Adding IPRs to the eluent increased peak widths significantly and decreased the resolution of the poly(dT) ONs, with the adverse effect becoming more pronounced as IPR hydrophobicity increases. Peak capacities of 243, 201, and 134 were obtained for DEAA, TEAA, and TPAA, respectively (a similar trend is observed for the effective peak capacities (i.e., the time between the first and last eluting peak as tG): 35, 22, 14, and 7 for AA, DEAA, TEAA, and TPAA, respectively). Peak capacity values could not be calculated for TBuAA as co-elution of the poly(dT) ONs prevented the determination of peak widths. But clearly, the peak widths obtained with TBuAA are broader than obtained with the other IPRs (Fig. ). Interestingly, the effect of IPR hydrophobicity on IP-HILIC separations is the opposite of IP-RPLC, for which an increase in IPR hydrophobicity results in higher peak capacities. No PS modifications, and thus no diastereomers, are present in the poly(dT) sample, and therefore, the reduction in IP-HILIC separation performance can be attributed solely to the influence of the IPRs on ON retention. It may be related to secondary hydrophobic interactions disturbing the HILIC separation. These could take place in two different ways: (i) ON-IPR interactions decrease the apparent hydrophilicity leading to decreased interactions between the ON and HILIC stationary phase. Therefore, an increase in hydrophobicity of the IPR increases the disturbance of the polar interactions of the ONs with the HILIC stationary phase, leading to broader peaks; (ii) The positively charged IPR can interact with the hydrophilic stationary phase, reducing its hydrophilicity, where the longer the alkyl chain of the IPR is, the stronger the secondary hydrophobic interaction may be.