The study of separation of the enantiomers of chiral anesthetic isoflurane on a cyclodextrin based stationary phase using a gas chromatography simulated moving bed process is reported. Experiments have been performed to investigate the effect of major operating parameters such as switch time, flow rates, and temperature. The study demonstrated that it is possible to separate the enantiomers to a high level of purity for use in clinical trials. It has also been found that the separation is very sensitive to disturbances as compared to the enflurane system that was studied previously.