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.

Spectral characterisation of a traditional Mesoamerican dye: relationship between in situ identification on the 16th century Codex Borbonicus manuscript and composition of Justicia spicigera plant extract.

Characterisation of vegetal colourants in manuscripts is still a challenging task. Their identification using non-invasive techniques - since sampling is rarely possible - is valid only if there is evidence that the recorded signals are generated by compounds specific to the plant species. Otherwise, more extensive chemical characterisations are required to relate the non-invasive technique signals to the chemical composition of the dye extract and thus avoid misidentification. The case study of a traditional Mesoamerican dye found in the Codex Borbonicus is reported herein. The non-invasive identification of the colourant plant source and chemical characterisation of the coloured molecules were carried out through a multi-analytical technique approach. In situ Raman and UV-Vis emission fluorescence signals suggested the use of Justicia spicigera leaves to produce the brown paint layers of the manuscript. Analysis of the plant extract by liquid chromatography revealed that two compounds are mostly responsible for the colour, and these compounds were successfully isolated. Both contribute to the Raman spectra while only one of them is mainly responsible for the fluorescence recorded on Codex Borbonicus.