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Modifications of inner walls of electrophoretic capillaries using positively charged anchors
Stávek, Petr ; Křížek, Tomáš (advisor) ; Coufal, Pavel (referee)
Electroosmotic flow plays a crucial role in capillary electrophoresis, as it can impact both the resolution and duration of the separation process. Altering the electroosmotic flow can be achieved through dynamic coating of the inner capillary surface. The primary objective of this work was to investigate the potential of various positively charged anchors in providing a suitable dynamic coating for capillary electrophoresis. At first, experiments were carried out with neopentyl anchors PK 1291, PK 941, PK 1300 and PK 1294 using four different coating methods. Obtained coatings demonstrated the ability to suppress the electroosmotic flow, although the stability of the obtained coatings varied significantly among the individual anchors. PK 941 demonstrated the most promising results within this group. Subsequently, measurements were also performed using the cyclodextrin anchor PK 1281, which yielded the most favorable outcomes. It was observed that depending on the background electrolyte and the chosen coating method, this anchor could either reverse the direction of the electroosmotic flow or nearly completely eliminate it. Key words: capillary electrophoresis, electroosmotic flow, dynamic capilary coating, positively charged anchors.
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Determination of pharmaceuticals in wastewaters
Horkelová, Simona ; Sobotníková, Jana (advisor) ; Coufal, Pavel (referee)
The issue of pharmaceutical accumulation in the environment is a pressing concern today. Annually, the consumption and misuse of pharmaceuticals increase, with a significant portion ultimately being discharged into wastewater. Unfortunately, wastewater treatment processes often prove inadequate in effectively removing these substances, as they were not originally designed for this purpose. Consequently, pharmaceuticals gradually infiltrate water systems, resulting in heightened pollution of surface water, groundwater, and even drinking water sources. Due to insufficient legislation, there is a need for stricter monitoring and elimination of pharmaceutical substances containing. This master's thesis investigates the presence of pharmaceuticals and some metabolites in wastewater samples using an accredited method at ALS Czech Republic laboratory. It compares the input and output concentrations of drugs in wastewater treatment plants of varying sizes and assesses the efficacy of their removal. Additionally, it contrasts municipal sewage treatment plants serving communities with hospital wastewater treatment plants. Key words: LC-MS, pharmaceuticals, metabolites, wastewaters, environment
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Investigation of calibration factor stability for GC-FID analysis of alcoholic products quality by internal standard methods
Čermáková, Veronika ; Čabala, Radomír (advisor) ; Coufal, Pavel (referee)
This thesis focuses on measuring calibration factors and the dependence of their stability on experimental conditions for the purpose of quality control and the safety of alcoholic beverages using gas chromatography with flame ionization detection (GC-FID). Ten volatile compounds (acetaldehyde, methanol, methyl acetate, ethyl acetate, 1-propanol, 1-butanol, 2-butanol, isobutanol, acetal, 3-methyl-1-butanol) were monitored according to the legal regulations of the European Commission. These target compounds were analysed in prepared solutions under 27 different measurement conditions. Taguchi's multifactorial statistical method was used to determine the influence of individual measurement parameters on the stability of calibration factors. The chosen variables for measurements were matrix composition, concentration of volatile compounds, GC injector temperature, sample injection volume, injection split ratio, and detector temperature. Within the experiments, each of these variable factors took one of three possible values. Relative response factors (RRF) for each analysed compound were compared using two internal standard (IS) methods. One method utilised commonly used 1-pentanol as the internal standard, while in the other, ethanol, the main component of all alcoholic beverages, was used as the...
