GABA (neurotransmitter) and its isomers are polar zwitter-ionic compounds. Due to the position of amino-groups, all three compounds show different polar and basic properties. The isomers of aminobuturic acid are separated on an Obelisc N HILIC/cation-exchange column. Buffer concentration has a different effect on retention of alpha-, beta-, and gamma-aminobutyric acid. This general and robust method can be used for separation of other polar and ionizable compounds and isomers by mixed-mode chromatography.