Shellfish Purple Research Articles

Identification of Degradation Products and Components in Shellfish Purple by Ultrahigh Performance Liquid Chromatography Coupled with Tandem Mass Spectrometry

Ultrahigh performance liquid chromatography coupled to tandem mass spectrometry (UHPLC-MS/MS) was used to analyze a colorant and silk, which were prepared and dyed using shellfish (Hexaplex trunculus L.) purple. Solutions of colorant and silk extracts were analyzed immediately after preparation (fresh samples) and after storing them in the dark for thirty days (aged sample I). Moreover, a silk sample was subjected to artificially accelerated ageing under UV radiation (aged sample II). The application of the UHPLC-MS/MS method leads to the detection of (i) the major coloring components of shellfish purple, which are indigotin, indirubin, 6-bromoindigotin, 6′-bromoindirubin, 6-bromoindirubin, 6,6′-dibromoindigotin, 6,6′-dibromoindirubin; (ii) four minor indigoid components in shellfish purple (compounds A, B, C and D), which belong to the same structural class as indirubin, and whose identification has been reported only once in the past; and (iii) eight degradation products (isatin, degradation products DP3, DP4, DP5, DP6, DP7, DP9 and DP10). The latter were also detected in stored indigotin solution, except for DP 6, which was used as reference sample. The method development was assisted by a new solution preparation approach for investigating compound fragmentation, using a solvent system compatible with direct infusion ESI. This system replaceddimethyl sulfoxide, which inhibits detection through electrospray ionization.

Multiscale characterization of shellfish purple and other organic colorants in 20th-century traditional enredos from Oaxaca, Mexico

Enredos are iconic textiles that have been produced for centuries by Mexican communities to celebrate pregnancy, weddings, etc. They have been traditionally dyed with three natural dyes, namely indigo, cochineal, and shellfish purple. In this study two modern enredos produced in the 1980s, now in the collection of the National Museum of Mexican Art, were investigated with the aim of understanding the colorants used and informing on the use of traditional dyeing practices in modern Oaxaca.The results were obtained by a combination of non-invasive analytical techniques, namely reflectance imaging spectroscopy (RIS) and macro X-ray fluorescence (MA-XRF), and high pressure liquid chromatography coupled to diode array detector and tandem mass spectrometry (HPLC-DAD-MS/MS). In addition to representing the first example of this combination of techniques applied to the study of textiles, this study also reports on the first characterization of the purple dye extracted from the Plicopurpura pansa G. mollusk by HPLC-DAD-MS/MS. Moreover, this study reveals the simultaneous use of synthetic red dyes with naturally-derived colorants such as indigo and shellfish purple. The limitations of the various techniques to provide an ultimate identification of certain dyes are emphasized together with the need for additional foundational research to expand molecular databases for the identification of synthetic dyes. Nevertheless, the results shed light on a dynamic dyeing scenario in modern Oaxaca (Mexico), in which traditional materials and methods are used alongside new ones in an attempt to preserve meaningful local practices.

Analytical characterization of 5,5\u2032-dibromoindigo and its first discovery in a museum textile

The exploitation of natural sources and later synthetic molecules to generate blue to purple coloration in textiles has a long history in the dyer’s craft. Natural indigoids such as indigo, woad, and Tyrian or shellfish purple served this purpose for millennia, but in the late 1800s synthetic analogs, in particular indigotin, quickly replaced natural sources. Halogenated versions of the dye were also created, and some like 5,5′-dibromoindigo were brought to market. Interestingly, these have not been significantly discussed in the literature, nor have they been found in forensic or technical art history investigations of textiles until now. This paper reports the first identification in a museum context of this unusual synthetic brominated analog of indigo, discovered on three twentieth century Japanese yukata. Analytical data collected on reference materials using liquid chromatography-mass spectrometry, UV–visible spectroscopy, Raman microspectroscopy, Fourier transform infrared spectroscopy, and X-ray fluorescence spectroscopy are provided to assist with future identifications of this relatively unknown colorant. Density functional theory applied to 5,5′-dibromoindigo was used to confirm the experimental Raman spectrum.

