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Automation of liberation tests for releasing biologically active substances from nanofibers
Kodedová, Marie ; Sklenářová, Hana (advisor) ; Chocholouš, Petr (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of analytical chemistry Candidate: Marie Schneidrová Supervisor: doc. PharmDr. Hana Sklenářová, Ph.D. Title of the diploma thesis: Automation of liberation tests for releasing biologically active substance from nanofibers The main goal of this diploma thesis is an automation of TTC liberation from nanofibers, using non-separation flow method sequential injection analysis. UV-VIS spectrophotometer was used for the detection of signal. Measurements were carried out using 1-3 connected Franz cells in parallel, palced in the water bath. Buffer solutions with pH 7.4 and 4.5 were used as liberation media. The liberation of TTC was realized from two different nanofiber carriers - types "A" and "AK", Difference was in their preparation process. Because of this, each of them revealed various characteristics mainly related to the amount of bound TTC and the way of its binding to the nanofiber carrier. Production of these nanofibers was realized by Technical University Liberec using the technology NanoSpiderTM . Measurements were performed under various predetermined conditions, representing the state of the human skin - two different pH (7.4; 4.5) and temperature (32řC; 37řC) values. Four different types of membrane filters were also used,...
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Development of supercritical fluid extraction for isolation of biologically active substances
Kuda, Lukáš ; Pilařová, Veronika (advisor) ; Sklenářová, Hana (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Lukáš Kuda Supervisor: PharmDr. Veronika Pilařová, Ph.D. Title of Diploma Thesis: Development of Supercritical Fluid Extraction for Isolation of Biologically Active Substances The main aim of this study was to optimize the method for extraction of quercetin - flavonoid with antioxidant activity - from dried crushed quince fruit (Cydonia oblonga Mill.) using supercritical fluid extraction (SFE). The supercritical fluid extraction is a method in which supercritical fluid is used to extract active compounds. Neat CO2 is the most common used solvent. Due to its physicochemical properties, it is suitable solvent for the extraction of non-polar compounds. While extraction of polar compounds id required, the organic solvents such as ethanol, methanol or isopropanol are added to the supercritical fluid. Depending on the amount of added organic solvent, SFE, SFE with modifier, CO2 expanded liquid extraction and pressurized liquid extraction are distinguished. Temperature and pressure are other key parameters affecting extraction and need to be optimized as well. The optimization of the quercetin extraction method was carried out in several following steps. First, the key parameters were tested using...
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Development of on-line solid phase extraction using flow methods
Vinklárek, Jan ; Chocholouš, Petr (advisor) ; Sklenářová, Hana (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Analytical chemistry Candidate: Bc. Jan Vinklárek Supervisor: doc. PharmDr. Petr Chocholouš, Ph.D. Title of the diploma thesis: Development of on-line solid phase extraction using flow methods The goal of this diploma thesis was to create and validate method using combination of SPE extraction and flow methods (in this case method SIA). Creatinine was chosen as extracted analyte and urine was chosen as biological material. At first, the method was optimized (concentration and flow rate of wash reagent and flow rate of sample and standards). Then the limits of the created method were tested. Finally, the trueness and repeatability of results was tested and also stability of diluted samples. All of these tests (in thesis named experiments) had good results. Final parameters of method are: Sorbent - SPH Iontosorb 100 Wash reagent - 50% acetonitrile, aspirated 0,5 ml Elute reagent - 1% water solution of ammonia hydroxide, aspirated 0,5 ml Sample diluted by acetic acid with pH 2,4, aspirated 20 µl, or 10 µl according to sample concentration (method alone makes decision about changing aspirated volume, decision algorithm is in the program) Method of standard adding is used for measuring creatinine concentration in...
