Evaluation of the Electrostatic Contribution to the Retention of Modified Nucleosides and Nucleobases by Zwitterionic Hydrophilic Interaction Chromatography

This work explores the benefits and limitations, on a quantitative basis, of using zwitterionic hydrophilic interaction chromatography (ZIC-HILIC) for the separation of several modified nucleosides and nucleobases of clinical interest. The target compounds were hydroxylated and methylated derivatives: 8-hydroxy-guanine, 8-hydroxy-guanosine, 8-hydroxy-2′-deoxyguanosine, 1-methyl-guanine, 7-methyl-guanine, and 9-methyl-guanine. A quantitative evaluation of the electrostatic interaction based on a systematic study of the nature and concentration of the salts in the mobile phase has been carried out. From the obtained results, it may be concluded that separation is based on a mechanism of partition and interaction through weak electrostatic forces: the contribution of the electrostatic interaction to the retention of the charged analytes reaching values between 25 and 52% at low salt concentration. However, the electrostatic contribution decreased progressively as the salt concentration rose.