Separation of Nucleobases, Nucleosides, Nucleotides and Oligonucleotides by Hydrophilic Interaction Liquid Chromatography (HILIC): A State-of-the-Art Review

The polar nature of nucleobases, nucleosides and nucleotides makes hydrophilic interaction chromatography (HILIC) a good choice of technology for separation. Both naturally occurring and modified nucleosides and nucleotides have been successfully separated in HILIC. A wide range of stationary phases with different retention and selectivity are suitable for the separation of nucleobases, nucleosides and nucleotides; and a sufficient knowledge base is also available to guide method development. Although oligonucleotides are significantly different from nucleotides in terms of polarity and charges, HILIC has been shown to be a viable alternative to ion-pairing reversed-phase liquid chromatography (IP-RPLC). Only a few polar stationary phases have been shown to provide satisfactory performance; however, the requirements for the mobile phase composition including organic solvent, mobile phase pH and salt concentration are sufficiently understood. This review provides a comprehensive evaluation of the chromatographic conditions with a historical perspective on adopting and developing HILIC for the separation of nucleobases, nucleosides, nucleotides and oligonucleotides. The areas for more research and potential directions for future development activities are identified and discussed.