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Hydrophilic Interaction Liquid Chromatography in the Analysis of Intact Glycopeptides
Molnárová, Katarína ; Kozlík, Petr (advisor) ; Coufal, Pavel (referee) ; Cvačka, Josef (referee)
The analysis of glycoproteins represents a significant challenge in glycoproteomics, primarily due to the macro- and microheterogeneity of protein glycosylation. Hydrophilic interaction liquid chromatography (HILIC) is a convenient alternative to reversed-phase chromatography, commonly used in glycoproteomic analysis. This dissertation thesis discusses the potential of HILIC in glycoproteomic analysis, ranging from the separation of glycopeptides on polar stationary phases to the use of HILIC in sample preparation processes. First, the effect of acetonitrile concentration on glycopeptide precipitation was investigated, depending on the type of glycan attached. Subsequently, three commercially available stationary phases were tested: a column containing a silica gel modified with five hydroxyl groups, an amide stationary phase, and a zwitterionic stationary phase. Their efficiency in separating glycopeptide isomers, differing only in branching and/or linkage position, was compared. Further research was devoted to the separation of human immunoglobulin G glycopeptides using relatively new columns that have not yet been characterized in glycoproteomic analysis. These columns, provided by Advanced Chromatography Technologies, included unmodified silica gel (HILIC-A), aminopropyl- modified sorbent...
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Development and testing of capillary columns based on ion exchanger
Vojta, Jiří ; Coufal, Pavel (advisor) ; Bosáková, Zuzana (referee)
In this work, capillary monolithic columns based on styrene, divinylbenzene and methacrylic acid were prepared by free radical polymerization inside fused sillica capillaries of 320 µm inner diameter. The columns were prepared by very simple process from polymerization mixtures with variable amount of methacrylic acid. Effect of electrostatic interactions between methacrylic acid, as weak cation-exchanger, and analytes on their retention was showed. Based on chromatographic measurements, effect of amount of methacrylic acid in polymerization mixtures on the morphological properties of monoliths and on the retention of small organic amines and benzene was discussed.
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Development of Electrochemical Thin-layer Degradation Cell for Oxidation Stress Testing of Active Pharmaceutical Ingredients
Šefčík, Martin ; Kubíčková, Anna (advisor) ; Coufal, Pavel (referee)
In this thesis, the oxidation conditions in a new electrochemical "thin-layer" flow-through cell for the study of the oxidative degradation of active pharmaceutical ingredients were optimized. In this field, it is an innovative approach to studying the oxidation properties of substances. The cell was manufactured using 3D printing technology. Oxidation was carried out in a two-electrode arrangement with a working boron-doped diamond electrode (BDDE) and an auxiliary stainless steel electrode. Optimization of conditions was performed using salicylic acid as a model active pharmaceutical ingredient. The electrochemical approach was used to degrade this active pharmaceutical ingredient by the required approximately 20% of the original amount in only 2.3 min. Thus, the degradation time was significantly reduced compared to the oxidative stability stress tests of active pharmaceutical ingredients commonly used today. Two major degradation products were observed, namely gentisic acid and 2,3-dihydroxybenzoic acid. The degradation products obtained by electrochemical oxidation and chemical oxidation using hydrogen peroxide are identical.
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On-line derivatization of saccharides in capillary electrophoresis
Mareš, Vít ; Křížek, Tomáš (advisor) ; Coufal, Pavel (referee)
Saccharides chains may suffer from various inaccuracies during their biosy- thesis, that effects their biologic functions. Therefore, they can be used for dia- gnosis of some diseases or quality control in biopharmaceutics. Capillary electro- phoresis has its place in diagnosing these occuring errors. In this thesis a method of on-line derivatization of saccharides in fused-silica capillary with 50 cm in lenght and 50 µm in inner diameter was developed. Gluco- se was used as a model molecule, which was labeled by a fluorescent derivatization reagent, 7-aminonaphthalene-1,3-disulfonic acid. Derivatization of glucose and its separation needed optimalization. First was determined an electrophoretic mo- bility of the derivatization reagent in 0.5; 1.0 and 2.5M acetic acid and 20mM phosphate buffer (pH = 3.5). Both, 2.5M acetic acid and 20mM phosphate buffer, were tested as solvents for 7-aminonaphthalene-1,3-disulfonic acid. The reaction with glucose was the best in acetic acid solution. On the contrary, phosphate buf- fer was found to be the best background electrolyte for the separation. Derivatized glucose was detected by UV detector at 229 nm. Henceforth, the reaction con- ditions were adjusted such as reaction time, voltage and time of its application. Input voltage was applied negative due to the...
