The above findings are used to investigate the possible biological origin of Tyrian purple detected in historical -initially studied using X-ray fluorescence spectroscopy- and archaeological samples. trunculus samples are slightly separated from the other two species. haemastoma species are not separated and H. and Stramonita haemastoma), using all the HPLC quantitative results reported up until now by various researchers. In particular, principal component analysis (PCA) is applied, to investigate if it is possible to achieve a distinction between the three Mediterranean molluscan species (H. The results are investigated in the light of previously collected (published and unpublished) analytical data. The improved method (treatment with DMSO at 80 ☌ for 15 min) is used to treat more molluscan samples which are then subjected to HPLC analysis. The effects of treatment temperature and time are investigated, suggesting that the best conditions correspond to 80 ☌ and 15 min. DMSO resulted in improved HPLC signals (peak heights) over DMF and is therefore selected for further studies. However, the relative composition of the purple dye is not affected by the solvent used for sample treatment. It is shown that pyridine results in poor yields compared to the quantities solubilised using DMF or DMSO. HPLC identifications are achieved as these compounds were synthesized in pure forms and characterized using 1H NMR, elemental analyses and IR spectroscopy. For the comparative study, samples of Hexaplex trunculus, collected from the area of Carthage, are treated with the three solvents and the following compounds are monitored with HPLC: indigotin, indirubin, 6′-bromoindirubin, 6-bromoindirubin, 6-bromoindigotin, 6,6′-dibromoindigotin and 6,6′-dibromoindirubin. The efficiencies of dimethyl sulfoxide (DMSO), N,N-dimethylformamide (DMF), and pyridine to treat and solubilise Tyrian purple are compared using high performance liquid chromatography (HPLC).
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