Chromatography of substituted benzoic acids with methanol–water–carbon dioxide mixtures

The impact of the proportion of CO2 concentration in methanol–water–CO2 mobile phases on the separation of several substituted benzoic acids was explored by studying the variation of retention with mobile phase pH in these mixtures. As the amount of CO2 in methanol–aqueous buffer–CO2 mixtures increased, a more basic buffer was needed to control the dissociation of these acids. Differences in terms of retention, separation efficiency and peak asymmetry were shown for substituted benzoic acids with methanol–water–CO2 and methanol–aqueous buffer–CO2 mixtures. Variations of these chromatographic parameters with mobile phase pH were related to the dissociation of these acids and their interaction with methanol–aqueous buffer–CO2 mobile phases and the stationary phase. The addition of a buffer into methanol–aqueous solution–CO2 was an effective means to optimize separations of acidic analytes with high fluidity liquid mobile phases. The substituted benzoic acids had baseline separation in the least amount of time using the high fluidity liquid mobile phases.