Chlortoluron is a herbicide from the group of substituted ureas and it is used for weed control in production of cereals and poppy. Substituted ureas are widely used but these substances are known for their high mobility in soil, persistence and contamination of underground water. Therefore, the development of analytical methods suitable for residual concentrations determination is necessary. This thesis deals with the determination of chlortoluron using carbon paste electrode (CPE) by the methods of differential pulse voltammetry and high-performance liquid chromatography with electrochemical (HPLC-ED) and spectrophotometric (HPLC-UV) detection. The aim of the work was to find optimal conditions suitable for chlortoluron determination. Further, the possibility of using miniaturized carbon paste electrode (mCPE) and usability of tested methods for determination of chlortoluron in model samples of river water and soil were studied. Concentration dependences in deionized water were measured by the method of DPV in the Britton-Robinson (BR) buffer (pH = 3) and methanol (10 %, v/v). The limit of detection was 3,7∙10-7 mol∙dm-3 in the case of CPE and 8,7∙10-8 mol∙dm-3 in the case of mCPE. Also, concentration dependences were measured using HPLC with mobile phase containing BR buffer (pH = 4) and...

Lipidomics as a part of metabolomics is a fast-growing area of research due to the improvement in analytical techniques. This master thesis is focused on lipid extraction techniques optimization, using liquid liquid extraction and solid phase extraction as pre-separation methods and ultra performance liquid chromatography coupled with mass spectrometry for extraction and subsequent identification of branched-chain fatty acid esters (FAHFA - branched-chain Fatty Acid esters of Hydroxy Fatty Acids). This newly discovered class of lipid molecules is associated with insulin secretion, which could improve whole body and local glucose metabolism, providing potential for diabetes 2 type treatment. Solid phase extraction of biological samples was optimized on columns regarding to sorbent composition using reversed phase columns with modified styrene divinylbenzene polymer or octadecyl-bonded polymer and normal phase columns packed with silica gel. Column Strata SI-1 Silica was the most effective for FAHFA separation from biological samples. Chromatographic separation of FAHFA was performed on UPLC Ultimate 3000 RSLC equipped with Kinetex C18 1,7 µm, 2,1 x 150 mm column using gradient program. UPLC was coupled to QTRAP 5500/SelexION, a hybrid, triple quadrupole, linear ion trap mass spectrometer equipped...

(EN) Cellulose tris-(3,5-dimethylphenylcarbamate) chiral stationary phase was used for separation of selected 24 analytes. Enantioseparations were realized using two systems, high performance liquid chromatography and supercritical fluid chromatography. Effect of mobile phase composition was studied. Five different aditives (isopropylamine, diethylamine, triethylamine, trifluoroacetic acid, isopropylamine combined with trifluoroacetic acid) and their influence on enantioseparation were tested. Influence of two different modifiers (methanol, propan-2-ol) combined with all aditives was also tested in supercritical fluid chromatography system. The aim of this work was to find optimized composition of mobile phase which was suitable for separation of the analytes studied and to compare separation potential among all mobile phases and also between used separations systems. The supercritical fluid chromatography was shown to yield better results, i.e. better resolution in shorter analysis time. However examples of analytes better resolved under optimized conditions in high performance liquid chromatography system have also been found. Keywords (EN) Chirality, enantiomers, enantioselective separation, chiral stationary phase, high performance liquid chromatography, supercritical fluid chromatography.

This diploma thesis deals with the determination of creatinine using a combination of flow injection analysis (FIA) or high-performance liquid chromatography (HPLC) with pulse amperometry, an electrochemical technique based on the application of potential pulses on a gold working electrode. The determination was performed in a basic environment of borate buffer with creatinine concentration of 1∙10-4 mol∙l-1 . The lenght of the cleaning and activation pulse was optimized as well as the pH of the running buffer. A cleaning pulse of +1.8 V was first applied to the electrode for 100 ms, then an activation potential of -0.5 V was applied for 150 ms and then a measuring potential of +0.2 V for 300 ms. The optimal pH was selected as pH=9,4. Methanol and acetonitrile were added to the borate buffer to test whether creatinine could be determined in presence of these organic solvents and whether flow injection analysis could be transformed into HPLC. Methanol in the system caused peak deformation, acetonitrile did not cause the peak deformation in the system, at higher contents the baseline was destabilized. Furthermore, the calibration dependence in the range of concentrations from 2.5∙10-4 mol∙l-1 to 5∙10-6 mol ∙ l-1 was measured using PAD in combination with FIA. At higher concentrations, peaks splitted....

To make chemical analysis available both practically and economically, one approach is to miniaturise the equipment needed for the analysis. High-performance liquid chromatography (HPLC) is an example of a flow chemistry analysis system where active work is performed to achieve miniaturised systems. In this thesis, the focus is on creating a miniatyrised pump system constructed of pressurised CO2 (PCO) and a microfluidic chip with a restriction channel. The assignment of the PCO is to force a separate medium, which in this case is water, through the remaining system. The pump system will therefore be defined as pressure-driven, which has advantages as pulse-free flows. Utilising the latent energy from the PCO also reduces the need for electrical power, hence allowing a smaller battery. However, the pressure from the carbon dioxide source will gradually decrease as the content is consumed. To obtain continuous pressure, heaters have been integrated into the chip, and thus, the pressure drop can be controlled by changing the viscosity and density of the through-flowing fluid. A cooling table was also used to enable the cooling of the chip and thus further increase the pressure drop. PID control was implemented for the temperature to be adjusted to maintain a constant pressure downstream of the chip. By using this technology, runs of just over 80 minutes have been achieved with a pressure of 60 bar and a flow of 100 µl/min downstream, with a maximal error of around 0.03 bar. Then a chip adapted for water was used to control the water flow. Chips adapted for carbon dioxide placed right after the carbon dioxide source were also tested andruns of just over 10 minutes at 75 bar and 100 µl/min could be achieved with a maximal error closer to 1 bar. The pressure vessel used held a maximum of 100 ml of CO2 at 60 bar. The idea is that the pump system, in the end, will be applied for portable HPLC, and the PCO will then be stored in a cartridge, but in the experiments, a turned-off ISCO pump functioned as a carbon dioxide source.

The naturally occurring dipeptide carnosine (-alanyl-L-histidine) specifically attenuates tumor growth. Here, we ask whether other small imidazole-containing compounds also affect the viability of tumor cells without affecting non-malignant cells and whether the formation of histamine is involved. Patient-derived fibroblasts and glioblastoma cells were treated with carnosine, L-alanyl-L-histidine (LA-LH), -alanyl-L-alanine, L-histidine, histamine, imidazole, -alanine, and L-alanine. Cell viability was assessed by cell-based assays and microscopy. The intracellular release of L-histidine and formation of histamine was investigated by high-performance liquid chromatography coupled to mass spectrometry. Carnosine and LA-LH inhibited tumor cell growth with minor effects on fibroblasts, and L-histidine, histamine, and imidazole affected viability in both cell types. Compounds without the imidazole moiety did not diminish viability. In the presence of LA-LH but not in the presence of carnosine, a significant rise in intracellular amounts of histidine was detected in all cells. The formation of histamine was not detectable in the presence of carnosine, LA-LH, or histidine. In conclusion, the imidazole moiety of carnosine contributes to its anti-neoplastic effect, which is also seen in the presence of histidine and LA-LH. Despite the fact that histamine has a strong effect on cell viability, the formation of histamine is not responsible for the effects on the cell viability of carnosine, LA-LH, and histidine.

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