Hydrophilic interaction chromatography of nucleotides and their pathway intermediates on titania

Nucleotides and their pathway intermediates play important roles in all living species. They are essential cellular components in energy transfer, metabolic regulatory processes and biosynthesis. Titania (TiO2) has strong Lewis acid sites which have an affinity for the strongly electronegative phosphonate group of nucleotides. Herein a bare titania column (150 mm × 4.6 mm I.D., 3 μm) with UV detection at 254 nm was used for the separation of a set of nucleotides (AMP, ADP, ATP, UMP, UDP, UTP, GMP, GDP, GTP, CMP and CTP) and their intermediates (NAD, NADH, UDP-Glu and UDP-GluNAc). Addition of phosphate to the eluent suppresses the ligand-exchange interactions with the titania surface such that hydrophilic interaction chromatography (HILIC) separations may be performed. Increasing the %ACN resulted in increasing retention and efficiency (up to 13,000, 9500 and 4500 plates/m for AMP, ADP and ATP, respectively). The effects of pH, buffer concentration and other eluent anions (fluoride and acetate) were also studied. Fifteen nucleotides and their intermediates were separated in 26 min (Rminimum > 1.3) using an one-step gradient.