To assess the effect of IPR concentration in the eluent on the separation of PS ONs in IP-HILIC, TEA concentrations of 5, 15, and 25 mM were tested (Fig. ; chromatograms in Fig. ). The temperature and pH of the eluent were 60 °C and 7, respectively. When the concentration of TEA is increased, the retention of the FLP increases. This is somewhat counterintuitive, as TEA causes a reduction in ON retention as compared to AA. A possible explanation is that TEA suppresses electrostatic repulsion with unreacted silanols groups on the stationary phase, as also suggested by Wei et al. when AA is used in the eluent. Lowering the concentration of TEA in the eluent leads to broader peaks (Table ). Moreover, when using a concentration 5 mM TEA in the eluent, an additional peak appeared after the main peak. This minor peak is likely to be an artifact not an impurity and probably a result of peak splitting due to insufficient ion-pairing. When the TEA concentration in the eluent was increased, the resolution of ONs comprising the same number of PS moieties (i.e., Switch and DA) from the FLP did not significantly improve. However, a higher TEA concentration resulted in a small improvement of the separation of the N-1, N+1, and PO impurities from the FLP (Fig. ). This minor improvement in resolution may be related to the decrease in the peak widths, but higher concentrations might also increase ON-IPR interactions. This results in more pronounced differences in ONs with varying numbers of nucleotides or ON-IPR interaction strengths (i.e., PO vs. PS groups). However, high concentrations of IPRs in the eluent cause ion suppression, detrimentally affecting MS detection. Therefore, no higher concentrations of TEA were tested and 25 mM was chosen as the optimal working condition.