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Determination of cholesterol, 7-dehydrocholesterol and cholecalciferol by HPLC with spectrophotometric and electrochemical detection
Dostálková, Lucie ; Schwarzová, Karolina (advisor) ; Vyskočil, Vlastimil (referee)
The aim of this thesis is to verify the possibility of simultaneous determination of three structurally related biologically active substances (cholesterol, 7-dehydrocholesterol and cholecalciferol) after previous separation by high-performance liquid chromatography followed by electrochemical (on a boron-doped diamond electrode) and spectrophotometric detection, which were compared in this work. The influence of the water content in the mobile phase (in the range of 0-10 %) on the retention factor of the analytes and their response for both types of detectors was investigated. Using all mentioted water contents, the analytes baseline-separated. As the water content increased, the retention factor of the analytes increased as well as the background current for higher positive detection potentials. Based on the recorded hydrodynamic voltammograms, the optimum detection potential + 1.8 V was chosen. The optimum wavelength for spectrophotometric detection was 200 nm. The concentration dependence was measured in an of 50 mmol l−1 sodium perchlorate in acetonitrile mixed with water (95:5, v/v). The spectrophotometric detector provided lower detection limits compared with the electrochemical detector, which had detection limits of 2.3 μmol l−1 for cholesterol, 1.4 μmol l−1 for 7-dehydrocholesterol and...
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Analysis of selected nutraceuticals by liquid chromatography
Ibrahim, Laureen Reda Youssef ; Jenčo, Jaroslav (advisor) ; Opletal, Lubomír (referee)
39 ABSTRACT Charles University, Faculty of Pharmacy in Hradec Králové Department: Department of Pharmacognosy and Pharmaceutical Botany Candidate: Laureen Reda Youssef Ibrahim Supervisor: RNDr. Jaroslav Jenčo, Ph.D. Title of dissertation thesis: Analysis of selected nutraceuticals by liquid chromatography The extract obtained pellets of Humulus Lupulus L. var Columbus was subjected to a phytochemical study and isolation of its bitter acid constituents. The summary extract was separated into individual fractions by liquid-liquid extraction. After performing HPTLC, GC-MS, and LC-MS analysis, the fraction HL-Hex B was selected and purified further by preparative chromatography. A monocomponent pale yellow solid substance of lupulone was obtained after the final preparative separation. Based on instrumental analysis by reversed phase HPLC with core-shell particles, the analyte was compared to the extract of dietary supplement HopsVital where the presence of bitter acids in the supplement was shown. Key words: nutraceuticals, high performance liquid chromatography, sample preparation, modern stationary phases
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Study of retention and separation of peptides in HPLC
Boudová, Hana ; Kalíková, Květa (advisor) ; Kubíčková, Anna (referee)
This diploma thesis deals with the study of retention and separation of structurally different peptides and digest products of cytochrome c using reverse phase and mixed modes of liquid chromatography. As the model peptides, five unmodified dipeptides, nine dipeptides containing a protective benzoyl group on the N-terminal amino acid and eight therapeutic peptides containing five to ten amino acids were selected. The chromatographic behaviour of the model peptides was tested on three different columns: XBridge C18, XSelect CSH C18 and Atlantis Premier BEH C18 AX under different conditions. The influence of the pH of the aqueous part of the mobile phase, i.e. 2,1; 3,0; 4,7 and 6,8, and the amount of acetonitrile in the mobile phase on retention and separation of model peptides was tested. The effect of the concentration of ammonium acetate buffer, pH = 6,8 in the mobile phase on the retention and peak symmetry in the concentration range of 10-50 mmol L-1 was assessed. The chromatographic conditions for the baseline separation of 20 different model peptides were optimized. The conditions were as follows: XSelect CSH C18 column, mobile phase composed of acetonitrile/10mM ammonium acetate, pH = 6,8 (v/v) under gradient elution. The suitable chromatographic conditions for the analysis and identification...
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Determination of antibiotics in the soil ecosystem
Brož, Tomáš ; Zlámalová Gargošová, Helena (referee) ; Mravcová, Ludmila (advisor)
This work deals with a current issue of increasing drug concentrations and thus the growing number of resistant bacteria in the soil. One of the problem helping this issue is the fertilization of agricultural fields with animal faeces, in which antibiotics occur in an unchanged and still active form, as well as the insufficient effectiveness of conventional methods used in wastewater treatment plants. In an effort to improve the monitoring of this problem, a method for the determination of these antibacterial agents in the soil matrix, using solid phase extraction, and also a UPLC/MS method for their evaluation have been developed and optimized. From the group of sulfonamide antibiotics, sulfacetamide, sulfadiazole, sulfamerazine, sulfamethazine, sulfamethoxazole, sulfapyridine, sulfasalazine, sulfathiaosal and trimethoprim were selected. The lowest limit of detection for the method is 0.3 µg/g for sulfathiazole and the highest of 8.1 µg/g for sulfasalazine. The highest average yields were achieved with sulfamethoxazole, namely 63 ±8 %.
