However, as compared to using AA, the separation between DA and FLP is improved when DEAA, TEAA, or TPAA are added to the eluent. ON deamination involves the conversionof 5-methylcytosine (log P, -0.8) to thymine (log P, -0.6), resulting in a decrease of the hydrophilicity of the nucleobase. We suggest that when using AA in the eluent (HILIC), this minor difference in hydrophilicity only slightly affects resolution as retention is dominated mainly by the thiophosphate groups. However, when IPRs are added to the eluent, the polar interactions of the phosphate backbone is diminished and the relative contribution of the nucleobases on retention is increased. The DA impurity is slightly less hydrophilic and elutes before the FLP, which is also in line with reported results for HILIC of nucleosides, whereas for the WAX method DA elutes after the FLP due to the additional charge on DA. For the nonconjugated impurity (NC), the resolution with its deaminated form (DA-NC) is even better as compared with the GalNAc-conjugated FLP (see Fig. ), suggesting that the GalNAc group, which is close to the deamination site, reduces DA-FLP resolution. This may be due to its contribution to the FLP hydrophilicity or because it causes steric hindrance, affecting interactions. For all tested conditions, DA-NC was better resolved from NC when compared to the separation of DA from FLP. Overall, TEAA offered the best results in terms of resolving DA impurities. The addition of IPRs to the eluent also significantly improved the resolution between the FLP and NC (Fig. ). With AA in the eluent, NC co-elutes with the FLP. In contrast, by adding IPRs to the eluent, a resolution of 5 or higher can be obtained, indicating that the relative contribution of the GalNAc on HILIC retention is also increased by IPRs. Interestingly, the PO impurity is better separated from the FLP when AA is replaced by smaller IPRs. However, increasing the IPR hydrophobicity further than TEAA decreases resolution between the PO impurity and the FLP. A similar separation was observed by Gong. The ability to separate the PO impurity from the FLP in the presence of IPRs might be related to differences in acidity between the PO and PS groups affecting interaction strength between ON and IPR. Also steric effects may play a role. An ON for which the G nucleotides were switched with T nucleotides (Switch impurity) comprising the same number of nucleotides as the FLP was also measured using different IPRs. As can be seen in Fig. , Switch is partly separated from the FLP with most separation using TBuAA. Overall, from our study of the influence of IPR type on ON impurity separation, we concluded that TEAA provides the most favorable IP-HILIC conditions for impurity profiling, with DEAA as a close second, as it resulted in the smallest peak widths and a slightly better separation of deamination impurities compared to DEAA, which are the most challenging to identify. We also compared the IP-HILIC method employing TEAA in the eluent with IP-RPLC and AEX methods using standard conditions. All methods provide separation of N-1, N+1, and NC impurities from the FLP, while IP-HILIC and AEX also separated the PO impurity (Figs. ). However, using IP-RPLC and AEX, the DA impurity fully co-eluted with the FLP, whereas IP-HILIC separates DA from the FLP, highlighting the complementary separation selectivity and benefits of IP-HILIC.