**Figure 3.** Detail of Figure 1 showing: part of the angel in the scene of *St. John the Evangelist in a Cauldron of Boiling Oil*; the inscription above this scene contains indigo; and the newly revealed border with a yellow foliate design painted on the red background. The inset shows a detail of this border. © The National Gallery, London.

**Figure 4.** Hirox microscope image of a detail of the yellow border showing craquelure of the yellow paint layer/s. © The National Gallery, London.

#### **2. Materials and Methods**

*2.1. Analytical Techniques*

#### 2.1.1. HPLC

The lake samples were extracted using 4% BF3 in MeOH (*ca*. 3–6 μL). The solutions were left overnight, before being centrifuged for 1 min at 3000 rpm and the supernatant collected and analysed using a standard protocol developed at the National Gallery for the analysis of lake pigments extracted from historical paint samples. An Agilent Technologies High-Performance Liquid Chromatography system, comprising a 1200 series capillary pump and vacuum degasser and an HP 1100 series column oven and Photo Diode Array (PDA) detector, was used. The samples were injected manually via a Cheminert Valco lowdispersion 6-port injector with a 2 μL sample loop. Agilent OpenLab CDS (Chemstation edition) was used to control the equipment and process the data. The flow cell path length was 10 mm (0.5 μL volume) and the bandwidth (resolution) of the detector was 8 nm. The method used a Phenomenex LUNA C18(2) reverse phase column, 5 mm particle

size, 150 × 0.5 mm (length × i.d.). The total run time was 80 min (+15 min post-run at initial conditions) at a flow rate of 10 μL min−1 and the column was maintained at a temperature of 40 ◦C. A binary solvent system was used; A = 0.1% aqueous trifluoroacetic acid, B = 94.9% acetonitrile, 5% methanol, 0.1% trifluoroacetic acid. The elution program was a linear gradient from 95A:5B to 70A:30B (0 to 25 min), followed by a linear gradient to 5A:95B (65 min). Isocratic conditions were maintained (75 min) before a linear return to the starting conditions, 95A:5B (80 min).

#### 2.1.2. (ATR) FTIR

Spectra were acquired using a Bruker Tensor 27 FTIR Spectrometer connected to a Hyperion 3000 Series microscope, fitted with a mid-band MCT detector (range = 12,000–600 cm−1) and a 64 × 64 (4096 pixels) FPA detector (range = 4500–900 cm−1). Both detectors were cooled with liquid nitrogen. The microscope was fitted with a CCD camera, x-y stage (adjustment accuracy of 0.1 μm), knife-edge apertures and two glass objectives (4× and 20× magnification), a 15× Cassegrain objective and a dedicated ATR objective (20× magnification). The ATR had a germanium crystal with a tip size of 250 μm. Both the spectrometer and microscope were purged with water- and CO2-free air. For transmission measurements, samples were compressed between the windows of a Spectra-Tech micro-compression diamond cell, then spectra acquired with the uppermost window removed. Bruker Opus software, version 8.7 was used to control the equipment and process the data. Spectra were collected with 4 cm−1 spectral resolution and were the sum of 128 scans.

#### 2.1.3. XRD

Five small fragments from a sample taken from the glaze layer on the yellow foliage design on the red border of the painting were mounted on carbon fibres using small drops of epoxy. The mounted samples were fixed on a goniometer head in the XRD instrument. XRD measurements were carried out using a Rigaku Dmax Rapid II Micro-XRD. The samples were analysed using copper radiation at 40 kV and 36 mA. The X-ray beam was conditioned to a size of 300 μm. The samples were moved during the measurements along two axes (omega and phi-rotation) to improve particle statistics. Measurement times varied between 1 and 5 h.

The collected 2D data were converted to conventional 1D XRD patterns using the DP software (Rigaku). Phase identification was carried out using HighscorePlus (Panalytical) in combination with the PDF-2 database from ICDD. The XRD patterns were background subtracted before phase identification.

#### 2.1.4. SEM-EDS

Samples were mounted as paint cross-sections and analysed in a Zeiss EVO MA10 variable pressure scanning electron microscope (VPSEM) with Oxford Instruments X-max large area Energy Dispersive X-ray Spectroscopy (EDS) and Silicon Drift Detector (SDD) at 25 keV, 200 pA, 44 Pa (Air) working distance 8.48 mm.