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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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Analysis of adenosine triphosphate and adenosine diphosphate by HPLC-MS/MS
Černá, Martina ; Coufal, Pavel (advisor) ; Bosáková, Zuzana (referee)
In this bachelor thesis, ADP and ATP samples were analysed and detected with HPLC- MS/MS method. Approximate limit of detection (LOD) for these particular substances were found and their values were compared with the LOD values published in the literature obtained via the same methods and under very similar experimental conditions. Our limits of detection for nucleotides were comparable with the limits described in the literature. Mass spectrometry analysis was performed in the positive and the negative mode of multiple reaction monitoring analysis and electrospray was used for the analyte ionization. The optimal conditions for high performance liquid chromatography of ATP and ADP analysis were acquired on a ZIC - HILIC column with the mobile phase of 75:25 (v/v) acetonitrile / 10 mM ammonium acetate. Ammonium acetate buffer was adjusted to pH of 7.15 and the separation was done under the isocratic elution.
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Study of peptide digestion kinetics by trypsin and chiral separations of biologically active compounds by HPLC
Šlechtová, Tereza ; Tesařová, Eva (advisor) ; Čabala, Radomír (referee) ; Mikšík, Ivan (referee)
This dissertation thesis composes of two parts; the first part focus on the characterization of trypsin, enzyme frequently used in proteomic research for the investigation and identification of protein sequences, and its peptide digestion kinetics. The second part is aimed to the enantioseparations of biologically active compounds. First part of this project focus on tryptic digestion of synthetic peptides and the development of HPLC method for the identification of synthetic peptides and their fragments. Using the in-solution digestion and HPLC method, relative kinetic constants were determined for problematic sequences. Amino acids responsible for the decrease in trypsin catalytic activity and their location towards the cleavage site were studied. Certain slight exopeptidase activity of trypsin was noted, especially at the end of peptide chain. Furthermore, three columns with immobilized trypsin used in HPLC were compared concerning their catalytic activity. The immobilization of enzymes on solid support is used to elevate the amount of enzyme present during digestion and to assure better repeatability and reproducibility of obtained results. Activity of a new trypsin column synthesized at the University of North Carolina at Chapel Hill was compared to two commercially available trypsin columns....
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Characterization and comparison of new reversed-phase HPLC columns
Putzová, Klára ; Kalíková, Květa (advisor) ; Kozlík, Petr (referee)
The aim of the bachelor thesis was to evaluate and compare interaction/separation properties of three chromatographic columns, namely XBridge C18, XSelect CSH C18 XSelect HSS C18, designed for reversed-phase high performance liquid chromatography. All the columns contain the same ligand, i.e. octadecyl but they differ in the stationary phase support. The columns were compared in terms of silanol activity, hydrophobicity, shape selectivity and efficiency. For this purpose, the simple chromatographic tests designed by Walters, Engelhardt and Galushko were used. Furthermore, the separation of five selected pesticides, namely acetochlor, fomesafen, isoproturon, metazachlor triflumuron, were optimized on tested columns in simple mobile phases composed of acetonitrile or methanol as organic modifiers and water. All the tested columns showed low silanol activity and medium to high hydrophobicity acording to the chromatographic tests used. The low silanol activity corelates with endcapping procedure used for all the columns. All pesticides were more retained in methanolic mobile phase due to the lower elution strength of methanol compared to acetonitrile in reversed-phase high performance liquid chromatography. The analysis time of separation varied for each column under the same chromatographic conditions. The...
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Speciation Analysis of Selected Mercury Compounds Using HPLC, UV-Photochemical Cold Mercury Vapor Generation and its AAS Detection
Linhart, Ondřej ; Červený, Václav (advisor) ; Spěváčková, Věra (referee)
The mercury occurs in the environment in a variety of forms. Mercury compounds can be found in the soil, atmosphere, water and living organisms. Although some of the mercury substances are very toxic, they are often used in various sectors of industry, agriculture and medicine. Mercury compounds differ in their toxicity, so it is necessary to do speciation analysis. The aim of this diploma thesis was to develop and validate a new analytical method for the determination of mercury compounds in different samples. This method involves the combination of high performance liquid chromatography, UV-photochemical cold vapor mercury generation and atomic absorption spectrometry. Effective separation of mercury(II), methylmercury(I), ethylmercury(I) and phenylmercury(I) ions and subsequent comparable efficient of mercury cold vapor generation from all of forms was achieved using these techniques. The reached detection limits were 8 µg l-1 , 31 µg l-1 , 16 µg l-1 and 38 µg l-1 . At the end of experimental work, the proposed method of RP-HPLC-UV-CVG- QTAAS was used for the determination of mercury compounds in real samples (fish tissue and water samples: Labe, Vltava and tap water) and in certified reference materials (DORM-3 and DOLT-4). Several methods for extraction of mercury species from solid samples...
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