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Preparation and testing of capillary monolithic columns for hydrophobic interaction chromatography
Vlková, Michaela ; Sobotníková, Jana (advisor) ; Zima, Jiří (referee)
In frame work of this diploma thesis, monolithic stationary phases based on hydroxymethylmethacrylate were prepared in fused silica capillaries of 320 μm innerdiameter. Monolithic columns were synthesized by a simple procedure using a polymerization mixture, consisting of a monomer N-(hydroxymethyl) methacrylamide (HMMAA), a croslinking agent ethylene dimethacrylate (EDMA), porogenic solvents butane- 1,4-diol, propane-1-ol and an initiator α,α′-azobisisobutyronitrile (AIBN). Prepared HMMA monolithic columns were utilized for separation of mixtures of biologically active compounds, namely peptides with small number of amino acids. Mechanical strength and specific permeability were determined for selected monolithic columns. Keywords: HPLC, HILIC mechanism, hydroxymethyl methacrylate (HMMA) monolithic columns, amino acid, enkephalins.
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Analysis of potassium tetrachloroaurate by high performance liquid chromatography
Petržilková, Eliška ; Sobotníková, Jana (advisor) ; Kozlík, Petr (referee)
The theoretical part describes the HPLC method and its components. The following are basic information about the analyte, specifically about the behavior of gold and its reactivity. The experimental part describes the procedure of optimizing the composition of the mobile phase in the HILIC system on two stationary phases, namely on Luna NH2 columns (5 μm, 100 Å, 2 x 250 mm) and XBridge Amide (3.5 μm, 2.1 x 150 mm). The best chromatograms were recorded on a Luna NH2 column in a mobile phase composed of 94% ACN and 6% 5.0 mmol⸱dm-3 HCl and its flow rate 0.4 ml⸱min-1 . For the XBridge Amide column, the most suitable measurement conditions were a mobile phase composition of 94% ACN and 6% 10.0 mmol⸱dm-3 HCl and a flow rate of 0.7 ml⸱min-1 . Subsequently, the repeatability of the measurements was verified under these conditions, which was evaluated from 10 repeated injections of a tetrachloroaurate solution of a concentration of 1.0 mmol⸱dm-3 in 100% ACN. Repeatability did not give satisfactory results, so the reason was looked for. Verification of the functionality of the injector and detector with the column disconnected was successful. After reconnecting the column, it was found that the cause was right here, due to the use of a mobile phase containing HCl and it was no longer possible to work with...
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Analysis of potasium tetrachloroaurate by capillary electrophoresis
Lebertová, Lucie ; Sobotníková, Jana (advisor) ; Křížek, Tomáš (referee)
This bachelor thesis describes the optimalization of condition for the analysis of potassium tetrachloroaurate by capillary zone electrophoresis with spectrophotometric detection. The theoretical part at first introduces the principle of capillary electrophoresis. The following are basic information about gold and its tetrachloroaurate compound. The experimental part at first describes the procedure of optimization the composition of the basic electrolyte and problems with the instability of the base line. As the most suitable conditions for the analysis of tetrachloroaurate, have been chosen the subsequent experimental conditions: fused silica capillary with a total length of 80 cm, effective length 71.6 cm and inner diameter 50 µm; applied separation voltage -20 kV; applied pressure during analysis 20 mbar; injection with 20 mbar for 20 s; background electrolyte containing 0.01 mol dm-3 HCl and 0.04 mol dm-3 NaCl; detection vawelength at 230 nm. Subsequently, the repeatability of the measurement, and the stability of the tetrachloroaurate stock solution were evaluated. Also the calibration dependence were measured and the limit of detection and the limit of quantification were determined for the optimized experimental conditions. The value of LOD was 7.49 ∙ 10-6 mol dm-3 and the value of LOQ was...
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Electrophoretic determination of saccharides in flower nectar
Hurychová, Nikola ; Křížek, Tomáš (advisor) ; Sobotníková, Jana (referee)
This work deals with the development and basic validation of a method for determination of sucrose, glucose and fructose content in floral nectar. It uses capillary zone electrophoresis with contactless conductivity detection. The theoretical part describes the functions of carbohydrates in nectar, principles of capillary zone electrophoresis and contactless conductivity detection. There is also a brief overview of applications in the determination of carbohydrates. In the experimental part, sample injection at the short and at the long end of capillary were tested. In further experiments, the background electrolyte was optimized. The tested concetrations of NaOH were from 10 to 50 mmol l-1 . Optimal conditions for the experiments were injection at the short end of the capillary, 40mmol l-1 NaOH as background electrolyte and separation voltage of 15 kV. Then, the repeatability, limit of detection and quantification and recovery of the method with a real sample of nectar were verified. Lactose was used as an internal standard. Relative standard deviation was below 2 %. Limits of detection were below 0.003 mg ml-1 and limits of quantification were below 0.009 mg ml-1 . Recovery of the method was 97-105 %. Furthermore, the calibration dependences were measured, from which the concentration of...
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Analysis of a human body odour using comprehensive two-dimensional gas chromatography
Bušovská, Radka ; Sobotníková, Jana (advisor) ; Žáček, Petr (referee)
Body odour perception plays an important role in human mate choice, especially in women. It was previously proposed that women select partners whose body odour resembles that of woman's fathers. Yet, this phenomenon has only been confirmed using ethological studies based on subjective perception of body odour similarities. Therefore, the aim of my diploma thesis was to test this hypothesis instrumentally using comprehensive GC×GC-TOFMS and subsequent multidimensional analyses of body odour chemical profiles of male partners and fathers of adult women. Body odour sampling from left and right axilla of fathers and partners of 41 women (altogether 164 samples) was performed using cotton swabs, which were then frozen and extracted into hexane. Typical human volatile substances, such as hydrocarbons, carboxylic acids, esters, alcohols, aldehydes, ketones, sterols and terpenes were detected in all examined samples. Using a newly available "tile-based" chromatographic alignment algorithm, we obtained a set of 341 compounds systematically occurring in male axillary odour. The principal component analysis was used to calculate Euclidean distances for all pairs of the studied male subjects. These estimates of "chemical distances" revealed to be significantly smaller for father-partner pairs of individual...
