To assess the effect of ion-pairing on ON retention in HILIC, IPRs of different physicochemical properties and hydrophobicity (DEAA, TEAA, TPAA, and TBuAA) were tested by analyzing a poly(dT) ladder applying a linear gradient from 90% ACN to 41% ACN (i.e., 100% to 30% B) on a BEH amide column. This sample does not contain any PS modifications and, therefore, no diastereomers, allowing us to evaluate the effect of IPRs in the eluent on ON peak widths and separation resolution. Using a concentration of 15 mM of IPR in the eluent, (partial) separation of the five ONs was obtained (Fig. ), where the HILIC retention of the poly(dT) ONs decreased with increasing IPR hydrophobicity. Compared with an eluent containing no IPR but 15 mM AA instead, a retention-time reduction of approx. 30 min was observed for the 15-mer poly(dT) when using TBuAA in the eluent. The interaction of the positively charged IPRs with the negatively charged phosphate groups decreases the apparent hydrophilicity of the ONs and thus their retention. Along these lines, the retention increases with ON size, as every additional nucleotide adds a phosphate group, increasing the overall hydrophilicity of the ON. In addition to affecting the retention of the poly(dT) ONs, the IPRs also had a noticeable difference in peak widths and resolution. The mobile phase with 15 mM AA (no IPR) resulted in the highest resolution and smallest peak widths, and thus the most favorable peak capacity (nc) of 258 (Table ).