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Sample Preparation for Determination of Alpha-tocopherol in Erytrocyte Membrane by High Performance Liquid Chromatography
Denková, Barbora ; Šatínský, Dalibor (referee) ; Solich, Petr (advisor)
In this thesis, a new method for isolation of α-tocopherol from human erythrocyte membranes, using ultracentrifugation and solid phase extraction (SPE), for next HPLC analysis, was developed. Rightly SPE is nowadays one of the most effective and reliable modern methods permitting appropriate preparation of biological material sample before HPLC analysis. The developed SPE procedure is the following: 1) Washing of taken erythrocytes: 2,5 ml of blood is centrifugated 10 min at 1300×g, 4žC and the upper layer of plasma is separated. 0.01% saline BHT solution is added to the erythrocytes to a final volume of 2.5 ml and the centrifugation is repeated. The supernatant is removed and the whole operation is repeated three times. Washed erythrocytes are stored at a temperature of -86žC. 2) Proper α-tocopherol extraction from washed erythrocytes: 500 μl of 50 mmol/l D-mannitol solution and 500 μl of 5 mmol/l HEPES are introduced in the ultracentrifuge cuvette. Next, 100 μl of defrost washed erythrocytes are taken using a piston pipette. The pipette is carefully cleaned using cellulose and erythrocytes are introduced into the cuvette. 11 μl of 1 mol/l CaCl2 solution are then added and the sample is centrifugated for 3 min, at 90 000 rpm (288 000×g, rAV = 31,8 mm), at 4žC. Afterwards, 500 μl of supernatant is...
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HPLC Determination of Lutein, Zeaxanthine and Betacarotene in Dietary Products
Dvořáková, Petra ; Šatínský, Dalibor (advisor) ; Chocholoušová Havlíková, Lucie (referee)
This rigorous thesis deals with modification of method for separation carotenoids lutein, betacarotene and zeaxanthin borrowed from diploma thesis (Optimization of chromatographic conditions for HPLC determination of chosen carotenoids, Petra Dvořáková, 2009). HPLC with VIS detection is the basic method. This method was modified by gradient elution for compressed time of analysis. Four ways of gradient elution were tested during optimization of new procedure. The second aim of this rigorous thesis is summary of extraction ways for carotenoids published in special literature and proposition of extraction procedures for carotenoids from dietary products. Extraction with organic solvents was used during searching for optimal way for extraction. Simple extraction with organic solvent was tested as basic method, but extraction with previous modification of sample (saponification or acidification of extraction medium) was tested too. In spite of testing of many various ways for extraction and many various organic solvents, there wasn't find optimal and universal extraction way.
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Application of SPE and technology of monolithic columns for HPLC analysis of biologically active compounds.
Horčičková, Šárka ; Šatínský, Dalibor (referee) ; Solich, Petr (advisor)
The solid phase extraction (SPE) is an effective, reliable and selective modern method for sample preparation of biological material before chromatographic analysis. In this graduation thesis the new SPE method for isolation of vitamin D2 (ergocalciferol), D3 (cholecalciferol) and its metabolite 25 - (OH) D3 (calcidiol) simultaneously with vitamin A (retinol) and E (α-tocopherol) in human serum was developed. The vitamins were subsequently determined by validated high performance liquid chromatography method (HPLC) using tocol as internal standard. The developed SPE procedure: Firstly 250 l of human serum was pipetted to glass tube and 1000 l of cool ethanol (4řC) was added. Sample was shaken and left 10 minutes in a refrigerator at 4řC for deproteinisation. After centrifugation (4 000 x g, 15 minutes, 4řC) all supernatant was carefully removed and applied into pre-treatment SPE column (firstly washed with 1 ml of methanol, then with 1 ml of distilled water). During these two steps the sorbent must not be dried up. The vitamins were eluted by 1.5 ml of methanol and then 2.0 ml of n-hexane. The organic solutions were evaporated in the vacuum concentrator at a temperature of 45 ř C. The residue was dissolved in 250 l of methanol and analyzed by using HPLC method. HPLC method: Mobile phase - A:...
