Heavy water enhancement of fluorescence signal in reversed-phase liquid chromatography

Liquid chromatography with fluorescence detection has been used in analyses demanding high sensitivity and selectivity. As majority systems rely on reversed-phase columns with water being the main component of the mobile phase, fluorescent compounds with emission maxima higher than 500 nm might be dynamically quenched. A simple replacement of H₂O with D₂O enhanced the sensitivity for selected compounds by 10-200%. Affected compounds included an anti-cancer drug doxorubicin, a luminescent probe fluorescein, and naturally occurring forms of vitamin B₂. Similar levels of enhancement were obtained by fluorescence spectrometry. Such simple yet effective approach may greatly improve HPLC analyses coupled to fluorescence detection.