For a standard four-zone simulated moving bed (SMB) chromatography, several process modifications have been made in previous studies such that its application scope could be extended to a ternary separation. One of the effective modifications reported was to (1) replace its closed-loop configuration with an open-loop configuration and (2) utilize the extract port for collecting both the intermediate-affinity and the highest-affinity components in regular sequence in every switching period. Most of previous researches on such a modified four-zone SMB (MF-SMB) have been limited to process simulation and optimization. The experimental validation of the MF-SMB process with linear isotherms was attempted in this article for the first time using a ternary amino acid mixture as a model system. First, the intrinsic parameters of three amino acids were estimated from a series of multiple-frontal experiments. The estimated parameter values were then used in the stage of the MF-SMB optimization, which was assisted by an up-to-date genetic algorithm. Based on the optimized conditions, the MF-SMB experiment was conducted and the assay results for product samples verified the attainment of a ternary separation. The experimental purities and concentrations were also found to agree closely with the model predictions. (C) 2010 Elsevier Ltd. All rights reserved.