The pH of the eluent affects the molecular charge of the ON, particularly when it is deaminated. For the tested FLP, deamination involves the conversion of 5-methylcytosine (MeC) to thymine (T), which goes along with the loss of an aromatic primary amine group (pKa 4.6) and a gain in acidity of the imide group (pKa changes from 12.4 for MeC to 9.9 in T). The difference in charge between the FLP and its deaminated form is a crucial parameter that contributes to their separation in WAX methods. Therefore, we studied the performance when using an eluent containing 25 mM TEA with a pH of 4.8, 7.0, and 9.0 (Fig. ; chromatograms in Fig. ). An increase of the eluent pH leads to a decrease in retention of the ONs. This effect can be explained by the number of charges on the ON at different pH values. At pH 4.8, the amine group of the MeCs and adenines (A) (pKa of 4.1) on the ON is partially positively charged, which decreases the net charge of the ON. However, the total number of charges on the ON is higher than at pH 7 (where all nucleobases are neutral), making the ON more hydrophilic and thus more strongly retained. At pH 9, T, G (pKa 9.2), and A (pKa 9.8) are partially negatively charged, increasing the number of IPR interaction sites. This increases the apparent hydrophobicity of the ON and thus decreases its retention. Small variation of peak widths is observed at different pH conditions, with the smallest peak widths on average at pH 7 (Table ).