The steviolglycosides are the natural, sweet substances from Stevia rebaudiana Bertoni. It affect human health positively and its sweetness is 300x stronger than the sweetness of sucrose. That's why it's used to sweetening the commercial products. Because of its potential properties it's good to have an appropriate method to isolate it. High performance liquid chromatography ( HPLC) is based on the separation of analyte between two immiscible phases with high pressure pump and appropriately chosen stationary phase in the column. Than the analytes come out from the column in different retention times. This master´s thesis follows up selection of the best HPLC system for isolation of the main steviolglycosides and its analysis in commercial products. In the theoretical part of the thesis is described the origin, basic characteristics, botanical description, cultivation and affect to the human health of Stevia rebaudiana Bertoni and its use in food industry. There are also concisely characterized the sweet substances contained in the plant, so called steviolglycosides. Than there are given the theoretical basics of high performance liquid chromatography, HPLC instrumentation and its specific applications at sleviolglycosides with the basic chromatographic parameters. The object of the first experimental part was to research the optimum conditions for time and separation effective chromatographic analysis and select the best chromatographic system for isolation of steviolglycosides. In the second experimental part, I have compared and defined the main steviolglycosides (stevioside, rebaudioside A) in nine selected products, commercially available in Czech republic, with the best chromatographic method. In these products was the contain of the stevioside or rebaudioside A confirmed or refused.

Novel sol-gel titania-poly(dimethylsiloxane) (TiO2-PDMS) and titania-silica-N-(triethoxysilylpropyl)-O-polyethylene oxide urethane (TiO2-SiO2-TESP-PEO) coatings were developed for capillary microextraction (CME) to perform on-line preconcentration and HPLC analysis of trace impurities in aqueous samples. Due to chemical inertness of titania, effective covalent binding of a suitable organic ligand to its surface is difficult via conventional surface modification methods. In this research, sol-gel chemistry was employed to chemically bind hydroxy-terminated poly(dimethylsiloxane) (PDMS) and N-(triethoxysilylpropyl)-O-polyethylene oxide urethane (TESP-PEO) to sol-gel titania and sol-gel titania-silica network, respectively. A method is presented describing in situ preparation of the titania-based sol-gel PDMS and TESP-PEO coatings and their immobilization on the inner surface of a fused-silica microextraction capillary. To perform on-line CME-HPLC, the sol-gel TiO2-PDMS or TiO2-SiO2-TESP-PEO capillarywas installed in the HPLC injection port as an external sampling loop, and a conventionalHPLC separation column was used for the liquid chromatographic separation. The sol-gel TiO2-PDMS-coated microextraction capillary was used for on-line CME-HPLC analysis of non-polar and moderately polar analytes, and the sol-gel coatings showed excellent pH (1-13), and solvent (acetonitrile and methanol) stabilities under elevated temperatures (150 C) over analogous non-sol-gel silica-based coatings. Extraction of highly polar analytes, especially from aqueous phases is not an easy task. However, the sol-gel TiO2-SiO2-TESP-PEO-coated capillaries showed excellent capability of extracting underivatized highly polar analytes from aqueous samples. This opens the possibility to employ sol-gel titania-based polar coatings for solvent-free extraction and trace analysis of target analytes in environmental and biomedical matrices. To our knowledge, this is the first research on the use of sol-gel titania (or titania-silica)-based organic-inorganic materials as a sorbent in capillary microextraction. The newly developed sol-gel titania (or titania-silica)-based organic-inorganic hybrid extraction media provides an effective solution to coupling CME with HPLC (CME-HPLC), and this can be expected to become a powerful analytical tool in environmental investigations, proteomic research, early disease diagnosis and biomarker research. Being a combination of a highly efficient solvent free sample preconcentration technique (CME) and a powerful separation method (HPLC), CME-HPLC poses to become a key analytical tool in solving complex chemical, environmental, and biomedical problems involving complex matrices.

