AbstractThe use of mixed surfactants for the separation of nine phenylthiohydantoin amino acids (PTH amino acids) by micellar electrokinetic chromatography (MEKC) was examined. The potential of a mixed surfactant system, consisting of cationic and anionic surfactants, for enhancing the selectivity of MEKC separations was explored. Both sodium dodecyl sulfate (SDS) and cetyltrimethylammonium bromide (CTAB) in the electrophoretic medium were found to improve the separation of the PTH‐amino acids compared to the use of CTAB or SDS alone. Complete separation of the PTH amino acids was achieved using a buffer containing 2.5 mM CTAB and 40 mM SDS. The analysis was performed without the need to reverse the polarity of the electrodes, typically required if oppositely charged micelles are used. The migration behavior of the nine PTH amino acids using the mixed surfactant system was compared with MEKC in presence of SDS alone.