The formation of decreasing internal pH gradients in carboxylic cationexchange columns, with a single buffering component in the eluent, was investigated. Two types of carboxylic cation exchangers (hypercrosslinked polystyrene and polymethylmetacrylate based) were used. To develop a pH gradient, the column was pre-equilibrated at pH 7.5 with Tris buffer, followed by washing with different organic acid solutions, including acetic, oxalic, tartaric, citric, and glutamic acids. The smoother quasi-linear gradients in the pH range from 7.5 to 3.5 were obtained with 0.2 -1 mM citric acid as eluent. The noticeable role of ionic strength of the starting buffer and of the eluent on the profile of pH gradient was demonstrated. The optimal pH gradient profiles were obtained at equal ionic strengths of both mobile phases.

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