A Review on the Archaeological Chemistry of Shellfish Purple

Shellfish purple, also known as Tyrian purple and royal purple, has a long history, which has been revealed and documented in recent years through valid physicochemical studies using sophisticated techniques. The aim of the work was to summarize the conclusions of these studies and to describe the results of two unpublished investigations regarding the (i) identification of shellfish purple in a textile (4th century BCE) from ancient Macedonia and (ii) dramatic effect of the dyeing conditions on the composition of the purple dye. Moreover, a critical discussion is included about the discovery of the shellfish pigment and dye based on the available scientific evidence. Previously published reports describing the identification of the shellfish colorant in objects of the cultural heritage were carefully summarized. Shellfish purple was not used only as colorant, but it served other purposes as emphasized in this review. In particular, examples for the use of shellfish purple in medicine, grave goods and fillers and plasters in walls, were described. Examples of materials and methods that were used in the past to produce “fake” purple, imitating the aesthetic result of the valuable royal marine material were summarized. Finally, the solubility of indigoids was discussed using modern approaches of physical chemistry.

Bromoperoxidase Producing Bacillus spp. Isolated from the Hypobranchial Glands of A Muricid Mollusc Are Capable of Tyrian Purple Precursor Biogenesis

The secondary metabolite Tyrian purple, also known as shellfish purple and royal purple, is a dye with historical importance for humans. The biosynthetic origin of Tyrian purple in Muricidae molluscs is not currently known. A possible role for symbiotic bacteria in the production of tyrindoxyl sulphate, the precursor to Tyrian purple stored in the Australian species, Dicathais orbita, has been proposed. This study aimed to culture bacterial symbionts from the purple producing hypobranchial gland, and screen the isolates for bromoperoxidase genes using molecular methods. The ability of bromoperoxidase positive isolates to produce the brominated indole precursor to Tyrian purple was then established by extraction of the culture, and analysis by liquid chromatography–mass spectrometry (LC–MS). In total, 32 bacterial isolates were cultured from D. orbita hypobranchial glands, using marine agar, marine agar with hypobranchial gland aqueous extracts, blood agar, thiosulphate citrate bile salts sucrose agar, and cetrimide agar at pH 7.2. These included 26 Vibrio spp., two Bacillus spp., one Phaeobacter sp., one Shewanella sp., one Halobacillus sp. and one Pseudoalteromonas sp. The two Bacillus species were the only isolates found to have coding sequences for bromoperoxidase enzymes. LC–MS analysis of the supernatant and cell pellets from the bromoperoxidase producing Bacillus spp. cultured in tryptone broth, supplemented with KBr, confirmed their ability to produce the brominated precursor to Tyrian purple, tyrindoxyl sulphate. This study supports a potential role for symbiotic Bacillus spp. in the biosynthesis of Tyrian purple.

UV-induced degradation of wool and silk dyed with shellfish purple

The effects of harsh UV radiation (λ = 254 nm) on wool and silk dyed with Hexaplex (Murex) trunculus extract are investigated using HPLC-DAD, colourimetry, SEM and FTIR. Indigotin, 6-bromoindigotin, 6,6′-dibromoindigotin and 6,6′-dibromoindirubin were quantified by an efficient HPLC-DAD method which was validated in terms of linearity, detection and quantification limits, precision and accuracy. Under optimal conditions, the developed analytical scheme offers limits of detection in the range 0.02–0.12 μg mL−1 and satisfactory linearity up to 5.0 μg mL−1. The purple dyes, extracted from the wool and silk fibres, were analysed by the established HPLC method, using the standards addition approach. To evaluate the matrix effect, the matrix-induced signal suppression/enhancement was determined. The accuracy was evaluated by recovery experiments.The effect of the UV light was initially dramatic, leading to (i) a rapid decrease in the amounts of the four shellfish compounds in the wool/silk fibres and (ii) severe colour changes of the dyed fibres. After extensive (>10 days) exposure in harsh UV light, only slight changes in the dye composition and the colour of the fibres were observed. Overall, larger reductions are reported in the amounts of the dibrominated molecules, whereas indigotin and 6-bromoindigotin appear to be more stable against the UV radiation. However, it is reported that the % relative composition of the shellfish dye is practically unaffected by the degradation process.Finally, the UV-induced structural and chemical changes of the wool and silk fibres were investigated using SEM and FTIR, respectively.

