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Posttranslational modification of proteins - their analysis and physiological aspects
Mikulíková, Kateřina ; Mikšík, Ivan (advisor) ; Bílková, Zuzana (referee) ; Glatz, Zdeněk (referee)
This thesi s is eoneerned with the deteetion of posttranslational modifieation of proteins. These proteins are identified using liquid ehromatography and eapillary eleetrophoresis, their eoupling and eoupling to mass speetrometry. The first part of the theoretieal ehapter is devoted to the strueture, classifieation and metabolits ehanges of proteins in the human body. Major attention is foeused on eollagen, its strueture and individual eollagen types. The seeond part eonsiders the problems of nonenzymatie glyeation and its influenee on the body and engage in produets of posttranslational modifieation. The third part summarizes the individual separation proeedures and analysis of posttranslationaly modified proteins. The seeond ehapter deseribes experimental parameters, including ehemieals, instrumentation, materials and methods. The preparation of individual samples is also deseribed. All results and findings are summarized and diseussed in the last ehapter. This ehapter is divided in two parts. The first part foeuses on problems of in vivo experiments. The seeond part foeuses on problems of in vitro experiments. Powered by TCPDF (www.tcpdf.org)
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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 pesticides enantiomers in water
Šlechtová, Tereza ; Lokajová, Jana (referee) ; Tesařová, Eva (advisor)
This Barchelor's project deals with chiral herbicide mecoprop and its determination by high-performance liquid chromatography method with teicoplanin based chiral stationary phase. The teoretical part focus on basic facts conserning chirality, chiral pesticides and problems related to their usage and effects to the enviroment. It also involves the ways of their determination by the above mentioned method and gives several examples of previous separations. The experimental part is based on method optimalization and determination of changes in mecoprop enantiomeric ratio in various water samples during different storage conditions.
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Determination of carvacrol using HPLC with electrochemical detection
Mužíková, Jana ; Schwarzová, Karolina (referee) ; Dejmková, Hana (advisor)
This thesis deals with determination of a mixture of carvacrol with thymol and eugenol by HPLC with electrochemical detection. Carbon paste electrode was used as the working electrode. The separation was performed on Kromasil-C18, 250x4,6 mm column. For the comparison, UV spectrophotometric detection at 275 nm was used besides electrochemical detection. Optimal separation conditions were found: mobile phase consisting of acetonitrile and phosphate buffer in ratio 60:40, the optimal buffer pH was pH 4. As the optimum potential of working electrode during electrochemical detection, potential +1.1 V was found. Under the obtained optimal conditions, calibration dependences were measured. Limit of quantification for carvacrol was found to be 9,6·10-7 mol dm-3 using UV spectrophotometric detection and 4,0·10-8 mol dm-3 using electrochemical detection.
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Speciation analysis of chromium in particulate matter of urban dust
Rybínová, Marcela ; Hraníček, Jakub (referee) ; Rychlovský, Petr (advisor)
Anion-exchange chromatography with inductively coupled plasma - atomic emission spectrometry (ICP-AES) has been used for the speciation of chromium (Cr). Chromium speciation has attracted attention because of the different toxicity, Cr(III) is relatively non-toxic and Cr(VI) has been classified as a human carcinogen. The aim of the present study was to develop simple method for the speciation analysis of Cr (Cr(III) and Cr(VI)) in particulate matter of urban dust. A combination of 2% KOH + 3% Na2CO3 has been chosen as the optimal reagent for the extraction of chromium from particular matter. It was found that there was no conversion of Cr(VI) into Cr(III). The effect of separation parameters such as acidity of mobile phase was also studied. The detection limit for Cr(VI) was about 12 ng.ml-1 . Results for the determination of Cr(VI) were confirmed by the analysis of standard reference material (BCR CRM 545, Cr(VI) in welding dust loaded on a filter) with good agreement between certified (40,16 ± 0,60 μg.g-1 ) and found (37,83 ± 1,14 μg.g-1 ) values.
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Determination of enantiomers of amino acids by HPLC
Michalčíková, Regina ; Janečková, Lucie (referee) ; Tesařová, Eva (advisor)
This Bachelor's thesis deals with determination of amino acids enantiomers, namely alanine in the experimental part, by high performance liquid chromatography using chiral stationary phases based on teicoplanin. The theoretical part is focused on summarizing the fundamental issue of chirality and chiral amino acids in foods, the characteristics of high-performance liquid chromatography, as one of the most appropriate methods to enantioseparation amino acids, and description of the methods of derivatization, which are used in determination of enantiomers of amino acids. The experimental part deals with the separation of alanine enantiomers, with the choice of the best separation system for the separation and determination of D-and L-alanine in fruit juice.
