The published method allows the separation of the four isomers of the anxiolytic 2,3-diazepine derivative, tofisopam. Assignment for the peaks of the two central and two helical isomers is performed by using the chromatogram of the pure tofisopam enantiomers and also considering the conformer-equilibrium was suggested by previous authors. The detection has been made by CD. The in situ CD spectra of the peaks, obtained by stop-flow method, definitely show different chiral character.