Compared to conventional C18 phases, polar-modified phases have distinct differences with regards to chromatographic behavior. In the present study, ODS phases and polar-modified phases were synthesized. The columns containing these new packings demonstrated satisfactory stability under both acidic (pH 1.5) and basic (pH 10) conditions. We evaluated the selectivity differences between alkyl and polar-modified alkyl RP columns by using a range of neutral analytes. The polar-modified alkyl phases showed excellent peak shapes for almost all compounds. We also compared the selectivity differences between them for separating nucleotides by using 100% aqueous mobile phase and tricyclic antidepressants in the intermediate pH mobile phases. The results demonstrated that polar-modified phases display a significantly reduced hydrophobic nature and a significantly reduced silanol activity compared to the conventional C18 phases.