Separation of highly charged compounds using competing ions with hydrophilic interaction liquid chromatography - Application to assay of cellular nucleotides

Separation of highly charged compounds has always been a challenge in chromatography. Ion-pair reversed phase chromatography has been the most successful approach to date. Although polar reversed phase and HILIC columns have been introduced, they have limitations with highly charged compounds. Competing ions have been used, in addition to ion-pair reagent, to achieve better resolution with reversed phase columns. Herein, we explored the use of competing ions with HILIC columns to demonstrate the effects on retention and separation of mono-, di-, and tri-nucleotides, introducing a new tool to improve resolution with HILIC columns. HILIC columns that had irreversibly retained highly charged tri-nucleotides became capable of successfully separating the same compounds, by using this approach. The optimised method was used to successfully resolve a mixture of 12 nucleotides with charges ranging from 1− to 3−. The method was applied to quantify nucleotides in blood cell extracts.