Oligoacetic acid characterization by isocratic and linear salt gradient anion-exchange chromatography

A homologous series of four oligomers of acetic acid (namely acetic acid, succinic acid, tricarballylic acid, and tetracarboxylic acid) is characterized using isocratic and linear salt gradient anion-exchange chromatography. The double logarithmic plot of the isocratic retention factors versus the salt concentration gives straight lines for all samples. These straight lines (with the exception of the line for the strongly retained succinate peak) have a common intersection point--something which is proved to be a direct consequence of the stoichiometric mass-action ion-exchange model. The characteristic charge and the equilibrium ion-exchange constant (and the corresponding Gibbs free energy of ion exchange, or deltaG(exchange)) are determined from the isocratic experiments. The characteristic charge agrees satisfactorily with the number of carboxylic acid groups in the samples, and the deltaG(exchange) value decreases linearly with the characteristic charge. Succinic acid always gives two chromatographic peaks despite the proven chemical purity of the sample. The characteristic charge that is calculated for both of the succinic acid peaks is approximately two. The deltaG(exchange) value calculated for the weakly retained succinic acid peak falls in the free energy versus characteristic charge straight line defined by the other homologues. The deltaG(exchange) value of the strongly retained peak is lower than that of the weakly retained peak by 1.85 kJ/mol. The two succinic acid peaks are explained in terms of an equilibrium between two conformers in solution--one binding the solution counterions tightly and the other loosely. An analysis of all samples under a linear salt gradient provides retention times that increase linearly with the number of functional groups. Using an appropriate model (along with the isocratically determined characteristic charge and ion-exchange constant), we predict theoretically the linear gradient retention times, which agree reasonably well with the experimental ones.