The (IP-)HILIC chromatograms obtained for the PS ONs without and with IPRs added to the eluent are depicted in Figs. and. Using the same eluent compositions and linear gradient as used for the poly(dT) ladder, the PS ONs eluted earlier relative to the poly(dT) 15-mer in all cases, even though the PS ONs contained more negative charges. Most probably, phosphorothioation creates more ON net hydrophobicity than phosphodiester phosphate bonds due to P=O being more electronegative than P=S. The effect of the IPRs on the peak widths is shown in Fig. and Table and S4, where the replacement of AA by IPRs lead to a decrease in peak widths up to TEAA. A further increase of IPR hydrophobicity presented increased peak widths compared to TEAA. The PS ONs contain diastereomers that could partially be resolved and which can be structurally affected by IPRs in the eluent. The decrease in peak width observed when going from AA to TEAA suggests that diastereomer separation is decreased when the hydrophobicity of the IPR is increased, which is also observed for IP-RPLC. This was further confirmed when analyzing an 8-mer ON with 2 PS linkages and 4 possible diastereomers, which clearly showed decreased diastereomer separation with IPRs of increasing hydrophobicity in the eluent (Fig. ). We concluded that DEAA and TEAA provided a good compromise between peak broadening due to the IPR's effect on HILIC retention and due to diastereomer separation.