Separation of purine and pyrimidine bases and nucleosides by hydrophilic interaction chromatography

The separation of 12 model compounds chosen among purine and pyrimidine bases and nucleosides was studied by using hydrophilic interaction chromatography (HILIC). The compounds investigated were small molecules with relevant properties for biomedical and pharmaceutical studies. The mixture of pyrimidines and purines was applied on a ZIC-HILIC 150 x 2.1 mm, 5 microm, and two TSKgel Amide-80 150 x 2.0 mm, 5 microm and 3 microm particle size columns. The retention of the analytes was studied by varying ACN%, ammonium formate concentration, pH, and column temperature. The results obtained confirmed the elution order of nucleobases, nucleosides, and nucleotides based on their hydrophobicity. The retention mechanism of the columns was studied considering the models used for describing partitioning and surface adsorption. The influence on retention of chromatographic conditions (ACN%, salt concentration, pH, and temperature) was described and discussed for both columns. The optimization of the conditions studied allowed to assess a gradient method for the separation of the 12 analytes. The developed method is a valuable alternative to existing methods for the separation of the compounds concerned.