Non-Destructive X-ray Spectrometric and Chromatographic Analysis of Metal Containers and Their Contents, from Ancient Macedonia

This work describes a holistic archaeometric approach to ancient Macedonian specimens. In the region of the ancient city Lete, the deceased members of a rich and important family were interred in a cluster of seven tombs (4th century BC). Among the numerous grave goods, there was also a set of metal containers preserving their original content. The physico-chemical analysis of the containers and their contents was performed in order to understand the purpose of their use. For the containers, Energy Dispersive micro-X-Ray Fluorescence (EDμXRF) spectroscopy was implemented taking advantage of its non-invasive character. The case (B35) and the small pyxis (B37) were made of a binary Cu-Sn alloy accompanied by a slight amount of impurities (Fe, Pb, As) and the two miniature bowls were made of almost pure Cu. For the study of the contents, a combination of EDμXRF, X-Ray Diffraction (XRD), and Gas Chromatography—Mass Spectrometry (GC-MS) was carried out. Especially for the extraction of the volatile compounds, the Solid Phase Micro-Extraction (SPME) technique was used in the headspace mode. Because of the detection of Br, High Pressure Liquid Chromatography coupled to a Diode-Array-Detector (HPLC-DAD) was implemented, confirming the existence of the ancient dye shellfish purple (porphyra in Greek). The analytical results of the combined implementation of spectrometric and chromatographic analytical techniques of the metal containers and their contents expand our knowledge about the pharmaceutical practices in Macedonia during the 4th century BC.

First evidence of purple pigment production and dyeing in southern Arabia (Sumhuram, Sultanate of Oman) revealed by mass spectrometric and chromatographic techniques

Abstract Archaeological excavations carried out in the ancient settlement of Sumhuram (3rd century BC–5th century AD) in the area of Khor Rori (Dhofar Governorate, southern Oman), brought to light pottery showing a pink-violet substance. In order to reveal the nature of this pink-violet colour, the substance was chemically examined by laser desorption-ionization mass spectrometry (LDI-MS) and high performance liquid chromatography-diode array detection (HPLC-DAD). The analytical investigations provided a detailed molecular composition of the organic fraction of the pink-violet material, highlighting the presence of 6,6′-dibromoindigo, 6-monobromoindigo, 6,6′-dibromoindirubin, 6- and 6′-monobromoindirubin, indigo and indirubin. The results revealed that shellfish purple was the source. In addition, in some of the pottery fragments, alizarin and purpurin, which are the molecular markers of madder type dyestuffs, were also ascertained by LDI-MS. The analytical results enabled us to draw hypotheses not only on the possible function of such vessels as dye baths, but also that, since Sumhuram was one of the most important harbours in southern Arabia during the pre-Islamic time, it was also possibly a centre for the production and trade of purple pigments.

Positive and negative‐mode laser desorption/ionization‐mass spectrometry (LDI‐MS) for the detection of indigoids in archaeological purple

Laser-based ionization techniques have demonstrated to be a valuable analytical tool to study organic pigments by mass spectrometric analyses. Though laser-based ionization techniques have identified several natural and synthetic organic dyes and pigments, they have never been used in the characterization of purple. In this work, positive and negative-mode laser desorption/ionization mass spectrometry (LDI-MS) was used for the first time to detect indigoids in shellfish purple. The method was used to study organic residues collected from archaeological ceramic fragments that were known to contain purple, as determined by a classical high-performance liquid chromatography-based procedure. LDI-MS provides a mass spectral fingerprint of shellfish purple, and it was found to be a rapid and successful tool for the identification of purple. In addition, a comparison between positive and negative mode ionization highlighted the complementarity of the two ionization modes. On the one hand, the negative-ion mode LDI-MS showed a better selectivity and sensitivity to brominated molecules, such as 6,6'-dibromoindigo, 6-monobromoindigo, 6,6'-dibromoindirubin, 6- and 6'-monobromoindirubin, thanks to their electronegativity, and produced simpler mass spectra. On the other hand, negative-ion mode LDI-MS was found to have a lower sensitivity to non-brominated compounds, such as indigo and indirubin, whose presence can be established in any case by collecting the complementary positive-ion LDI mass spectrum.

