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Analysis of defensive substances of Diving beetles (Dytiscidae) by ultra-high performance liquid chromatography with mass spectrometry
Bosáková, Tereza ; Kozlík, Petr (advisor) ; Nesměrák, Karel (referee)
Obranný sekret potápníků (Dytiscidae) produkovaný jejich prothorakálními žlázami obsahuje řadu steroidních látek. Pro analýzu obranného sekretu potápníka druhu Ilybius fenestratus byla využita ultra- vysokoúčinná kapalinová chromatografie ve spojení s detekcí diodovým polem a hmotnostní detekcí. Analýza methanolického extraktu sekretu probíhala na koloně Acquity BEH C18 za podmínek gradientové eluce s dvousložkovou mobilní fází tvořenou methanolem s 0,1% obsahem amoniaku a 0,1% vodným roztokem amoniaku. V sekretu byla zjištěna přítomnost dvou steroidních hormonů, a to boldenonu a testosteronu a jejich identifikace byla potvrzena na základě shody jejich retenčních časů i hmotnostních spekter s příslušnými standardy.
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Comparison of contactless conductivity detection and direct UV detection sensitivity for electrophoretic determination of N-acetylaminosaccharides
Malý, Michal ; Křížek, Tomáš (advisor) ; Kozlík, Petr (referee)
is work deals with development and optimization of a method for separation and detection of N- acetylglucosamine and its dimer and trimer using capillary zone electrophoresis with contactless conductiv- ity detection. Separation and detection of these substances is possible if sodium hydroxide is used as back- ground electrolyte. Usage of another electrolyte such as disodium phosphate leads to unacceptable baseline noise level. Optimal conditions of this method are a background electrolyte concentration of 40 mmol dm−3 and voltage of 15 kV. e method is very sensitive to background electrolyte pH, used background electrolyte also causes rapid degradation of the inner capillary wall which is why this method has proven difficult to cali- brate properly. e developed method was compared to an already existing method which uses UV detection. e existing method has lower limits of detection and quantification and is less sensitive to working condi- tions. Limits of detection and quantification of the developed method are 34 µmol dm−3 and 104 µmol dm−3 respectively. Limits of detection and quantification of the method based on UV detection are 7 µmol dm−3 and 21 µmol dm−3 respectively. Limits of quantification are that of the analyte whose detection was the least sensitive which was the dimer in both...
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Spectrophotometric Determination of Chondroitine Sulphate by Sequential Injection Analysis
Hrubá, Lucie ; Hraníček, Jakub (advisor) ; Kozlík, Petr (referee)
This thesis deals with the optimization determination of chondroitin sulfate sequential injection analysis with spectrophotometric detection. The reaction proceeds in the presence of phenothiazine cationic dyes (Azure A, Azure B and methylene blue) and measuring the decrease absorbance dyes in their absorption maximum after the addition of chondroitin sulfate. Have found the optimum conditions for this determination: dye concentration 5 10-5 mole dm-3 , the dosing volume 100 µl CS + 50 µl dye + 100 µl CS, reaction time 0 s, flow rate 40 µl s-1 . For the determination of the CS in a static arrangement was found the lowest limit of detection and quantification using metylene blue (LOD = 0,23 mg l-1 a LOQ = 0,76 mg l- 1 ). The best repeatibility was achieved also using methylene blue 2,4 %. On the other side the best sensitivity was achieved using azure A. For the determination of the CS in SIA arrangement was found the lowest limit of detection and quantification using azure (LOD = 0,34 mg l-1 a LOQ =1,01 mg l-1 ). The best repeatibility was achieved also using azure A 1,9 %. And the best sensitivity was achieved using azure B. Based on these findings was chondroitin sulphate determined in food supplements, the more suitable method were the calibration curve .The determination was also carried out...
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Modern Trends in High Performance Liquid Chromatography and Their Application
Kozlík, Petr ; Bosáková, Zuzana (advisor) ; Pacáková, Věra (referee) ; Cajthaml, Tomáš (referee)
(EN) The dissertation thesis is focused on major trends in high performance liquid chromatography such as miniaturization of separation systems in hyphenation with high- sensitivity detection or characterization of new types of stationary phases for the separation of polar compounds in systems suitable for mass detection. Application of recently developed stationary phases in hydrophilic interaction liquid chromatography (HILIC) is also considered. Capillary liquid chromatography with tandem mass spectrometry (cLC-MS/MS) method was developed for determination of five estrogenic pollutants in samples of water. Several new sorption materials for solid phase extraction (SPE) were compared to obtain sufficient recovery of all the tested analytes. Discovery DSC-18Lt column provided the highest recovery (95 - 100 %). The optimized cLC-MS/MS with SPE was used for determination of estrogens in water samples in the order of units to tens of ng/L. HILIC separation systems with silica gel, cyclofructan and isopropyl cyclofructan modified silica stationary phases were tested and compared. Ability to donate protons and dispersion interactions are the main interactions that affect retention in HILIC with cyclofructan-based columns while they are less important in separation systems with bare silica stationary...
