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Preparation and testing of capillary packed column sephadex G-10
Tolasz, Jakub ; Coufal, Pavel (advisor) ; Sobotníková, Jana (referee)
Sephadex is a common sorbent in biochemistry for separation of molecules of a wide range of molecular masses. Sephadex is also used for purification or desalination of isolated proteins. Up to now, the gel chromatography has been performed in wide separation columns. Therefore, the isolation techniques for proteins had to be very effective to isolate enough proteins as a sufficient amount of the sample. The preparation and application of a capillary separation column packed with sephadex could decrease the needed sample amount and enable work with hardly isolable proteins obtained only in small quantities. In this bachelor project, the capillary column was packed with sephadex using the slurry packing procedure. The principle of monolithic columns was used to create a gel in the column. The column was packed in organic solvent firs, and then hydrated when flushing with water to generate the gel.
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Determination of adenosine triphosphate and adenosine diphosphate in real samples
Černá, Martina ; Coufal, Pavel (advisor) ; Zima, Jiří (referee)
The aim of the diploma thesis was to find optimal conditions of high pressure liquid chromatography for the detection and quantification of two common nucleotides, namely adenosine diphosphate and adenosine triphosphate, as well as to perform an analysis of these in real life samples of citrus fruits and plant extracts. Further aim of the project was to determine the limits of detection and quantification of adenosine diphosphate and adenosine triphosphate under the optimized conditions and using these to compare the sensitivity of given detectors. To achieve this HPLC-UV, capillary HPLC-DAD and HPLC-MS apparatus were used. With the help of HPLC with UV detection and capillary HPLC with diode array detector, the calibration curves of the mixture of analytes were measured and the limits of detection as well as quantification of adenosine diphosphate and adenosine triphosphate were determined. Separation of the analytes up to the base line using HPLC-UV and capillary HPLC-DAD was achieved under the conditions of ion pairing chromatography. Column C18 was chosen as an appropriate column. The mobile phase included phosphate buffer, acetonitrile and tetrabutylammonium bisulphate as an ion pairing reagent. The separation was performed with gradient elution. Conditions for analysis using LC-MS were...
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Determination of double bond position in wax esters by dimethyl disulfide derivatization and mass spectrometry
Háková, Martina ; Schwarzová, Karolina (advisor) ; Coufal, Pavel (referee)
Wax esters are substantial constituents of natural waxes, which can be found in many living organisms. Properties of lipids, including wax esters, may be significantly influenced by the position of double bond. In this diploma thesis the location of double bonds was determined by dimethyl disulfide (DMDS) derivatization followed by detection using tandem mass spectrometry with atmospheric pressure chemical ionization (APCI) and electrospray ionization (ESI). We managed to measure the APCI and ESI MS/MS spectra of 8 different wax esters with different position of double bond. Diagnostic ions determining double bond position were identified. This method could be used in HPLC/MS analysis of wax esters, which cannot be analyzed by GC/MS. It was shown that the DMDS derivatization reaction and mass detection with APCI ionization is also suitable for locating double bonds in alkenes.
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Analysis, characterization and separation of 2,6-diaminopimelic acid derivatives by capillary zone electrophoresis and micellar electrokinetic chromatography
Vítovcová, Miloslava ; Coufal, Pavel (advisor) ; Čabala, Radomír (referee)
In this work, capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) with UV-photometric detection were used for the determination of degree of chemical purity, limit of detection (LOD) and limit of quantitation (LOQ) of N-acyl derivatives of 2,6-diaminopimelic acid (DAP). The characterization of DAP derivatives was performed by determination of their effective electroforetic mobilities in several background electrolytes in acidic and alkalic pH range. The mobilities were corrected to reference temperature 25 řC. Separation of the mixture of DAP derivatives were achieved by MEKC in acid background elektrolyte (500 mmol·dm-3 acetic acid, pH 2.54) using anionic surfactant 60 mmol·dm-3 sodium dodecylsulfate (SDS) as a constituent of the micellar pseudostationary phase. Subject words: analytical chemistry, separation methods, capillary electromigration methods, derivatives of amino acids Key words: capillary zone electrophoresis, micellar electrokinetic chromatography, derivatives of 2,6-diaminopimelic acid
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The use of ambient ionization techniques in mass spectrometry
Rejšek, Jan ; Cvačka, Josef (advisor) ; Coufal, Pavel (referee)
Keywords: ambient ionization techniques; mass spectrometry; desorption electrospray ionization; desorption atmospheric pressure photoionization; thin layer chromatography; lipids, mass spectrometry imaging Ambient ionization technique in mass spectrometry is an ionization, which carries out in open space outside the machine and which does not require any, or only a minimal sample pretreatment. DESI (desorption electrospray ionization) and DAPPI (desorption atmospheric pressure photoionization) equipped with software control of the spray emitter position for analysis of low molecular organic compounds were investigated in this thesis. These methods use a spray of solvents for desorption and ionization molecules from solid substrate. Conditions for the successful analysis of phospholipids, wax esters and some other compounds were developed. Ambient ionization techniques were quantitatively compared. The application was HPTLC/DESI-MS of lipid's mixture and HPTLC/DAPPI-MS of vernix caseosa. DAPPI-MS was applied for the analysis of termites of Prorhinotermes genus (Isoptera, Rhinotermitidae). Pilot experiments of two dimensional analysis and mass spectrometry imaging were realized.
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Chiral separation of acyclic nucleoside phosphonates and determination of association constants of their complexes with cyclodextrins by capillary electrophoresis
Mikysková, Hana ; Coufal, Pavel (advisor) ; Jelínek, Ivan (referee)
Capillary electrophoresis (CE) has been applied to the chiral separation of acyclic nucleoside phosphonates, which belong toof the most important already registered or potential antiviral drugs. For enantioseparation of acyclic nucleoside phosphonates, the following native and derivatized cyclodextrin were used as chiral selectors: cyclodextrin, cyclodextrin, cyclodextrin, heptakis(2,6dimethyl) cyclodextrin, 2hydroxypropylcyclodextrin and quaternary ammonium cyclodextrine). The best resolution of (R,S)enantiomers of acyclic nucleoside phosphonates was achieved in basic backgroung electrolyte composed of 35 mM sodium tetraborate; pH 10, with addition of 17.6 mM cyclodextrin or 10 mM quaternary ammonium cyclodextrin chiral selectors. Capillary electrophoresis has been employed for determination of association constants of complexes of acyclic nucleoside phosphonate enantiomers with above mentioned chiral selectors. The association constants were determined by nonlinear regression analysis from the experimentally measured dependence of effective electrophoretic mobilities of the analytes on concentration of the chiral selector in the background electrolyte. Prior to association constant calculation, the effective electrophoretic mobilities were corrected to reference temperature,...
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System peaks in chip electrophoresis
Hezinová, Markéta ; Gaš, Bohuslav (advisor) ; Coufal, Pavel (referee)
This work investigates the behavior of system peaks in chip electrophoresis. The aim of this work was to propose a suitable separation environments with regard to the position and number of system peaks. In the theoretical introduction we first deal with processes taking place in the separation of proteins by SDS-PAGE method, its miniaturization and provide an overview of current application of this method. Then there is briefly summarized the issue of system zones. System peaks were observed in the three separation systems. The behavior of the system zones was first investigated theoretically in PeakMaster program and results were then verified experimentally by means of Agilent 2100 Bionalyzer. In the experiments there was recorded larger number of system peaks than should correspond to the theoretical composition of the systems. Due to this fact it was not possible to fully identify the theoretical results from PeakMaster with the results from real experiments.
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