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Study of sensory-active compounds of hops in dry-hopped beers
Zlochová, Tereza ; Sobotníková, Jana (advisor) ; Jelínek, Ivan (referee)
Study of sensory-active compounds of hops in dry-hopped beers Key words: hop, hop oils, dry-hopping, beer, beer-aging, beer sensory analysis Nowadays, dry-hopping is a widely used method of beer making, with its pros and cons. The main purpose of dry-hopping is to derive specific hop aroma to beer in its very unchanged form. The essential part of this work consists of a study of hop essential oils, as the main providers of desired hop aroma. Firstly, the validation of methods for isolating essential oils from hop material and beer is carried out. Secondly, the transfer of these volatiles in process of dry-hopping is studied and the impact on beer aroma profile is discussed. Subsequently, changes of essential oils profile during beer-aging are observed and evaluated with a valuable tool, the sensory analysis. This study was realized on beer samples from Brewers' Choice program, thanks to the Research Institute of Brewing and Malting and Lenka Strakova, the Pilsner Urquell brewer.
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Utilization of polynailine in separation methods
Riečan, Martin ; Sobotníková, Jana (advisor) ; Křížek, Tomáš (referee)
Submitted master thesis is by its character focused on development of polyaniline which would possess attributes typical for monolithic stationary phase. Developed polyaniline aims to be used in capillary liquid chromatography. Accompanying target of submitted master thesis is to provide solutions for variety of complications which follow the preparation of polyaniline in its desired form such as consistency in a form of solutions, gels and pastes, extremely fast polymerization reaction, limited solubility of cross-linking agents, solubility of monolithic polyaniline in polar mobile phases and insufficient bond of polyaniline to the inner wall of fused silica capillary tubing. Solutions for this complications are selection of ideal oxidation agent (ammonium persulfate), cross-linking agent (tris(4-aminophenyl)amine) and porogenic agent (methanol). Also, setting ideal ratio between oxidation agent, cross-linking agent and porogenic agent, heat treatment, choice of suitable mobile phase (tetrahydrofuran), adjustment of capillary wall using silanization agent (3-[3-(trimethoxysilyl)propyl]-aniline) and construction of equipment needed for quick filling of capillaries. Described development concluded in the preparation of solid polyaniline monolith which had a steady bond to the inner wall of fused...
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Oxidative degradation study of abacavir
Šušová, Nikola ; Kubíčková, Anna (advisor) ; Sobotníková, Jana (referee)
The aim of this study is forced oxidative degradation of active pharmaceutical ingredient abacavir, used to treat HIV-infected patients. A fast and sensitive method for the determination of abacavir and its degradation products by ultra-high performance liquid chromatography has been developed and validated, that made it possible to evaluate the oxidation stability of abacavir and Ziagen tablets. Suitable chromatographic separation was achieved using a Kinetex C18 column and gradient elution with a mobile phase consisting of acetonitrile and ammonium acetate (c = 20 mmol dm−3 , pH = 7.0). The total run time was 11 minutes. The determination of abacavir and its degradation products was performed by a photodiode array detector at λ = 254 nm. The optimized method for the determination of abacavir and its degradation products was applied to study the oxidation of abacavir by both traditional and electrochemical approaches. The forced degradation study in solution revealed abacavir instability in the presence of 3% hydrogen peroxide and during electrochemical oxidation. The study found that excipients in the tablet suppress the degradation of abacavir by approximately 10 %. Abacavir is oxidized by 15 % by hydrogen peroxide after 24 hours at 25 řC, after 1.5 hours at 50 řC and after 5 minutes at...
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Forced degradation study of canagliflozin with the use of HPLC
Máchalová, Jitka ; Křížek, Tomáš (advisor) ; Sobotníková, Jana (referee)
In this work a method for determination of canagliflozin and its degradation products by HPLC with UV and MS detector was developed. The developed method was used to study the forced degradation of canagliflozin and to investigate the major degradation products resulting from exposure of canagliflozin to oxidative stress. Canagliflozin is a phenolic glycoside derivative and a glucose-sodium transporter 2 inhibitor that stimulates urinary glucose excretion by suppressing glucose reabsorption from the proximal tubule in the kidneys. Canagliflozin is used to control blood glucose levels in patients with type 2 diabetes. In an optimized method, an Agilent Poroshell 120 SB-Aq (2.1 × 100 mm, 2.7 µm) column was used and a mixture of buffer (10mM HCOOH adjusted with ammonium hydroxide to pH 3.5) and acetonitrile as a mobile phase. The method validation included testing of accuracy, repeatability, the limit of detection and quantification, linearity and linear dynamic range, the robustness of the method, and testing of sample stability. The limit of detection of the method was 8.9·10-5 mg ml-1 (2.0·10-7 mol l-1 ) and the limit of quantification was 3.0·10-4 mg ml-1 (6.8·10-7 mol l-1 ). At a concentration of 0.3 mg ml-1 , the repeatability (n = 7) was 0.17 % and 0.75 % for the retention time and the peak...
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