Precursor carboxy- and chloro-silica supports for functionalization with various chromatographic ligands: Polar neutral and ionizable, nonpolar alkyl and aryl, ionic liquid and bio- affinity ligands

The development of novel stationary phases to separate and analyze complex mixtures is an ongoing task of relevance in the field of high performance liquid chromatography (HPLC). From this perspective, the work presented in this dissertation seeks to address the introduction of novel functionalities to silica-based matrices in order to develop stationary phases suitable for different HPLC separation modes. The functionalization of the carboxy-silica surface was carried out using carbodiimide conjugation reactions for grafting retentive ligands via stable amide bond formation. On this basis, aromatic-based reversed-phase stationary phases were prepared by immobilizing 2-naphthylamine and 2-aminoanthracene by on-column reactions via carbodiimide reaction. The retention behavior of these columns was studied for various aromatic analytes and compared with a conventional C18 column. The same precursor was used to prepare a neutral polar stationary phase for hydrophilic interaction liquid chromatography (HILIC) by grafting D-glucamine ligands onto the activated surface carboxyl groups. Unique retention behavior with high separation efficiencies were observed for polar analytes and some derivatized sugar samples. In addition, mixed mode retention properties were investigated using the carboxy-silica precursor itself via changing mobile phase conditions. Proteins were separated under weak cation and hydrophobic interaction modes (WCX and HIC) whereas the HILIC mode was used in separating nucleotides, nucleic acid bases and other polar solutes. With the aim of capturing glycoproteins with different glycoforms, three lectin-based affinity columns were prepared by successfully immobilizing concanavalin A (Con A), wheat germ agglutinin (WGA), and Phaseolus vulgaris leucoagglutinin (PHA-L) on a carboxy-silica surface. Firstly, the columns were characterized for their specific binding ability using standard proteins followed by proteomics-based analysis. Con A and WGA columns were used in the identification of differentially expressed proteins (DEPs) present in adenocarcinoma cancer and disease-free sera for the recognition of potential cancer biomarkers. Similarly, two immuno-affinity columns were prepared by immobilizing anti-apolipoprotein and anti-haptoglobin antibodies and analyzed for their specific binding potential using the corresponding antigens. Further, a proteomic-based study was carried out in identifying DEPs. In a different approach, hexadecylimidazole-based reversed phase column and 1-ethyl imidazole-based ion exchange column were prepared and their separation performances were evaluated.