Methodology for dyes purification by preparative bidimensional offline reversed‐phase high‐performance liquid chromatography separation based on mobile phase pH change: Case study of Justicia spicigera

The extensive characterization of the natural dyes involves the purification of their colored compounds for their structural analysis and stability studies. As most natural compounds being ionizable, herein is presented the optimization of an easy and affordable preparative bidimensional offline reversed‐phase high‐performance liquid chromatography purification based on mobile phase pH change. At the analytical scale, several combinations of stationary phases and mobile phases at different pH values were investigated first. The orthogonality between the dimensions was quantitatively evaluated using the nearest‐neighbor distance approach. The optimized separation method was transferred to the preparative scale for the successful isolation of two colored compounds from Justicia spicigera , a traditional dye plant from Central America. They were identified as perisbivalvine B (2‐amino‐8‐hydroxy‐7‐methoxy‐3H‐phenoxazin‐3‐one) and one of its derivatives, also found in another tinctorial plant species, Peristrophe bivalvis growing in Asia.