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Determination of propyl gallate on carbon paste electrode
Vysoká, Marie ; Fischer, Jan (referee) ; Zima, Jiří (advisor)
Propyl gallate (PG) is a significant synthetic antioxidant and preservative. Its determination has been studied at carbon paste electrode (CPE) using differential pulse voltammetry (DPV) and high performance liquid chromatography with electrochemical detection (HPLC-ED) and with UV spectrometric detection (HPLC-UV). Concentration dependences were measured in the media of Britton-Robinson buffer (pH 5) and methanol (20 %, v/v) by DPV and the limit of detection 0,6110-7 moldm-3 was obtained. Using HPLC with a mobile phase consisting of 0,01 moldm-3 phosphate buffer (pH 4) and methanol (50 %, v/v) with potential of working electrode E = +0,8 V and detection wavelength λ = 280 nm, concentration dependences were measured. Limit of detection was determined to 0,39 moldm-3 for HPLC-ED and 4,95 moldm-3 for HPLC-UV. After verification of the extraction procedure PG was determined in vegetable oil. The resulting value of 3,2 mgkg-1 corresponds with permited limits.
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HPLC separation of chlorophenoxypropionic acid derivatives
Horová, Kateřina ; Kalíková, Květa (advisor) ; Šlechtová, Tereza (referee)
This Bachelor's thesis focuses on the separation of chlorophenoxypropionic acids and their derivatives by a high-performance liquid chromatography. The separations were performed in a reversed phase mode. These compounds are employed as herbicides, i.e. chemical substances to control unwanted plants, to kill weeds. The herbicides studied for the purposes of this thesis were 2-(4-chloro-2-methylphenoxy)propionic acid, 2-(2,4- dichlorophenoxy)propionic acid, 2-(2-chlorophenoxy)propionic acid, 2-(3- chlorophenoxy)propionic acid and 2-(4-chlorophenoxy)propionic acid. The study aimed at finding and optimizing chromatographic conditions for simultaneous separation of all five herbicides mentioned above. The analysis employed two types of columns, that is ZORBAX SB-C8 column and ZORBAX Eclipse XDB-C18 column whose level of polarity differ. As mobile phases, there were used acetonitrile/water, acetonitrile/formic acid, pH 2.1 and methanol/formic acid, pH 2.1 in various volume ratios. ZORBAX SB-C8 column, mobile phase methanol/formic acid (concentration of 0.365 mol/l, pH 2.1) 40/60 (v/v), temperature of 25řC, and flow rate of 1 ml/min delivered the most convenient separation conditions. Given these isocratic conditions, all the herbicides were baseline separated within 80 minutes. Keywords: HPLC,...
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Biochemical methods as tool for study of reproductive proteins
Postlerová, Pavla ; Zigo, Michal ; Pohlová, Alžběta ; Jonáková, Věra
Study of molecular mechanisms in reproduction is essential for the understanding of this outstanding process. Our lab studies proteins secreted by reproductive organs and sperm using various biochemical methods for a long time. We have expertise in protein extraction from spermatic cells using different approaches, and by kits for proteins from the sperm surface and distinct subcellular compartments. The proteins of reproductive organ fluids are separated by chromatographic methods, such as size exclusion chromatography, high-performance liquid chromatography with reverse phase (RP-HPLC) and affinity chromatography on matrices with various ligands. Proteins are subjected to SDS- or 2D-electrophoresis for their characterization and comparison of various extraction methods, different mammalian species, and sperm in different functional development. Electrophoretically separated proteins may be transferred onto nitrocellulose membrane (Western blot) for antibody detection or binding studies with lectin-labelled ligands (lectins, polysaccharides, zona pellucida glycoproteins). We use immunoprecipitation method with specific antibody for protein determination followed by the MALDI identification. Proteins are localized by immunofluorescent techniques on/in spermatic cells and tissue sections of reproductive organs. Isolation of proteins from reproductive tissues and fluids, and the antibody detection is crucial for the studying of reproductive protein origin.
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