HPLC Separation of Caffeine, 3- Methylxanthine, 1- Methylxanthine, Xanthine on Zodiac HST HB Column

Xanthines are polar, neutral compounds that are difficult to retain and separate using conventional reversed-phase chromatography. However, the Zodiac HST HB column enables their effective separation through hydrogen-bonding interactions.

Retention on the Zodiac HST HB column correlates with the number of available hydrogen donors in the xanthine structure. Compounds lacking hydrogen-bond donors exhibit lower retention, while those with multiple hydrogen donors show stronger interaction and higher retention times.

Retention and selectivity can be fine-tuned by adjusting the acetonitrile (ACN) to methanol (MeOH) ratio in the mobile phase. Additionally, both protic and aprotic solvents can be used to further control separation behavior, making this method highly versatile.