (xylenes). The microextraction yield was varied from 39.5% (tetrachlorethylene) to 59.1% (p-xylene and m-xylene). The new method was tested by analyzing real samples of tap water, river water and water from the sewage treatment plant. The samples were...
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Post-column derivatization in HPLC for analysis of amino acids
Vaňkátová, Petra ; Coufal, Pavel (advisor) ; Sobotníková, Jana (referee)
Proteinogenic amino acids are the basic structural building units of proteins. Their analysis is important in many fields, especially in medicine. This thesis deals with one of the methods of derivatization of amino acids in order to increase their absorption in the UV - the post-column derivatization using copper (II) oxide. It is quick, robust and easy-to-use method. Following the nowadays trend in the analysis of free amino acids, this thesis is focused on the HILIC mode separation environment.
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Preparation and testing of capillary packed column sephadex G-10
Tolasz, Jakub ; Coufal, Pavel (advisor) ; Sobotníková, Jana (referee)
Sephadex is a common sorbent in biochemistry for separation of molecules of a wide range of molecular masses. Sephadex is also used for purification or desalination of isolated proteins. Up to now, the gel chromatography has been performed in wide separation columns. Therefore, the isolation techniques for proteins had to be very effective to isolate enough proteins as a sufficient amount of the sample. The preparation and application of a capillary separation column packed with sephadex could decrease the needed sample amount and enable work with hardly isolable proteins obtained only in small quantities. In this bachelor project, the capillary column was packed with sephadex using the slurry packing procedure. The principle of monolithic columns was used to create a gel in the column. The column was packed in organic solvent firs, and then hydrated when flushing with water to generate the gel.
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Separation of phospholipids by high pressure liquid chromatography
Erdeová, Karolína ; Čabala, Radomír (advisor) ; Sobotníková, Jana (referee)
Fosfolipidy jsou součástí buněčných membrán všech živých organismů. Jedná se o nejvýznamnější skupinu polárních lipidů, vyznačující se amfifilními vlastnostmi. Tyto vlastnosti vyplývají z jejich molekulární struktury, která se skládá z hydrofobních mastných řetězců a polární hlavice. Podle polární hlavice se dělí fosfolipidy do příslušných tříd. Poměr fosfolipidových tříd, zastoupených v bakteriální membráně, značně odráží životní pochody mikroorganismu. Pomocí změn v zastoupení těchto tříd lze sledovat vliv vnějšího prostředí, čehož se využívá v mikrobiologickém výzkumu. Pro sledování fosfolipidového složení vyšlechtěného kmene Bacillus subtilis, který je intenzivně zkoumán pro produkci antibiotik, se stále využívá tenkovrstvá chromatografie. Její časová náročnost výzkum značně zpomaluje, a proto byly podmínky tohoto uspořádání přeneseny na HPLC. Pro tyto účely byla metoda optimalizována formou nelineárního gradientu. Vhodnost metody byla testována na vzorku bakteriálních membránových lipidů, který obsahoval pět fosfolipidových tříd, z čehož dvě majoritní. Jejich poměrné zastoupení lze stanovit, nicméně je nutno pečlivě volit objem nastřikovaného vzorku, aby se zabránilo překročení úzkého koncentračního rozsahu metody. Klíčová slova: fosfolipidy, HPLC, optimalizace, validace, HILIC, NP-HPLC
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Quantitative Structure-Retention Relationships of Salicylthioamide Derivatives
Kalužíková, Aneta ; Nesměrák, Karel (advisor) ; Sobotníková, Jana (referee)
The aim of the thesis is to study the relationship between the structure of N-benzylsalicylthioamide derivatives and their retention behavior in high-pressure liquid chromatography. Furthermore, the influence of various substituents on electron absorption spectra in ultraviolet spectral range was studied. All studied derivatives have two absorption maxima in ultraviolet spectral range at 260 nm and 293 nm. The substitution of an auxochrome on the acyl ring leads to bathrochromic shift and hyperchromic shift, especially of the first absorption maxima. The presence of an auxochrome on the amide ring leads to hyperchromic shift as well. HPLC reversed-phase chromatography, using XDB-C18 ZORBAX column and mobile phase with various composition of acetonitrile, was used to obtain retention times of the derivatives. Using this data, the correlation equations between retention factor (mobile phase with no acetonitrile), resp. slope of the dependence of the logarithm of retention factor on the volume fracture of acetonitrile in mobile phase, and partition coefficient octanol-water, resp. hydrophobic substituent constants, were derived. Key words: QSAR, HPLC, spectrometry, benzylsalicylthioamides
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Development of miniaturized extraction method used for GC-NCD screening of non-volatile nitroso compounds in malt
Malečková, Michaela ; Sobotníková, Jana (advisor) ; Kameník, Zdeněk (referee)
The aim of this diploma thesis was to develop a miniaturized extraction method for a fast screening of non-volatile nitroso compounds using gas chromatography with a nitroso specific chemiluminescence detection. According to a final methodology, the samples were prepared by extraction of grinded malt using a mixture of pyridine and acetonitrile in ratio 60:40 (v/v). To enhance volatility of the determined analytes, the two-step derivatization using hexamethyldisalazane and N,O-bis(trimethyl)-trifluoroacetamide was used. The total volume of the sample was 200 l and the preparation time after optimization was in total 80 min. The extraction method was connected to a classification method, which can divide chromatographic peaks into the groups of N-nitroso and C-nitroso compounds, and interfering substances. After application of the methods mentioned above to real malt samples, the specific chromatographic peaks of C-nitroso and N-nitroso compounds were selected. Description of their properties and structure suggestion will be a subject of the following study. Keywords Nitroso compounds, malt, extraction, derivatization, gas chromatography, chemiluminescence detector
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Use of comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometric detection for metabolomic analysis of Gloeophyllum trabeum fungus
Kuchler, Ondřej ; Sobotníková, Jana (advisor) ; Kozlík, Petr (referee)
Fungus Gloeophyllum trabeum (Agaricomytes: Gloeophyllates) is a brown rot wood-decay fungus which produces a vast spectrum of volatile secondary metabolites. Scientific publications state, that one of the metabolites produced by G. trabeum, can be the substance (3Z,6Z,8E)-dodecatrien-1-ol. This chemical substance is also the main component of trail-following pheromone of Rhinotermitidae termite family. In this diploma thesis, I was trying to verify whether various species of G. trabeum are in fact capable of producing the substance (3Z,6Z,8E)-dodecatrien-1-ol. I was also focusing on the effects of saccharides, present in nutrient solutions, on quantitative and qualitative change in composition of volatile secondary metabolites produced by G. trabeum. The saccharides I used for my research were - maltose, fructose, sucrose, xylose, and mannose. The analysis was made by using comprehensive two-dimensional gas chromatography separation technique with time-of-flight mass spectrometric detection (GC×GC-TOFMS). During my research I discovered that one of obtained species of G. trabeum can produce substance (3Z,6Z,8E)-dodecatrien-1-ol, but only under specific conditions. It is produced when cultivating on Petri dishes on agar - cellulose growth media. The measurement was further validated by...
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