The enantiomeric separation of methadone in the presence of multiple isomer mixtures of sulfated -cyclodextrin (S--CD) was studied experimentally with CZE and theoretically using computer simulation. Experiments were performed over many years with several lots of S--CD from the same manufacturer with a specified degree of substitution of 7-11. Large differences in the migration patterns were observed between certain lots and it was concluded that the extent of labelling in lots released after a transition time was higher than originally specified. The migration pattern was observed to be associated with (i) the ionic strength increase resulting from using S--CDs with a higher charge state and (ii) differences in buffer composition. Apparent binding constants between methadone and the S--CD and complex mobilities were determined for different lots of S--CD at varying ionic strength using phosphate and 3-morpholino-2-hydroxypropanesulfonic acid buffers. The obtained values were used as input for simulations. For a given ionic strength, agreement between predicted and experimentally observed behavior was obtained for different buffers. R-methadone has a stronger interaction with S--CD than S-methadone. For any given configuration there is a distinct S--CD concentration range which results in the cationic migration of S-methadone while the migration direction of R-methadone is reversed. This configuration was demonstrated to be applicable for micropreparative CZE separations.