#### 2.1.5. MA-XRF

MA-XRF scanning was undertaken using a Bruker M6 Jetstream macro X-ray fluorescence (MA-XRF) scanner which allows paintings to be scanned while secured on an easel in front of the scanner, with no contact being made with the paint surface. The measuring head consists of a 30 W Rhodium-target microfocus X-ray tube with a maximum voltage of 50 kV and a maximum current of 0.6 mA. With the Bruker M6, only elements heavier than silicon can be detected. The painting was scanned in 6 sections with acquisition parameters: Tube, 600 μA, 50 kV: detector, 275 kcps; spot size, 580 μm; step size, 580 μm; dwell time 10 ms; number of cycles, 1.

#### *2.2. Synthesis of Syngenite*

Syngenite was first synthesised using gypsum and potassium sulfate following an adapted version of a published method [3]. Gypsum (CaSO4·2H2O; 12.1 g) was added to an aqueous solution of potassium sulfate (K2SO4; 14.2 g in 100 mL) at room temperature with constant stirring. After 3 h, the suspension, which had become noticeably "fluffy", was filtered under vacuum, and the resulting solid dried in an oven at 80 ◦C overnight. Syngenite was then synthesised in an identical manner using gypsum (CaSO4·2H2O; 12.1 g) and an aqueous solution of potassium carbonate, K2CO3 (9.7 g in 100 mL) following an adapted version of a published method [4]. In both cases, analysis of the dried powder by FTIR confirmed the absence of gypsum (no bands present at 3560 and 3430 cm−1) and indicated the presence of syngenite (K2Ca(SO4)2·H2O; signature absorption bands at 758 and 658 cm−1) [5], with chalk (CaCO3; signature absorption bands at 1430 and 877 cm−1) also present in the second sample (Figure 5).

**Figure 5.** FTIR absorbance spectra indicating the presence of syngenite, synthesised from (**a**) gypsum and potassium sulfate and (**b**) gypsum and potassium carbonate (also containing chalk).

#### *2.3. Preparation of Reference Organic Pigments*

#### 2.3.1. Weld on Gypsum Support

The dried weld (2.5 g) was placed in distilled water (250 mL) and left to soak for *ca*. 48 h. The solution was then heated to between 60 ◦C and 80 ◦C for 30 min before being left to cool for another 30 min, then filtered. One aliquot of the filtered solution (100 mL) was used for the syngenite experiment (see Section 2.3.2), while a second aliquot (100 mL) was placed in a beaker and gypsum (1.2 g) added. This was left for *ca*. 24 h (with occasional stirring) before the solid, now slightly yellow in colour, was removed and placed on a watch-glass to dry in a desiccator. To increase the amount of dye adsorbed onto the surface and produce a more intensely coloured pigment, a portion of the pale-yellow solid (0.7 g) was then placed in a second weld dye solution, prepared in a similar fashion to the first, and left for *ca*. 24 h (with occasional stirring) before being removed and placed on a watch-glass to dry in a desiccator. The same procedure was then repeated with a portion of the solid from the second immersion (0.4 g), thus producing three separate pigments which had been immersed in one, two and three fresh dye solutions, respectively.

As expected, immersion in successive dye solutions produced pigments with slightly deeper shades of yellow, however, all three pigments were still relatively pale in colour, so a further experiment was conducted using a more concentrated weld solution. In this case, the dried weld (15 g) was placed in distilled water (200 mL) and left to soak for *ca*. 24 h. The solution was then heated to boiling for 60 min before being left to cool for another 30 min, then filtered. The filtrate was placed in a beaker and heated, allowing the solution to evaporate until only *ca*. 10 mL remained. The resultant solution, slightly syrupy in consistency, was left to cool for *ca*. 30 min before an aliquot (5 mL) was removed and added to a beaker containing gypsum (1.2 g). This was left for *ca*. 24 h (with occasional stirring)

before the solid was removed and placed on a watch-glass to dry in a desiccator. This produced a yellow pigment, deeper and darker in shade than those produced previously.

#### 2.3.2. Weld on Syngenite Support

An aliquot (100 mL) of the weld solution prepared above (Section 2.3.1) was placed in a beaker and syngenite (prepared using gypsum and potassium sulfate in Section 2.2; 4.0 g) was added. This was left for *ca*. 24 h (with occasional stirring) before the solid, now slightly yellow in colour, was removed and placed on a watch-glass to dry in a desiccator. Analysis by FTIR confirmed the substrate, now slightly yellow in colour, had remained as syngenite and had not decomposed back to gypsum.

