A Continuous Extraction Protocol for the Characterisation of a Sustainably Produced Natural Indigo Pigment.

The sustainable industrial production of indigo necessitates a unique extraction process to separate the plant-derived compounds. Calcium compounds are added to encourage hydrolysis of these precursors and to facilitate the isolation of the final form, resulting in an organic-inorganic composite pigment with unspecified characteristics. In this study, we devised a continuous solvent extraction procedure to fractionate the organic indigoid phase within the composite pigment. Overcoming challenges posed by limited solubility in the common organic solvents, this method allows for the analysis of individual fractions, significantly enhancing resolution. Comprehensive characterisation using spectroscopic analysis, thermogravimetry, and UHPLC-MS/MS revealed the potential for quantifying primary components of the natural pigment and distinct differentiation from the synthetic dye. This approach also holds promise for establishing robust manufacturing practices in the industrial production of natural indigo.