HPLC Separation of Adenosine and Adenine Using the Hydrogen Bonding Method

Nucleosides are glycosylamines composed of a nucleobase linked to either a ribose or deoxyribose sugar. As essential building blocks of DNA and RNA, these compounds are highly polar and contain functional groups that readily participate in hydrogen bonding interactions.

A robust HPLC method was developed using the Zodiac HST HB hydrogen-bonding column for the separation of adenine and adenosine. Due to their structural similarity and polarity, traditional reversed-phase approaches provide limited resolution. However, hydrogen-bonding interactions in the Zodiac HST HB column enable strong retention and effective separation.

Retention behavior of adenine and adenosine is highly dependent on the mobile phase composition, particularly the ratio of acetonitrile (ACN) to methanol (MeOH). The elution order of these nucleosides can be reversed simply by adjusting the proportions of ACN and MeOH. This tunability allows for method optimization based on target resolution.

The method is fully compatible with LC/MS detection and preparative-scale chromatography, making it suitable for both analytical and purification workflows.