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HPLC separation of p-aminophenol, paracetamol, caffeine and propyphenazone on polar stacionary phase.
Maroušková, Alena ; Sklenářová, Hana (referee) ; Šatínský, Dalibor (advisor)
This thesis deals with finding optimal chromatographic conditions for a separation of p- aminophenol, paracetamol, caffeine and propyphenazon by HPLC. These analytes have different polarity and acidobazic properties. The application of traditional octadecylsilane stationary phases for their simultaneous determination is problematical. The PEG column have proved to be suitable for an efficient separation of the analytes with different polarities, i.e. with different chromatographic behavior. The resulting method brings shortening of analysis time, which is important for analysis of big series . Within the optimization of chromatographic conditions: a wide range of mobile phases has been tested (various organic solvents, various ratio of organic components to water phase components) a various level of acidity of the mobile phase has been tested ranging from 4 to 7 pH units various buffers have been tested behavior of the analytes has been tested under an enhanced temperature two PEG columns with different lengths have been tested Optimal chromatographic conditions for a separation of the analytes mentioned above, as well as the fulfillment of requirements for simplicity, analysis time and high degree of reliability have been found. The final chromatographic conditions: Stationary phase:...
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Optimization of conditions for qualitative and quantitative analysis of farnesol and tyrosol using UPLC-MS/MS.
Greguš, Petr ; Šatínský, Dalibor (referee) ; Nováková, Lucie (advisor)
APLICATION OF UHPLC/MS/MS TECHNIQUE FOR QUALITATIVE AND QUANTITATIVE ANALYSIS OF FARNESOL AND TYROSOL, AS A PRODUCT OF METABOLISM OF CANDIDA ALBICANS Petr Gregus 1 , Lucie Novakova 1 1 Charles University in Prague, Faculty of Pharmacy in Hradec Kralove, Heyrovskeho 1203, 50005 Hradec Králové SUMMARY : Candida albicans is generally one of the most often isolated fungal pathogens in human body. It is a common cause of nosocomial infections, bloodstream infections, urinary infections and infections of digestive tract. One of the most infectious diseases is vaginal candidosis, which affects most of healthy population of women. Candida albicans can grow as hyphae, pseudohyphae, or budding yeast. The availability of this diversity - the variability of the internal morphology is of great benefit for the oraganism's lifestyle. Morphological change of yeast forms to pseudomycelium or mycelium (hyphae) characterizes the transition from colonial statute to invasive. In addition to the physical-chemical factors during these transformation procedures - farnesol and tyrosol participate as well as quorum sensing compounds. Farnesol which, if accumulated over a certain limit, it prevents the conversion of yeast to the mycelium. Tyrosol, which accelerates the conversion in to the form of the germ (germ tubes), namely the...
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A comparison of UV a CAD detection possibilities for selected groups of compounds.
Vrkočová, Kateřina ; Šatínský, Dalibor (referee) ; Nováková, Lucie (advisor)
This thesis was focused to a comparison of UV and CAD detection for High Liquid Performance Chromathography. The separation of estrogen steroids and phenolic acids was performed on Zorbax SB C18 (50x4,6 mm; 1,8 µm) analytical column. The determination was carried out using two type of detection: ultra - viollet detection (UV) and charged aerosol detection (CAD). The optimized conditions were carried out for determination of estrogen steroids and phenolic acids, especially it was focused on appropriate sensitivity of CAD. The effect of composition and concentration of aqueous part of mobile phase, flow rate of mobile phase and setting of CAD range to its response was monitored. The gradient elution was used for determination of estrogen steroids. The mobile phase was formed by 0,1% acetic acid and acetonitrile. The gradient elution was used for determination of phenolic acids. The mobile phase was formed by 0,5% formic acid and methanol. A part of this thesis was verification of Corona CAD detector applications. CAD enables simultaneous detection of β - estradiol and its five possible impurities at optimized conditions. Relevant results by CAD detection were not obtained in case of the latest eluted cinnamic acid in determination of ten phenolic acids. The method for estrogen steroids was validated...
