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Study of vortioxetin complexation by native cyclodextrins and its determination using capillary electrophoresis
Počtová, Žofie ; Křížek, Tomáš (advisor) ; Sobotníková, Jana (referee)
In this master thesis, the formation of supramolecular complexes between vortioxetine active substance and cyclodextrins (α, β, γ) is examined and the stability constants of these complexes are determined. The stoichiometric ratios of cyclodextrin and vortioxetine 1:1 and 2:1 are considered. The results suggest that the 1:1 ratio of cyclodextrin and vortioxetine is more accurate for each of used cyclodextrins and that the strongest interaction is between vortioxetine and β-cyclodextrin. Further, a method for vortioxetine assay in a tablet is developed by using non-aqueous capillary electrophoresis technique. The precision, accuracy, and robustness are validated and the values of detection and quantification limits are determined. This method can be used as a verification method for the results obtained by HPLC as the separation principle is different in capillary electrophoresis. Also, the stability of vortioxetine as a pure powder and as a mixture with excipients is examined under the conditions of elevated temperature and humidity.
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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 monolithic columns for hydrophobic interaction chromatography
Vlková, Michaela ; Sobotníková, Jana (advisor) ; Zima, Jiří (referee)
In frame work of this diploma thesis, monolithic stationary phases based on hydroxymethylmethacrylate were prepared in fused silica capillaries of 320 μm innerdiameter. Monolithic columns were synthesized by a simple procedure using a polymerization mixture, consisting of a monomer N-(hydroxymethyl) methacrylamide (HMMAA), a croslinking agent ethylene dimethacrylate (EDMA), porogenic solvents butane- 1,4-diol, propane-1-ol and an initiator α,α′-azobisisobutyronitrile (AIBN). Prepared HMMA monolithic columns were utilized for separation of mixtures of biologically active compounds, namely peptides with small number of amino acids. Mechanical strength and specific permeability were determined for selected monolithic columns. Keywords: HPLC, HILIC mechanism, hydroxymethyl methacrylate (HMMA) monolithic columns, amino acid, enkephalins.
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Determination of non-volatile N-nitrosamines in malt
Vavrová, Dominika ; Sobotníková, Jana (advisor) ; Čejka, Pavel (referee)
The aim of diploma thesis has been development and optimization of method determination of N- nitrososarcosine and N-nitrosoproline in malt by gas chromatography with chemiluminiscence detector. Optimization of extraction method has been performed by response surface method. Quantification has been performed by internal standard method (N-nitrosopipecolic acid, in which matrix effects has been studied. These has been verified in münchen and pilsen malt, therefore matrix-matched calibration has been constructed. The developed method has been aplicated on wheat, münchen and pilsen malt. N- nitrosoproline was detected only in münchen malt and in other cases has been under limit of detection (LOD=4,0 µg/kg). N-nitrososarcosine was in all cases under limit of detection (LOD=3,7 µg/kg). The matrix-matched calibration has been constructed for experimental münchen malt with N-nitrosoproline concentration at 13,2 ± 2,9 µg/kg. Powered by TCPDF (www.tcpdf.org)
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Determination of biologically active compounds in hops by LC/HR-MS method
Jandovská, Vladimíra ; Sobotníková, Jana (advisor) ; Spáčil, Zdeněk (referee)
This thesis describes development and optimalization of method for identification of polyphenols compounds present in hops (Humulus lupulus L.) that have potential antimicrobial activity. Within extraction of compounds from hop, minced hop cones were applied in a growth medium, where compounds diffused into the medium and created an inhibition zone after inoculated with Streptococcus aureus. The method combining extraction with acetone a salting out using QuEChERS principles was optimized for extraction of compounds from solid growth medium. The analysis was carried out using LC/HR-MS with a quadrupole-orbitrap hybrid mass analyzer. The MS method was developed as a screening method with a subsequent fragmentation of compound of interest on the base of inclusion mass list. The compounds extracted from inhibition zone were identified either by searching against a database or their structures have been elucidated on the basis of their fragmentation spectra.
