Tyrian purple in archaeological textiles: DMF extraction and recrystallization for the Raman identification of precursors and derivatives Brenda Doherty, a,b * Witold Nowik, c Costanza Miliani a,b and Catia Clementi d The prized ancient dyestuff, Tyrian purple, has been extensively and widely studied by multiple spectroscopic and chromato- graphic methods alike. In this paper, an extraction method adopted by liquid chromatography by using N,N-dimethylformamide is proposed as a sample pre-treatment for the characterization of minor reference brominated and unsubstituted isatin and indirubin components by micro-Raman spectroscopy. The method has been validated for the identification of these constituents in accordance with HPLC PDA measurements on a series of Tyrian purple-dyed wool, silk and cotton textiles and in archaeological samples from the Roman period. It results specifically effective in cases when high-scattering dibromoindigoids are not revealed and when main indigoid spectral features, seemingly of plant origin, can only be reasonably hypothesized by conventional Raman. This method also innovatively highlights the possible presence of carotenoids and porphyrin-type copper and iron complexes, noted in marine molluscs, and potentially opening a new perspective to the current diagnostics of Tyrian purple through its biomarkers in the cultural heritage domain. Copyright © 2017 John Wiley & Sons, Ltd. Keywords: Tyrian purple; bromoisatin; bromoindirubin; marine porphyrin Introduction Among the most ancient of dyes, Tyrian purple continues to be of high interest for scientists and historians alike in the cultural heritage field with regard to its identification in artefacts. Extensive scientific literature is available concerning the high lightfast natural indigoid class of vat dyes and pigments encompassing plant sources of indigo, principally Indigofera species in Asia and Isatis tinctoria L. in Northern Europe and many others as well as animal mollusc sources pertaining to Tyrian purple. The modern elucidation of the chemical structures and natural production mechanisms of both sources have been vastly studied highlighting production intermediates, precursors and by-products, as observed in Fig. S1. [1–6] Tyrian purple, variously known as true, genuine, royal or imperial purple, unlike indigo, was prevalently rare and costly and was notably obtained from multiple shellfish species, such as, Murex trunculus, Murex brandaris, Thais haemastoma and Nucella la- pillus, around the Mediterranean and Atlantic coastal areas. Analytical characterizations by optimized chromatographic techniques have highlighted Tyrian purple’s main brominated and unsubstituted indigotin, indirubin (Inr) and isatin (Isa) constituents [7,8] as well as extremely minor components, the iminoindirubins. [9] Similarly, non-invasive UV–Vis reflectance spectroscopic techniques have been able to characterize Tyrian purple on parchment [10] -illuminated manuscripts [11] and textile reference samples dyed with main purple components. [12–14] Newer data-handling methods, able to establish the relationship between HPLC and non-invasive UV–Vis reflectance spectroscopy data, have recently led to Tyrian purple’s identification through statistical analyses. [15] Wide vibrational spectroscopic studies by infrared and Raman have long reported on different reference Tyrian purple and indigoid spectral features. [16–18] Although for historic samples, the predominance of signals that arise from textile matrices in infrared has often led to the prevalence of Raman for investigations where weak scattering and simultaneous fluores- cence have in the last years facilitated the use of ulterior Raman techniques including subtracted shifted Raman spectroscopy [19] and surface-enhanced Raman spectroscopy for successful characterization. [20,21] However, it is often overlooked by Raman spectroscopic methods that the main components of Tyrian purple dye can inevitably observe compositional differences according to the relative amounts of precursors, enzymes and other substances present in the secretion from different molluscan species. It is to be expected that such components can differ even further * Correspondence to: Brenda Doherty, Centro di Eccellenza SMAArt, c/o Dipartimento di Chimica, Biologia e Biotecnologie, Università degli Studi di Perugia, via Elce di Sotto 8, I-06123 Perugia, Italy. E-mail: b.bd.doherty@talk21.com a Centro di Eccellenza SMAArt, c/o Dipartimento di Chimica, Biologia e Biotecnologie, Università degli Studi di Perugia, via Elce di Sotto 8, I-06123, Perugia, Italy b Istituto CNR di Scienze e Tecnologie Molecolari (CNR-ISTM) c/o Dipartimento di Chimica, Biologia e Biotecnologie, Università degli Studi di Perugia, via Elce di Sotto 8, I-06123, Perugia, Italy c Laboratoire de Recherche des Monuments Historiques (LRMH), 29 rue de Paris, 77420, Champs-sur-Marne, France d Dipartimento di Chimica, Biologia e Biotecnologie, Università degli Studi di Perugia, via Elce di Sotto 8, I-06123, Perugia, Italy J. Raman Spectrosc. 2017, 48, 744–749 Copyright © 2017 John Wiley & Sons, Ltd. Research article Received: 16 September 2016 Revised: 12 December 2016 Accepted: 12 December 2016 Published online in Wiley Online Library: 13 January 2017 (wileyonlinelibrary.com) DOI 10.1002/jrs.5096 744