Application of 2D-liquid chromatography for the separation of a mixture of isomeric and structurally related azatryptophan derivatives

The present study describes the critical achiral and chiral separation of a mixture of azatryptophan derivatives having positional isomers or isobars and their enantiomers, which is successfully achieved using 2D HPLC by employing two different stationary phases and mobile phase combinations in one instrument. The first-dimension experiments were carried out under reversed-phase conditions, where the best separation of racemates was obtained with a Kinetex F5 core-shell column with 10 mM ammonium acetate in water and ACN as a mobile phase in gradient elution mode. The racemate peaks of first dimension experiments were selectively heart-cut based on the time program set in its instrument method and parked in loops for the second-dimension analysis. The parked racemate peaks were sequentially sent into the second dimension to obtain the best separation of the respective isomers, which was achieved using a Chiralcel OD-H column with 0.1% DEA in MeOH : ACN (90 : 10, v/v) as a mobile phase in isocratic elution mode. The study provides valuable insights into the separation methods, where isobars and their respective isomers can be easily and quickly separated using various stationary and mobile phase combinations within a single instrument.