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Chiral and achiral separation using in-house coated β-cyclodextrin column, comparison with commercially available β-cyclodextrin column
Procházková, Hana ; Kalíková, Květa (advisor) ; Kozlík, Petr (referee)
The main aim of this work was to prepare a new chiral stationary phase by dynamic coating of an ion-exchange stationary phase by PEMPDA-β-cyclodextrin (N1 , N1 , N3 , N3 , N3 -pentamethylpropane-1,3-diamine-β-cyclodextrin) and to test column selectivity and separation abilities of the newly prepared sorbent for chiral and also achiral sets of analytes and to evaluate stability of the column. Commercial β- cyclodextrin stationary phase (Astec Cyclobond I 2000 column) was used for comparison. The set of chiral analytes contained five structurally different compounds - lorazepam, oxazepam, BNP, HDDP and Cl-HDDP. Six different groups of achiral analytes were used for testing the column selectivity. All measurements were carried out in reversed phase separation mode. Methanol and 10mM ammonium acetate (pH 4.00) were used as mobile phase constituents. Baseline separations of oxazepam enantiomers and five mixtures of achiral analytes were achieved with PEMPDA-β-cyclodextrin stationary phase. Partial separations of two chiral analytes (HDDP and Cl-HDDP) and one mixture of achiral analytes were obtained with this stationary phase. Enantiomers of BNP and oxazepam and four mixtures of achiral analytes were baseline separated on the Astec Cyclobond I 2000 column. Partial separations were obtained for the rest...
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The Comparison of Methods for the Determination of Ibuprofen in Medical Preparations
Kacerovská, Veronika ; Nesměrák, Karel (advisor) ; Kozlík, Petr (referee)
Tato bakalářská práce je určena k porovnání a zhodnocení čtyř vybraných analytických metod pro stanovení obsahu ibuprofenu v lékových formách z hlediska jejich finanční a časové náročnosti, pravdivosti a preciznosti. Byly vybrány čtyři analytické metody: acidobazická titrace s vizuální indikací konce titrace, UV spektrofotometrie, voltametrie a spektrofluorimetrie. Dále byly zvoleny čtyři léčivé přípravky: potahované tablety Brufen 400 mg, čípky Nurofen 60 mg, Dolgit gel 150 g a Dolgit krém 150 g. Nejpreciznější hodnoty poskytla acidobazická titrace a spektrofluorimetrie. Naopak nejméně precizní bylo voltametrické stanovení u vzorku potahované tablety Brufen a UV spektrofotometrické a voltametrické stanovení u vzorku Dolgit krému. Výsledky spektrofluorimetrického stanovení, voltametrického stanovení a UV spektrofotometrického stanovení se shodly s deklarovanou hodnotou, tj. povoleným rozmezím obsahu ibuprofenu v lékových formách. Z finančního hlediska byla nejvýhodnější spektrofluorimetrie, naopak nejnákladnější bylo voltametrické stanovení. Časově nejnáročnější bylo voltametrické stanovení, naopak nejrychlejší metodou byla acidobazická titrace. Klíčová slova: ibuprofen, acidobazická titrace, UV spektrofotometrie, voltametrie, spektrofluorimetrie
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HPLC study of pharmacological coupling reactions
Krouská, Hana ; Kubíčková, Anna (advisor) ; Kozlík, Petr (referee)
In this work decomposition of phenylisothiocyanate in aqueous and methanolic solutions was studied. RP-HPLC with UV detection was chosen to monitor these reactions. A simple method of isocratic elution has been developed. Due to a long total analysis time, it was necessary to develop also gradient elution method. Using these methods degradation rate of phenylisothiocyanate in methanol and in an aqueous solutions were monitored. Time course of decomposition exhibits exponential shape that is typice for the first-order kinetics. The measurements also showed that disintegration of phenylisothiocyanate is slower in water than in methanol. In both cases it is very slow - decomposition of half of the original quantity of phenylisothiocyanate occurs within days.
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Determination of estrogen pollutants in real water sample by HPLC-UV after solid phase extraction.
Kozlík, Petr ; Coufal, Pavel (referee) ; Bosáková, Zuzana (advisor)
4 Abstract Estrogens are considered to belong to chemicals that negatively affect the endocrine system, even if present at very low concentrations. They are discharged into environment as a result of an increasing application of drugs etc. This work is focused on the separation and quantification of five estrogens, namely estrone (E1), 17β-estradiol (βE2), 17α-estradiol (αE2), 17α-ethynylestradiol (EE2) and estriol (E3) in natural water samples by HPLC-UV method. The chromatographic system consisted of a C18 stationary phase (SunFire® C18, 150 x 4.6 mm, octadecyl bounded to silica gel, particle size 5 µm) and binary mobile phase of acetonitrile/water in various ratios in isocratic separation mode. The effect of acetonitrile content in the mobile phase and flow rate of the mobile phase on retention and separation parameters was tested. Under the optimized separation conditions (acetonitrile/water 40/60 (v/v), 1.3 ml/min), all the compounds were baseline resolved and eluted within 15 min. These experimental conditions were applied to the calibration measurements which were carried out within the concentration range from 0.001 to 1 mg/ml. Limits of detection (LOD) and limits of quantification (LOQ) for the individual estrogens and their mixture (standards dissolved in methanol) were determinated. The detection...
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