The invention is a method of preparing multimodal ligands for hydrophobic interaction chromatography (HIC), which comprises providing a cyclic scaffold comprising a thiol, an amine and a carbonyl group; derivatisation of the nitrogen with a reagent to introduce a primary interaction; and aminolysis of the resulting derivative, whereby a secondary interaction is introduced next to the carbonyl; wherein at least one of the primary interaction and the secondary interaction comprises a hydrophobic group and wherein non of said interactions comprises charged groups, i.e. ion exchange ligands. The invention also encompasses a separation medium comprising such multimodal ligands immobilised to a base matrix.