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The phi-plot optimization method showed great potential as a search routine for determination of the best operational conditions of solvent-gradient simulated moving bed process (SG-SMB) separation of the amino acids phenylalanine and tryptophan. The SG-SMB process can be used to improve the performance of separation of bio molecules if compared to the traditional isocratic solvent conditions. In the SG-SMB process the modulation of the solvent strength leads to significant increase in the purities and productivity followed by reduction in the solvent consumption. A negative aspect of such process is the increase in the complexity for determination of the best operational conditions of separation being it a difficult task. The phi-plot optimization method was applied to the separation of the amino acids tryptophan and tyrosine which has not been studied before in the context of SG-SMB process separation. The solvent gradient chromatographic data of the amino acids was utilized for mass transfer kinetic parameters determination which was incorporated in a stepwise SG-SMB simulator. The phi-plot optimization approach was applied to achieve the best operational conditions of separation being evaluated by the stepwise SG-SMB simulator. The new phi-plot optimization approach was able to determine the best configurations of the operational conditions to achieve separations with high values of purities in the extract and raffinate which was evaluated later by the simulator. The simulation results showed the viability of SG-SMB separation of the amino acids Tryptophan and Tyrosine despite the fact the biomolecules presented close retention times, comparing to the separation of phenylalanine and tryptophan.