Characteristics of the adsorption mechanism of acido-basic compounds with two pKa in reversed-phase liquid chromatography.

Overloaded band profiles of phthalic acid, nicotinic acid, and sodium nicotinate eluted with a methanol-aqueous buffer solution were recorded at the exit of a 150 mm x4.6mm column packed with 3.5 microm X-Bridge-C(18) porous particles. The WSpHs of the mobile phase was adjusted at different values, with addition of hydrochloric acid (WSpH approximately 1.37), phosphate (WSpH approximately 2.01, 2.85, and 7.32), formate (WSpH approximately 3.61), acetate (WSpH approximately 4.41 and 5.15), or succinate buffers (WSpH approximately 6.11). The ionic strengths of all the buffers were fixed at 0.1M by adding the necessary amount of potassium chloride when necessary. The compositions of the solutions at equilibrium were solved by using the extended Debye-Hückel theory, which estimates the activity coefficients of the ions in the bulk phase. The adsorption of the samples onto X-Bridge-C(18) assumed a simple bi-Langmuir isotherm for the neutral species (or zwitterion) and a Langmuir isotherm for the ionizable species. Competition for adsorption between the neutral and the ionic species was neglected. The band profiles were calculated based on the equilibrium-dispersive model of chromatography. An excellent agreement was observed between the experimental and the calculated band profiles for all buffer solutions. This work validates the adsorption mechanism assumed in earlier work for neutral and ionizable species. It is consistent with the neutral species adsorbing weakly (due to dispersive interactions) onto and/or into the C(18)-bonded layer (large number of sites) while the singly charged species adsorb strongly onto the few residual accessible silanol groups (due to charge-dipole interactions). Doubly charged species are not retained and are probably excluded from the pores of the hydrophobic stationary phase.