Chromatography of ionizable positional isomers with methanol–water–carbon dioxide mixtures Original Research Article

The separations of five groups of ionogenic disubstituted benzene isomers were achieved by taking advantage of the pH variation caused by proportions of CO2 in the methanol–water–CO2 mobile phases without the addition of an aqueous buffer. The retention behavior of acidic, basic, and zwitterionic isomers was predominantly affected by the dissociation of these analytes within methanol–water–CO2 mobile phases of different pH. A comparison with previously published reversed-phase high-performance liquid chromatography (HPLC) of these isomers revealed that resolution improvement for anisic acid, toluic acid and aminobenzoic acid isomers was achieved with methanol–water–CO2 enhanced-fluidity liquids. Significant reductions in the retention factors were also obtained. Meanwhile, the detrimental effect of the presence of an aqueous buffer on the stationary phase was avoided which made fast repetitive analyses possible.