HPLC Separation of Cytidine and Cytosine Using the Hydrogen Bonding Method

Nucleosides are glycosylamines composed of a nucleobase attached to a ribose or deoxyribose sugar. As fundamental components of DNA and RNA, nucleosides are highly polar and possess functional groups capable of hydrogen bonding.

A selective HPLC method was developed for the separation of cytosine and cytidine using the Zodiac HST HB column, which operates via hydrogen-bonding interactions. Retention times for these polar compounds are highly sensitive to the composition of the mobile phase, which consists of acetonitrile (ACN) and methanol (MeOH). The elution order of cytosine and cytidine can be manipulated by adjusting the ACN/MeOH ratio.

This method is fully compatible with LC/MS detection and preparative chromatography, and can be extended to separate other nucleobases, nucleosides, and nucleotides effectively.