Mass spectrometric identification of new minor indigoids in shellfish purple dye from Hexaplex trunculus

Analysis of natural dyes in historical objects is important for both conservation purposes and to determine the origin and culture that produced it. Identification of a particular dye is usually made based on the presence of its main components, while consideration of minor components is important for differentiating between dyes originating from closely related species. Tyrian purple is one of the oldest dyes known to man and derives from different species of marine molluscs. In all of these species, indigotin, indirubin and their brominated analogues are the main colouring compounds. Here, we describe the identification of minor indigoids found in extracts of the pigment obtained from one of the Tyrian purple species, Hexaplex trunculus. Identification of these compounds was made based on isotopic patterns and accurate mass measurements of protonated molecular ions and their high collision energy fragments obtained in LC–MS/MS experiments. The unknown compounds appeared to be analogues of indirubin and its mono- and dibrominated derivatives with one CO group in the indirubin backbone substituted by a CNH group. Identification of these compounds facilitates the detection of dyestuffs from H. trunculus in historical objects and increases our knowledge about the dye biosynthesis and technology of Tyrian purple production.

An investigation of paint from a mural in the church of Sainte Madeleine, Manas, France

The pigment in brown paint samples taken from a church in the Drôme region of France has been shown to be almost pure 6-bromoindigo and 6,6'-dibromoindigo. The composition of the pigment was established by comparison with 6-bromoindigo and 6,6'-dibromoindigo standards using atmospheric pressure photoionization combined with liquid chromatography and tandem mass spectrometry. The brown paint samples were taken from a frieze of ca 20 brown images having symmetric tree-like forms composed of five overlapping filled circles representing foliage. The tree-like images, simple rather than artistic, had a metallic luster. The molar ratio of 6-bromoindigo and 6,6'-dibromoindigo in the brown paint pigment (98:2) is remarkably similar to that of shellfish purple from the Mediterranean Murex brandaris (96.5:3.5) thus, it is possible that the origin of the indigoid compounds in the brown paint pigment is the eastern coast of the Mediterranean Sea. As the production of Tyrian purple ended with the fall of Byzantium in 1453, purple pigment had to have been produced prior to this time. We have conjectured about the circumstances that may account for the use of 'purple' in this manner in the Drôme region and how the pigment was transported there from the eastern coast of the Mediterranean Sea during the time of the Crusades.

High performance liquid chromatography of slightly soluble brominated indigoids from Tyrian purple

Attempts at identification of mollusc species producing Tyrian purple from archaeological material are usually done with high performance liquid chromatography in the reversed-phase system, but the peaks obtained are often wide and asymmetric. This is due to the low solubility of the indigoids and their brominated derivatives in the mobile phase, especially 6,6′-dibromoindigotin, which is soluble in only few, particular solvents. Our study focused on improving both symmetry and peak height for more precise quantification. The influence of various factors was evaluated: stationary phase characteristics, mobile phase composition, elution gradient parameters and temperature on the peak shape of the main components of Tyrian purple. The best results were obtained using highly retentive, but moderately bonded ODS stationary phases (about 2.8 μmol m −2), percolated with gradient of acetonitrile with acidified aqueous mobile phases (0.1% strong acid) at elevated temperatures (70 °C). The upper quantification limit for 6,6′-dibromoindigotin was improved by over 350%, between standard and optimised systems. Using them, the detection and quantification of trace Tyrian purple components (less than 0.15%) aside from major indigoids becomes possible. Consequently, for the first time, the new analogues of brominated and unbrominated indirubins were found in the shellfish purple from Hexaplex trunculus.