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Analysis of nutritionally important substances in fruit tree waste products by HPLC
Šírová, Karolína ; Šatínský, Dalibor (advisor) ; Sklenářová, Hana (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Bc. Karolína Šírová Supervisor: doc. RNDr. Dalibor Šatínský, Ph.D. Consultant: Mgr. Anežka Adamcová Title of Diploma Thesis: Analysis of nutritionally important substances in fruit tree waste products by HPLC This diploma thesis deals with the content of phenolic compounds in various parts of tree of the genus Pyrus. The theoretical part is focused on characterization, biosynthesis, chemical and biological properties of major phenolic compounds in pear-trees and the possibilities of their determination with the use of the latest scientific studies. The experimental part contains an extensive analysis of arbutin, chlorogenic acid, 1,5- dicaffeoylquinic acid, 3,5- dicaffeoylquinic acid and rutin in extracts of leaves, bark, buds, fruits, flowers and wood chip. Developed and validated HPLC-DAD method was used and the total antioxidant activity was determined by FIA method with electrochemical detection. An ASCENTIS Express RP-Amide 150×4.6 mm, 2.7 μm analytical column and gradient elution consisting of organic part of acetonitrile (ACN) and water part with 0,85% phosphoric acid (pH = 2.2) were used for analysis. The detection was performed by DAD detector at wavelengths 220 nm, 327 nm and 354 nm. The...
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Monitoring the profile of phenolic substances in different parts of apple trees by HPLC
Šilhavá, Kristýna ; Šatínský, Dalibor (advisor) ; Sklenářová, Hana (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Bc. Kristýna Šilhavá Supervisor: doc. RNDr. Dalibor Šatínský, Ph.D. Consultant: Mgr. Anežka Adamcová Title of Diploma Thesis: Monitoring the profile of phenolic substances in different parts of apple trees by HPLC This diploma thesis covers the performance of a quantitative analysis of phenolic compounds in ten different cultivars in Malus. Specifically, the presence of phloridzin, phloretin, rutin, quercitrin and chlorogenic acid was monitored. Extracts of buds, flowers, leaves and bark in each species were analyzed during the four different vegetation periods. Further, the presence of phenolic compounds in samples of wood chips and fruits was monitored. The determined analytes displayed a different content profile depending on the plant material, apple cultivar and the period in which they were collected. Except for fruits, phloridzin was the dominant component in all varieties and in all materials. The highest total concentration of phenolic compounds was found in the leaves in the spring March-April 2020 in species 'Rubinstep'. This diploma thesis used a developed and validated HPLC-DAD method for a large number of extracts. A YMC-Triart C18 ExRS column (150 × 4.6 mm × 5 µm, 8 nm) was chosen...
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Optimization of extraction of phenolic substances using carbon dioxide
Karola, Michal ; Sklenářová, Hana (advisor) ; Pilařová, Veronika (referee)
Charles University, Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Michal Karola Supervisor: doc. PharmDr. Hana Sklenářová, Ph.D. Advisor: Mgr. Marcela Hollá Title of diploma thesis: Optimization of extraction of phenolic substances using carbon dioxide This diploma thesis deals with the optimization of extraction of phenolic substances by supercritical fluid extraction, using carbon dioxide, from a sample of homogenized dried apple chips. To quantify and identify the analytes contained in the extract, 14 standards were analysed using a separation method in a UHPLC system with a diode array detector. In the identification of the extracts' components, 13 analytes were identified and after their quantification, 6 analytes were found to have major content. The difference in the sum of the concentrations of the 6 and 13 analytes was small, and therefore, optimization was carried out for these 6 majorly represented analytes, which are chlorogenic acid, epicatechin, catechin, phloridzin, quercitrin, and guaiaverin. The extraction optimization was carried out in 4 successive steps. The first step of the optimization was to determine the influence and significance of CO2 content in the extraction medium, H2O content in the ethanolic cosolvent, pressure and temperature...
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Monitoring of liberation tests for the release of clotrimazole from nanofibers
Ernest, Radovan ; Sklenářová, Hana (advisor) ; Chocholouš, Petr (referee)
Charles University Faculty of Pharmacy in Hradec Kralove Department of Analytical Chemistry Candidate: Radovan Ernest Supervisor: Doc. PharmDr. Hana Sklenarova, Ph.D. Consultant: PhamDr. Martina Hakova, Ph.D. Title of the diploma thesis: Monitoring of liberation tests for the release of clotrimazole from nanofibers The diploma thesis was focused on the analysis of release profiles of clotrimazole from different types of nanofibres using non-separation flow technique, sequential injection analysis (SIA). Simultaneously the diploma thesis dealt with finding ideal conditions for saturation of the fibres with a solution of clotrimazole. Nanofibers were produced at the Technical university of Liberec. The diploma thesis dealt with two types of nanofiber carriers - polymeric (polydioxanone and polycaprolactone) and ceramic (silica). Part of the fibre of polydioxanone and polycaprolactone was saturated directly during production in different ratios of polymer and clotrimazole. The saturated fibres were then analysed in the laboratory. The second part of polydioxanone and polycaprolactone together with ceramic nanofibers was produced in a pure state, i.e., without an active substance. These fibres were tested in the laboratory for saturation with an ethanolic solution of clotrimazole at a certain...
