HPLC Separation of Thymidine, Uridine, Adenosine, Guanosine, and Cytidine Using the Hydrogen Bonding Method

Nucleosides are glycosylamines composed of a nucleobase linked to either ribose or deoxyribose sugars and serve as the fundamental building blocks of DNA and RNA. Due to their high polarity and the presence of multiple functional groups capable of hydrogen bonding, they pose challenges for conventional chromatographic methods.

Using the Zodiac HST HB column, which operates via hydrogen-bonding interactions, effective separation of nucleosides such as thymidine, uridine, adenosine, guanosine, and cytidine was achieved. The retention time shows a strong dependence on the mobile phase composition, particularly the ratio of acetonitrile (ACN) to methanol (MeOH).

Retention strength increases with the number of hydroxyl groups present in the analyte, offering a predictable elution pattern. This method is fully LC/MS-compatible and suitable for preparative chromatography.