Characterisation of natural indigo and shellfish purple by mass spectrometric techniques.

Two analytical methods based on mass spectrometry were used in the characterisation of constituents of natural indigo prepared from the leaves of Indigofera tinctoria, and of shellfish purple prepared from the hypobranchial glandular secretions of Murex trunculus, following old recipes. On-line pyrolysis gas chromatography in the presence of hexamethyldisilazane followed by mass spectrometric analysis (Py-silylation/GC/MS), and direct exposure mass spectrometry (DE-MS), were used. Extensive fragmentation of indigoid dyes was obtained by Py-silylation/GC/MS. The following molecular markers were highlighted, which are useful for identification purposes: 1,2-dihydro-3H-indol-3-one for indigoid dyes, 1,3-dihydro-2H-indol-2-one for indirubine, and 6-bromo-1,2-dihydro-3H-indol-3-one for shellfish purple. Using DE-MS, 6,6'dibromoindigotine, monobromoindigotine and indigotine were identified as the main components, and the presence of tyrindoxyl, one of the dye precursors, was also assessed.

Integrated analytical techniques for the study of ancient Greek polychromy

The materials used in the decoration of three painted astragaloi (knucklebones) from the Koroneia cave (Greece) were investigated by means of sequential application of non-destructive and destructive techniques: optical microscopy, environmental scanning electron microscopy coupled with X-ray microanalysis (ESEM–EDX), Fourier transform infrared spectroscopy (FTIR) with micro-attenuated total reflection (μ-ATR) technique, high performance liquid chromatography (HPLC) coupled with UV-fluorescence and gas chromatography–mass spectrometry (GC–MS) were used.The main results highlighted that the three astragaloi were prepared with a ground of ochre or iron clay and painted with a proteinaceous matter such as binder egg tempera. Both FTIR and GC–MS agree in the detection of lipids that can be related to egg. Organic dyestuffs identified as madder lake and shellfish purple were used together with inorganic pigments.

Anglo-Saxon Manuscript Pigments

There has been a lack of analyses of early manuscript pigments that use reliable and unambiguous analytical techniques. An interdisciplinary methodology for identifying which pigments were used on Anglo-Saxon manuscripts (those from England, c. 600 — c. 1066 AD) was applied, and a pilot series of analyses was carried out. A list of likely pigments was compiled from contemporary written sources, from analyses of near-contemporary European manuscripts, and from archaeological finds of raw colourants or coloured artifacts. Pigments were analysed using micro-Raman spectroscopy and near-infrared imaging. Results are presented. Analysis provided data mainly on inorganic pigments, and information on organic pigments was derived principally from other indirect sources of evidence. Preliminary patterns of pigment use were identified, correlating with the places, dates and workshops of origin. Minium, verdigris, carbon black and orpiment were found throughout, with indigotin in the earliest examples, and shellfish purple and lead white in some de luxe books. Ultramarine started to be used c. 1000 AD but apparently only in Canterbury. There is some late use of azurite and red ochre. The results presented are the first reliable ones since 1885.

The Pigments used in Painting 'The Rosslyn Missal' in the Advocates' Library, and the Celtic Psalter, D.p.111,8, in the Library of the Univberrsity of Edinburgh

The pigments used on early Byzantine and early Celtic manuscripts are considered with particular reference to the difficulties involved in preparing ultramarine from lapiz lazuli. The Rosslyn Missal is believed to be of late thirteenth or early fourteenth century. The letters down the margin of the MS. are outlined in black, and are tinted with four pigments, vermilion, orpiment, ultramarine, and shell-fish purple. The Celtic psalter is thought to be of eleventh-century date. The pigments used are lead in place of vermilion as in the Lindisfarne Gospels, the Irish variety of orpiment, transparent copper-green, badly washed ultramarine, and shell-fish purple. The gold used on a single page consists of little rounded and\r\nkidney-shaped particles obtained as the fine gold dust from river gold.