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Monitoring the profile of phenolic substances in different parts of apple trees by HPLC
Šilhavá, Kristýna ; Šatínský, Dalibor (advisor) ; Sklenářová, Hana (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Bc. Kristýna Šilhavá Supervisor: doc. RNDr. Dalibor Šatínský, Ph.D. Consultant: Mgr. Anežka Adamcová Title of Diploma Thesis: Monitoring the profile of phenolic substances in different parts of apple trees by HPLC This diploma thesis covers the performance of a quantitative analysis of phenolic compounds in ten different cultivars in Malus. Specifically, the presence of phloridzin, phloretin, rutin, quercitrin and chlorogenic acid was monitored. Extracts of buds, flowers, leaves and bark in each species were analyzed during the four different vegetation periods. Further, the presence of phenolic compounds in samples of wood chips and fruits was monitored. The determined analytes displayed a different content profile depending on the plant material, apple cultivar and the period in which they were collected. Except for fruits, phloridzin was the dominant component in all varieties and in all materials. The highest total concentration of phenolic compounds was found in the leaves in the spring March-April 2020 in species 'Rubinstep'. This diploma thesis used a developed and validated HPLC-DAD method for a large number of extracts. A YMC-Triart C18 ExRS column (150 × 4.6 mm × 5 µm, 8 nm) was chosen...
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Monitoring of toxicological tests
Rosecká, Michaela ; Sklenářová, Hana (advisor) ; Horstkotte, Burkhard (referee)
Charles University Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Bc. Michaela Rosecká Supervisor: Doc. PharmDr. Hana Sklenářová, Ph.D. Title of Diploma Thesis: Monitoring of toxicological tests The aim of this work was to monitor kinetic profiles of permeation of fluorescent marker through cell monolayer (MDCKII-MDR) by sequential injection analysis. For this purpose, a new type of permeation module was prepared that could measure from 2 compartments - donor and acceptor. The module was thermostated at 37řC during the experiment and liquid in the acceptor compartment was continuously agitated. Experiments were repeated to observe difference in interaction of fluorescent marker with cell membrane transporter. MDCKII-MDR monolayer cell inserts were replaced after the experiments. The cell line used in this experiment include a genetically modified P-glycoprotein. Rhodamine 123, the fluorescent agent, was applied for on-line monitoring of extracellular transport. The permeability kinetics of this marker was monitored using a fully automated sequential injection analysis system with five minutes sampling intervals for the testing period of 120 minutes. The sample was taken from the donor compartment in one cycle, and from the acceptor one in the next cycle. Then,...
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Study of deproteinization of milk samples via salting-out induced phase separation and automation by Lab-In-Syringe
Machián, Daniel ; Horstkotte, Burkhard (advisor) ; Sklenářová, Hana (referee)
Charles University in Prague Faculty of Pharmacy in Hradec Králové Department of Analytical Chemistry Candidate: Daniel Machián Supervisor: M.Sc. Burkhard Horstkotte, Ph.D. Co-supervisor: PharmDr. Kateřina Fikarová, Ph.D. Diploma thesis title: Study of deproteinization of milk samples via salting-out induced phase separation and automation by Lab-In-Syringe A novel approach to sample deproteinization based on homogeneous liquid-liquid extraction with salting-out induced phase separation was explored. A methodology was developed, experimentally tested, and optimized that allows automated centrifuge-less deproteinization of milk samples using the flow-batch technique Lab-In-Syringe. As in other sample preparation methods, the aim was to eliminate the sample matrix and obtain an organic phase that could be submitted to analysis. The thesis includes a range of preliminary experiments performed offline, i.e. without the intended instrumentation, as well as optimization experiments performed online using a Lab-In-Syringe system. Salting out agents and three solvents were tested as well as phase ratios and operational parameters on the Lab-In-Syringe system, including system configuration. In the method optimized by the work reported in this thesis, milk proteins were found effectively denatured and...
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