Size exclusion chromatography (SEC) separates molecules based on their actual size rather than their molecular weight. This indicates that the smaller a molecule is, the longer it will be held in the column since it can penetrate the pores more effectively than a bigger molecule, resulting in a longer retention time, whereas the larger molecule has a shorter retention time. In order to achieve reliable and accurate results the asymmetry factor of the packed columns should be between 0.6-1.4. The aim for this project was to examine whether or not the batch variations of the base matrix used to derive WorkBeads™ 40S and 40Q displayed either hydrophobic or hydrophilic interactions by running different types of proteins through columns packed with the base matrices. The project was performed using an ÄKTA explorer equipped with an ultraviolet (UV) detector and a refractive index detector (RID). The data was gathered and analyzed with the Unicorn™ by Cytiva. The results from the first experiments showed that lysozyme did not elute as expected or not at all, thus leading to a concern that the there might be some hydrophobic interactions in the base matrix, which is a porous media in the form of spherical particles that have been selected for their physical stability and inertness (lack of reactivity and adsorptive properties), and lysozyme. With this suspicion in mind, the different batches of the base matrix underwent hydrophobic interaction chromatography (HIC), where the results may be interpreted in a way that there might electrostatic interactions instead of hydrophobic interactions. However, due to the gel not being suitable for HIC the results were unreliable. By subsequently running lysozyme and other proteins through the columns at different NaCl concentrations the results showed consistent elution at NaCl concentrations > 150 mM, yet inconsistent at a concentration of 150 mM for lysozyme. The elution order by size showed that although lysozyme has a larger hydrodynamic radius (Rh) than cytochrome c it eluted later, which is theoretically incorrect, but it might be owing to some of the base matrix's characteristics or the lysozyme's dual nature of expressing both hydrophobic and hydrophilic interactions on the base matrix. Because of the inconsistent results from lysozyme, another experiment just at 150 mM was conducted where lysozyme did elute consistently with a KD value < 1.  Lastly, a titration was performed on the base matrix where some of it was brominated and some of it was not, which was the reference. The results showed that there could be hydrophobic interactions on the brominated sample and hydrophilic interactions on the reference sample. However, what is more likely is that lysozyme is an unreliable protein to use to determine hydrophobicity on this type of gel. / Size exclusion chromatography (SEC) separerar molekyler baserat på deras faktiska storlek snarare än deras molekylvikt. Detta indikerar att ju mindre en molekyl är, desto längre kommer den att hållas i kolonnen eftersom den kan tränga sig in i porerna mer effektivt än en större molekyl, vilket resulterar i en längre retentionstid, medan den större molekylen har en kortare retentionstid. För att uppnå tillförlitliga och nogranna resultat bör asymmetrifaktorn hos de packade kolonnerna vara mellan 0.6-1.4. Syftet med detta projekt var att undersöka huruvida batchvariationerna av basmatrisen som användes för att ta fram WorkBeads™ 40S och 40Q uppvisade antingen hydrofoba eller hydrofila interaktioner genom att köra olika typer av proteiner genom kolumner packade med basmatriserna. Projektet utfördes med en ÄKTA explorer utrustad med en ultraviolett (UV) detektor och en refractive index detector (RID). Data samlades in och analyserades med Unicorn™ av Cytiva. Resultaten från de första experimenten visade att lysozym inte eluerade som förväntat eller inte alls, vilket ledde till en oro för att det kan finnas vissa hydrofoba interaktioner i basmatrisen, som är ett poröst medium i form av sfäriska partiklar som har har valts ut för deras fysikaliska stabilitet och inerthet (brist på reaktivitet och adsorptiva egenskaper), och lysozym. Med denna misstanke i åtanke genomgick de olika bactherna av basmatrisen hydrofob interaktionskromatografi (HIC), där resultaten kan tolkas på ett sätt att det kan förekomma elektrostatiska interaktioner istället för hydrofoba interaktioner. Men på grund av att gelen inte var lämplig för HIC var resultaten opålitliga. Genom att efteråt köra lysozym och andra proteiner genom kolonnerna vid olika NaCl-koncentrationer visade resultaten konsekvent eluering vid NaCl-koncentrationer > 150 mM, men ändå inkonsekventa vid en koncentration av 150 mM för lysozym. Elueringsordningen efter storlek visade att även om lysozym har en större hydrodynamisk radie (Rh) än cytokrom c, eluerade det senare, vilket är teoretiskt inkorrekt, men det kan bero på några av basmatrisens egenskaper eller lysozymets dubbla natur att uttrycka både hydrofoba och hydrofila interaktioner på basmatrisen. På grund av de inkonsekventa resultaten från lysozym, utfördes ett annat experiment enbart vid 150 mM där lysozym eluerade konsekvent med ett KD-värde < 1. Slutligen utfördes en titrering på basmatrisen där en del av den var bromerad och en del av den inte, vilket var referensen. Resultaten visade att det kunde finnas hydrofoba interaktioner på det bromerade provet och hydrofila interaktioner på referensprovet. Men vad som är mer troligt är att lysozym är ett opålitligt protein att använda för att bestämma hydrofobicitet på denna typ av gel.