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Analysis of polyphenolic compounds in selected brewery matrices by HPLC-HRMS method
Zušťáková, Veronika ; Sobotníková, Jana (advisor) ; Mikyška, Alexandr (referee)
The thesis has focused on the separation of proanthocyanidins (Pas) in beer, raw beer material and beer intermediates (wort, hopped wort, green beer) by using high performance liquid chromatography coupled to high resolution mass spectrometric detection. A chromatographic column XSELECT HSS T3 was used for separation of individual proanthocyanidins. Mobile phase was formed by a mixture of 0,1% formic acid in water (A) and 0,1% formic acid in acetonitril (B), separation was conducted in gradient mode. Calibration range for catechin and procyanidin B1 a B2 was linear in the range from 10 µg.l-1 to 200 µg.l-1 and for epicatechin from 5 µg.l-1 to 200 µg.l-1 . Limit of quantitation (LOQ) was set to the lowest point of calibration curve, hence for catechin, procyanidin B1 and B2 was LOQ = 10 µg.l-1 and for epicatechin was LOQ = µg.l-1 . Yield of method of the observed Pas was very good, where yield oscillated around 100%. The method also had excellent repeatability, where the relative standard deviation (RSD) was lower then 20%. After optimization of this method it was used for observation of profile changes during brewery process in catechin, epicatechin, procyanidin B1, B2 and B3. It was discovered, that catechin, epicatechin, procyanidin B1 and B2 originate mainly from hops and procyaniding B3 is...
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Electrophoretic determination of organic acids in industrial solutions
Taraba, Lukáš ; Křížek, Tomáš (advisor) ; Sobotníková, Jana (referee)
This work deals with the development and optimization of conditions of pretreatment of two industrial surface finishing baths containing chromium(III) ions and oxalic, maleic, acetic or citric acid and their electrophoretic analysis. Some model mixtures containing known amounts of components of industrial solutions have been made for simulation of complex matrices of the real samples. Prior to analysis a sample pre-treatment consisting of different dilution and addition of fluoride, hydroxide or EDTA anions as suitable agent releasing acid out of the stable chromium complex were studied. Determination of organic anions was accomplished by indirect UV detection at 350 nm with a reference at 230 nm. A commercially available background electrolyte, pH 5.7, was used for separation of analytes. The most appropriate pre-treatment to release acids have been achieved by precipitation of chromium(III) hydroxide. The method of standard additions was used for the quantification. The concentrations of oxalate and citrate in the real samples were calculated as 96,50 % (S.D. = 0,71 %) and 97,53 % (S.D. = 0,79 %), respectively, of declared amount. Satisfactory repeatabilities were obtained for both analytes with R.S.D. values (n = 5) for migration times lower than 0,51 %, R.S.D. for peak areas of oxalic acid were...
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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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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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Mobilization of electroosmotic flow markers in capillary electrophoresis
Martínková, Eva ; Křížek, Tomáš (advisor) ; Sobotníková, Jana (referee)
Capillary zone electrophoresis is a separation method used to separate charged substances in electric field. Substances migrate to detector due to their own electrophoretic mobility and electoosmotic flow in capillary. Separation is possible because of electroforetic mobility of substances, which is directly proportional to the charge and undirectly proportional to the hydrated radius of ion. This means that neutral molecules form just one neutral zone, which is detected together. Well absorbing neutral substances are thus used as markers of average (mean) EOF. It was found, that some of the markers have dispositions to be mobilized with respect to the EOF. This mobilization is caused by interactions of free electron pair on atoms of nitrogen or oxygen of marker with components of background buffer. The aim of the study was to measure mobilization of some commonly used markers with different cations of acetate buffer and thus find "ideal" marker, whose mobility is almost the same as EOF and find combinations of marker+buffer, which are better to be avoided. Eleven markers in combination with six cations of background electrolyte were studied. We have found, that relative mobilities of some of chosen markers come up to five percent of limit mobilities of small inorganic cations. An appropriate...
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