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The use of high-performance separation techniques for monitoring the bioaccumulation of selected micropollutants in plants of model root treatment plants
Modroczká, Karin ; Sobotníková, Jana (advisor) ; Coufal, Pavel (referee)
This bachelor's thesis deals with the determination of ibuprofen in model root refineries from Phalaris arundinacea extracts. The goal was to find out if the plant is able to accumulate ibuprofen from the aqueous solution which was added to the plant. A HPLC method with UV/VIS detection was also optimized to determine the ibuprofen content. A C18 column (4.6 mm × 150 mm × 3.5 µm) was used to analyse the samples. An isocratic program was used, in which the mobile phase consisted of methanol and an aqueous acetic acid solution of pH = 3,27. As part of the validation of the method, the repeatability, robustness, and yield (which was 99,04 %) of the method were verified. Furthermore, the linearity of the method, which was from 0,01 mg ml-1 to 2,0 mg ml-1 , the limit of detection (LOD = 0,049 mg ml-1 ) and the limit of quantification (LOQ = 0,16 mg ml-1 ) were determined. After the validation of the method, we can claim that the method is accurate, because the values of the relative standard deviations did not exceed 2 %. Infrared spectroscopy was used to verify the purity of ibuprofen batches. Last but not least, it was proven that Phalaris arundinacea has the ability to accumulate ibuprofen from an aqueous solution. Keywords: HPLC, ibuprofen, Phalaris arundinacea, root wastewater treatment plant,...
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Optimization of electrodeposition of polyaniline on platinum electrode for propanol oxidation
Dobiášová, Klára ; Schwarzová, Karolina (advisor) ; Coufal, Pavel (referee)
Optimization of electrodeposition of polyaniline on platinum electrode for propanol oxidation Dobiášová Klára 2023 Abstract Composites of polyaniline (PANI) with rare metal nanoparticles (metal/PANI) are a promising material for the oxidation of aliphatic alcohols. In this work, the deposition of a polyaniline (PANI) film on a platinum electrode (Pt-PANI electrode) was performed potentiostatically and using cyclic voltammetry (CV). For both methods, the parameters influencing the deposition were optimized. For cyclic voltammetry, electrodeposition in aniline solution is significantly influenced by the final potential Efin of the scan. The PANI film with the highest current response during the deposition itself, which correlates with the thickness of the formed film, was created in the case of Efin + 1.1 V; i.e. the peak potential of the aniline oxidation peak. However, this method proved unreliable due to the poor reproducibility of the current response corresponding to PANI formation. For the PANI film formed by the CV method, its color change related to its oxidation state was investigated, with the color change being imperceptible to the naked eye in a basic environment and the most visible colour change occurring between + 0.05 V and + 0.2 V in an acidic environment. Potentiostatic deposition of...
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Capillary monolithic columns based on copolymer of styrene, divinylbenzene and methacrylic acid and their application for separations of small molecules.
Musilová, Adéla ; Coufal, Pavel (advisor) ; Čabala, Radomír (referee) ; Sýkora, David (referee)
This work is focused on the preparation and characterization of capillary monolithic columns based on a copolymer of styrene, divinylbenzene and methacrylic acid for the chromatographic separation of small molecules. The capillary monolithic columns were prepared in the quartz capillaries by one-step radical copolymerization reaction of monomers of styrene, divinylbenzene and methacrylic acid in the presence of toluene and isooctane as porogenic solvents and azobisisobutyronitrile as an initiator. The effect of incorporation of methacrylic acid into the polymerization mixture on the separation behavior and efficiency in reversed phase capillary liquid chromatography (CLC) and capillary liquid electrochromatography (CEC) was studied using a test mixture of compounds containing thiourea, phenol, aniline, benzene, toluene, ethylbenzene, propylbenzene and butylbenzene. The Walters test for reversed stationary phases was employed to evaluate the hydrophobicity of the prepared columns. Based on the calculated value of hydrophobicity index of 10.76, the prepared monolithic columns can be classified as very hydrophobic. Excellent repeatability of measurements in CLC mode and very good repeatability of the monolithic column preparation were achieved. The effect of polymerization time on the chromatographic...
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