Effect of ion-pairing reagent hydrophobicity on liquid chromatography and mass spectrometry analysis of oligonucleotides

•We proposed an RP LC method for the classification of ion-pairing reagents.•Hydrophobic (stronger) alkylamine ion-pairing reagents give greater separation of oligonucleotides.•Ion-pairing RP LC of oligonucleotides behaves as a mixed mode chromatography.•The separation selectivity depends on the pairing reagent hydrophobicity.