Nükleozidlerin Zwitteriyonik Monolitik Kolonlar ile Kapiler Elektrokromatografi Sisteminde Ayrımı
xmlui.mirage2.itemSummaryView.MetaDataShow full item record
Boronic acids are important ligands for selective recognition and enrichment of compounds containing cis-diol groups such as nucleosides and glycoproteins. This feature makes boronic acids excellent ligands for use in molecular recognition, proteomics and metabolomics studies. Since boronate affinity is based on covalent binding, it provides high specificity by suppressing nonspecific interaction. The hydrophobic interaction is one of the most important secondary interactions affecting the selectivity of boronate affinity. Boronic acid ligands used as affinity materials are generally aromatic boronic acids and cause π-π interactions which leads to nonspecific adsorption of proteins. Monolithic silica columns have found high application in proteomics and metabolomics studies due to their superior advantages such as high column efficiency, hydrophilicity, high permeability and high surface areas. However, the preparation process is very difficult and post-modifications are time consuming compared to organic monolithic columns. The hybrid organic silica monolithic columns used in the thesis have significant advantages such as less shrinkage, more stable mechanical and pH stability as an alternative. In this study, organic-silica hybrid monolithic columns were synthesized for specific isolation of nucleosides. The basis of the mechanism is the reversible covalent complex formation between boronic acids and cis-diol groups which vary depending on the alkali/acidic conditions. Since boronic acid chromatography (BAC) is based on covalent binding, nonspecific interactions are suppressed when appropriate conditions are selected and high specificity is obtained. Ribozylated metabolites, particularly modified nucleosides, are used as cancer biomarkers. Boronate adsorbents are promising materials that can be used to extract nucleosides from complex matrix media. Despite that due to the limited capacity and low selectivity of boronate affinity materials, the determination of metabolites in low amounts is still a problem. For this aim, before modification by phenyl boronic acid (PBA), methacryl polyhedral oligomeric silsesquioxane- methacryloyl histidine ([POSS-MAH]) monolithic column was synthesized to form methacryl polyhedral oligomeric silsesquioxane- methacryloyl histidine-phenyl boronic acid ([POSS-MAH-PBA]) based BAC adsorbents. PBA, which is bound to [POSS-MAH] monolithic column with its numerous binding sites introduced by methacryloyl histidine (MAH) monomer, has a higher adsorption capacity in the column compared to other materials used for this purpose. The synthesized hybrid [POSS-MAH] and [POSS-MAH-PBA] monolithic columns were characterized by swelling test, fourier transform infrared spectrometry (FTIR), elemental analysis, scanning electron microscope (SEM), micro computed tomography (Micro-CT) and BET. The surface areas of the [POSS-MAH] and [POSS-MAH-PBA] monolithic columns determined by Brunauer–Emmett–Teller (BET) analysis were 110.79 and 507.60 m2/g, respectively. After functionalization with PBA, the [POSS-MAH-PBA] monolithic column with a large surface area showed a high binding capacity (13.38 mg/g) for adenosine nucleoside. The effect of different parameters such as pH, concentration, temperature, ionic strength, flow rate and interaction time on the adenosine nucleoside adsorption capacity of the prepared [POSS-MAH-PBA] monolithic column was investigated. pH 8.0, adenosine concentration of 0.1 mg/ml, NaCl salt concentration of 0.01 mg/ml, the flow rate of 0.5 ml/min and the interaction time of 60 min were determined as maximum values for adenosine adsorption. A mixture of 25 mM formic acid and methanol solution (1: 1, v/v) was used as the desorption solution. In order to show that the affinity of [POSS-MAH-PBA] monolithic column to cis-diol-containing molecules is due to PBA functional group, [POSS-MAH] monolithic column was prepared and the binding capacity of adenosine molecule was examined and estimated to be 1.89 mg/g. Adenosine nucleoside adsorption behavior of [POSS-MAH-PBA] monolithic column was investigated by applying Langmuir and Freundlich adsorption models. Experimental results showed that adenosine molecule was bound to the binding sites as a monolayered and equally. In reusability tests, the decrease in adsorption capacity was only 1.6% after 5 consecutive repeated adsorption. The selective affinity of the [POSS-MAH-PBA] monolithic column to cis-diol-containing nucleosides was demonstrated by examining the sample containing the deoxyadenosine triphosphate (dATP) molecule. In addition, to examine the nucleoside metabolites passed to the urine solution, the artificial urine solution was first spiked with adenosine (0.25 mg/ml) and adsorbed to the [POSS-MAH-PBA] monolithic column. The amount of adsorbed adenosine molecule was determined to be 10.05 mg/g due to the both ions that make up crowd effect and the competing molecules. Adsorbed adenosine was confirmed by HPLC. To demonstrate the group specific affinity of the prepared [POSS-MAH-PBA] monolithic column to the cis-diol containing compounds, in the same way, the nucleoside metabolites passed to the artificial urine solution was examined. For that, the artificial urine solution was spiked with nucleosides (0.25 mg/ml) and adsorbed to the [POSS-MAH-PBA] monolithic column and the desorbed nucleosides were determined by HPLC method. The [POSS-MAH-PBA] monolithic column's ability to adsorb nucleosides was demonstrated. Finally, in order to increase the sensitivity, electrochromatographic analyses were performed with the capillary [POSS-MAH-PBA] monolithic capillary column prepared in the same proportions.