A solvent-gradient simulated moving bed (SG-SMB) process for separation of two useful amino acids, phenylalanine and tryptophan, has been studied in previous researches, which confirmed that the SG-SMB outperformed the corresponding isocratic SMB for the same separation task. However, all of the previous studies on the SG-SMB for separation of the two amino acids have been limited to process simulation and optimization, including some batch-chromatography tests. The experimental validation of such an SG-SMB process was attempted in this article. This task began by assembling the experimental unit of the SG-SMB process. Its operating conditions were determined from the SG-SMB optimization tool based on genetic algorithm, in which the mass-transfer parameters as well as the adsorption isotherm parameters of the feed components (two amino acids) were entered as the mathematical models expressed as a function of liquid-phase composition (i.e., modifier concentration in the liquid phase). Based on the determined operating conditions, the relevant SG-SMB experiment was conducted until a cyclic steady-state was reached. The assay of all the resultant product samples verified that the SG-SMB separation of interest was performed successfully as designed. The experimental data were also found to agree closely with the model predictions. (C) 2011 Elsevier Ltd. All rights reserved.