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Determination of nitrate and nitrite by SIA method.
Kymlová, Helena ; Sklenářová, Hana (referee) ; Šatínský, Dalibor (advisor)
New SIA method was developed for simultaneous determination of nitrites and nitrates. Nitrites were analyzed by Griess reaction. Nitrates were determined after their reduction to nitrites in cadmium column. Absorbance was measured at maximum of absorption 540 nm. Optimal conditions for analysis: speed of peristaltic pump is 40 % of its maximal speed, speed of syringe pump is 50 µl.s-1 , sample volume of nitrites and nitrates is 150 µl. Optimal time for reduction of nitrates is 0 sec, it means during their flow through cadmium column. Method was used for analysis of nitrites and nitrates in samples of waters: Fromin, Bonaqua, Rajec, Mattoni and tap water in Hradec Králové. Obtained values were compared with values declared by producers. New SIA method for simultaneous determination of nitrites and nitrates is used in subject Special methods of instrumental analysis as a new practical task.
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Determination of active substances in lubricant gel using gas chromatography.
Ryšavá, Pavlína ; Matysová, Ludmila (advisor) ; Šatínský, Dalibor (referee)
This thesis deals with the topic The determination of content substances in lubrication gel by method of gas chromatography and with lubrication gel Tea Tree Fantasy, which includes natural substance of tea tree oil. Tea tree oil has strong antiseptic, antimycotic and antifungal effects, and the terpinene-4-ol is its important germicidal component. This thesis is divided into eight chapters; its main part consists of chapters 3 - 6. The third chapter deals with the theoretical part, where the gas chromatography is described. This method is suitable for determination of terpinene-4-ol in lubrication gel. This chapter also deals with the validation of analytical method and with individual parameters and contains the characteristics of lubrication gel, tea tree oil and terpinene-4- ol. In the fourth chapter, there are worked out the search of used methods. The fifth chapter deals with the experimental part; all chemicals, instruments and conditions of isolation are written down there. The sixth chapter describes results and discussion from the development of analytical method, from the system suitability test and from the validation. The main goal of this thesis is the validation of gas chromatography method for the determination terpinene-4-ol in lubrication gel.
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SIA determination of nitrites, nitrates and chlorides using valid ISO norms.
Dundová, Aneta ; Sklenářová, Hana (advisor) ; Šatínský, Dalibor (referee)
This diploma work deals with determination of nitrite, nitrate and chloride based on Sequential Injection Analysis methodology following valid ISO norms. CSN EN ISO 11 732 sets method suitable for determination of ammonia nitrogen based on the FIA and CFA techniques in water samples. Determination is based on injecting sample with nitrite and nitrate content into continual flow of the carrier stream, where it is mixed with alkaline solution. Ammonium is separated in diffusion segmentor unit through semi-permeable membrane and is introduced into the flow with pH indicator. In consequences changes of pH values change the indicator color, which is scanned by spectrophotometer. Transfer of the mentioned FIA determination into the SIA system was carried out meeting calibration ranges of the ISO norm. Nitrite was determined directly using the Griess diazo- coupling reaction and the formed azo dye was measured at 540 nm in the flow cell of the spectrophotometer. The calibration curves were linear over the ranges 0,01 - 0,1 mg/l and 0,1 - 1,0 mg/l; correlation coefficients were 0,9979 and 0,9962. Nitrate solution was aspirated to reducing column containing copperised cadmium. After reduction of nitrate into nitrite the sample was aspirated to the holding coil, where it was mixed with the reagent and passed...
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