Abstract A commercially available aminopropylsilyl bonded-phase high-performance liquid chromatography support has been modified with three different organic acids each containing an amine group and a hydrophobic moiety to produce anion-exchange supports with increased hydrophobicity. The chromatographic characteristics of these supports were examined using two oligonucleotides of equal chain length but differing hydrophobic character, (Ap) 3A and (Up) 3U. The resolution of these two solutes on the modified supports in the pH range 4.5–6.5 and an acetonitrile concentration of 0-48% is described. The most effective support contains a phenylalanine moiety (APS-PHE) bound through an amide to the commercial support. This support has been used to resolve oligonucleotides of the same size (6–12 residues) but different sequence prepared enzymatically or chemically. The difficulties inherent in parameter optimization in mixed-mode chromatography are discussed.