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Extraction of nucleotides from plant matrix and their determination by ion-exchange high performance liquid chromatography
Konečná, Markéta ; Sobotníková, Jana (advisor) ; Coufal, Pavel (referee)
In this thesis chromatographic conditions for the HPLC analysis of ribonucleotides AMP, ADP and ATP were optimized. For their separation chromatographic column, Nucleogel SAX 1000-8, 50 x 4,6 mm, from the German company Marcherey-Nagel was used. The mobile phase of a 0,2 mol/L solution of KH2PO4, its pH was adjusted with 1mol/L with potassium hydroxide to pH = 5,00. As the most suitable flow rate was selected 1,0 mL/min. The analysis was conducted using an isocratic elution. Detection was conducted by a DAD detector at a wavelength of λ = 260 nm. Substances were eluted in the following order AMP (tr = 0,911 min), ADP (tr = 1,667 min) and ATP (tr = 7,262 min). The total analysis time of the mixture of standards, under the conditions mentioned above, lasted for 10 minutes. For the extraction of adenosine-5-ribonucloetides the real matrix frozen and lyophilized leaves of tobacco virginia (Nicotiana tabacum L.) have been used. Two methods of extraction were applied. The first procedure was based on the extraction of AMP, ADP and ATP from the leaves of the tobacco plant by boiling deionized water. In the second method 0,07 mol/L HClO4 for the extraction instead of deionized water was used. Keywords: HPLC, adenosine ribonucleotides, AMP, ADP, ATP, tobacco virginia
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Analysis, separation and physico-chemical characterization of peptide hormones by capillary electrophoresis
Šolínová, Veronika ; Jelínek, Ivan (advisor) ; Feltl, Ladislav (referee) ; Coufal, Pavel (referee) ; Samcová, Eva (referee)
I 11 CoNcr,usroNs The work bas demonstrated that capinary zone elechophoresis and micelrar electokinetic cbromatography are high-perfomrance and high-sensitive methods for qualitative and quantitative anďysis and for physico-chemicď characÚerization ofpeptide hormones. using the strategy for the rational selection of experimental conditions for cE analysis and separations ofpeptides, ionogenic peptides were separated as cations in the classical and isoelectric buffers in the acidic region, pH 2- 2'5, and as anions in amphoteric buffer in weakry arkaline conditions, pH = 8'1. Non-ionogenic peptides were analyzed by miceilar electrokinetic chromatography with micelrar phase consisting of anionic detergent sodium dodecylsulphate in alkaline conditions, pH 8.8. The experimental conditions were selected with the aim to avoid the adsorption of peptides on the capillary wall, which was particularly reached with suppression of dissociation of si'anol gÍoups on the imer capillary wall in the acidic pH region and with electrostatic repursion negatively charged peptides from negatively charged dissociation silanol groups on the inner capillary wall in the alkaline pH region. The pH values of background electrolýes were chosen in the region, where the biggest differences in calculated specific charges of analyzed...
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High-performance chromatographic methods for determination of proteins
Vařilová, Tereza ; Pacáková, Věra (advisor) ; Feltl, Ladislav (referee) ; Coufal, Pavel (referee) ; Kučerová, Zdenka (referee)
Conclusions This Thesis gives an overview of high-performance riquid chromatography in various separation modes for a characterization of selected proteins. various affinity stationary phases were prepared for analysis of pepsin, seminal plasma proteins and selected glycoproteins' choice of the affinity system suitable for a particular purpose is always a comprex probrem in which number of aspects must be considered. properties of the proteins to be separated and ligands used had to be taken into an account. The separation system should not influence the biorogicai activity ofproteins separated. From this point of view it is clear that for any each individuar system is necessary to find specific conditions for I) immobilizationof ligand, and II) separation conditions. Stationary phases with immob'ized 3,5-diiodo-L-tyrosine exhibited sufÍicient selectivity, proved to be useful for analysis ofporcine pepsin A and are appricable to practical sampres of human pepsin' Reversed-phase crs corumn is suitable for anarysis ofpepsin fragments obtained after a-chymotrypsin digestion. A study oť interaction of various forms of pepsin and pepsinogen are essentiar because of their high importance as crinicar diagnostic markers and as stomach enzyme. "lA IŤ Aťfinity chromatography with heparin immobilized to Toyopearl...
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Polymethacrylate monolithic columns in capillary liquid chromatography
Grafnetter, Jan ; Coufal, Pavel (advisor) ; Pacáková, Věra (referee) ; Ventura, Karel (referee) ; Sýkora, David (referee)
3 Conclusions This Thesis gives chromatographic and structural characteristics of the butyl methacrylate monolithic columns prepared for the capillary liquid chromatography. Results of this Thesis clearly demonstrate the possibility of preparation of capillary monolithic columns with a good repeatability of the principal chromatographic parameters. A completely new technique for preparation of the monolithic columns for use in the on-column detection mode was introduced. Different photometric detection modes were applied to monolithic capillary columns in CLC. The advantages and disadvantages of the on-column detection mode in comparison to the additional detection capillary were studied and described. It was shown that the best separation and the most sensitive quantification in the short analysis time could be reached by using the monolithic columns with detection window placed directry on the separation capillary, just after the stationary phase or within the monolith. The attempts leading to preparation of chiral monolithic stationary phases, which would allow enantioseparation of selected chiral analýes' were not totally successful. No time was left to find the suitable chromatographic conditions for enantioseparations. \ I
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