The separation of quaternary mixtures of lactobionic acid, lactose, sorbitol and fructose using a gel-type ion-exchange resin loaded with H+, K+ and Ca2+ ions has been investigated. This separation is relevant in order to improve the productivity of lactobionic acid obtained from the enzymatic reaction catalyzed by Glucose-fructose oxidoreductase and glucono-delta-lactonase enzymes. Measurement of the single-component equilibrium isotherms for all four species was carried out in a preparative column packed with the selected resin at temperatures of 293, 313 and 333 K using the frontal analysis and column adsorption-desorption method. Mass transfer coefficients for all species at different temperatures have been estimated by solving the dynamic model of the chromatographic column. From pulse tests of single-component and mixture solutions, the K+ loaded resin was found to be suitable for this quaternary separation. The unfavourable adsorption isotherm for the lactobionic acid is described by the anti-Langmuir model. Other compounds have shown linear equilibrium in the concentration range up to 130 g L-1. Binary and quaternary fixed-bed experiments were well described by the dynamic model using the parameters obtained from the single-component adsorption data. (c) 2008 Elsevier B.V. All rights reserved.