In an identical manner to that described in Section 2.3.1, a second pigment was prepared with the aim of increasing the amount of dye adsorbed onto the surface. However, when a portion of the solid from the first immersion (1.9 g) was added to an aliquot of the second weld solution and left for *ca*. 24 h (with occasional stirring) then removed and placed on a watch-glass to dry in a desiccator, it became apparent that it had decomposed. Analysis by FTIR confirmed the substrate was now gypsum.

In order to produce a more intensely coloured pigment with only one immersion, a further experiment was conducted using a more concentrated weld solution. An aliquot of the concentrated weld solution described in Section 2.3.1 (5 mL) was left to cool for *ca*. 30 min before adding syngenite (prepared using gypsum and potassium sulfate in Section 2.2; 2.3 g). This was left for *ca*. 24 h (with occasional stirring) before the solid was removed and placed on a watch-glass to dry in a desiccator. While analysis by FTIR confirmed the presence of syngenite, it was no longer the only component within the substrate. It is therefore apparent that although, in principle, syngenite can be used as a substrate for a yellow organic pigment, it remains as a pure substrate only under certain conditions.

#### 2.3.3. Safflower

Safflower petals contain two dyes; the water-soluble yellow dye is generally washed out before the red dye (soluble in alkali) is extracted (see discussion in Section 4.1). However, the use of an alkali in the preparation of the dye solution makes safflower a particularly interesting dye source to consider in the present study as it provides a compelling reason for the addition of an alkali to the dye solution. It perhaps offers an explanation as to how a substrate such as syngenite might be produced from gypsum and an alkali such as potassium carbonate (following a synthetic route such as that outlined in Section 2.2) or potassium hydroxide. This first exploratory experiment was unsuccessful and did not produce syngenite, but the method is reported here nevertheless.

Safflower petals (10 g) were washed in distilled water and left to soak for 1 week to remove a large proportion of the yellow dye. They were then washed again five times with distilled water (the first and second washes used 500 mL and the petals were left to soak for *ca*. 1 h, then the third and fourth washes used *ca*. 250 mL before a final wash using *ca*. 200 mL). Approximately half the petals were then placed in a desiccator and the other half added to a potassium carbonate solution (100 mL; 9.7 g), warmed slightly, and left for one hour. The gypsum was then added (12.1 g) and the mixture stirred for *ca*. 30 min before being left overnight. The following day, the solution was stirred for several hours before citric acid (10%; 10 mL) was added and the solid, now yellowish-orange in colour, removed and placed on a watch-glass to dry in a desiccator. Before addition of the acid, a small sample of the solid was removed and allowed to dry in a desiccator. FTIR analysis indicated the presence of syngenite together with gypsum and chalk. However, analysis of the dried solid after the addition of citric acid indicated that syngenite was no longer present.

The second portion of washed and dried safflower petals (*ca*. 5 g) was mixed with solid potassium carbonate (0.64 g) in a process based on historical methods for testing or extracting the red dye (see Section 4.1) and then transferred to a Buchner funnel with filter paper. Deionised water (50 mL) was gradually added and allowed to drip through (not under vacuum). The same liquid was collected and allowed to drip through the petals again before a second portion of distilled water (50 mL) was gradually added and allowed to drip through. Any remaining liquid was collected by filtration under vacuum and the combined filtrate (*ca*. 80 mL) split into two equal portions. Citric acid (10%; 5 mL) was added to the first portion until a fine precipitate appeared, indicating that safflower red pigment was indeed present and had been soluble in the alkaline solution. The pigment was left to settle and then removed from the solution and allowed to dry in a desiccator. It is very possible that such a pigment could have been manufactured then ground with a suitable white substrate, such as gypsum or syngenite. However, another possibility is that the white material was added to the dye solution, thus syngenite (prepared from gypsum and potassium sulfate in Section 2.2; 1.0 g) was added to the second portion, followed by citric acid (10%; 5 mL). The mixture was stirred for *ca*. 30 min, then the solid, now red in colour, removed and placed on a watch-glass to dry in a desiccator. However, FTIR analysis of the dried solid confirmed that the syngenite had decomposed and was no longer present.

#### **3. Results**

The analytical investigations undertaken to establish the presence of syngenite in the glaze layer on the surface of the yellow foliate design are presented below.

#### *3.1. Analysis of Layer Structure of Yellow Foliate Pattern of Border*

A cross-section prepared from a paint sample taken from the yellow foliate pattern revealed the following stratigraphy (Figure 6a,b). Directly on top of the red earth paint of the background (1) is an opaque yellow paint layer (2) containing bright yellow particles with thin tabular and foliated fragments. These are clearly visible in the back-scattered electron (BSE) image (Figure 7) and SEM-EDS analysis of these yellow pigment particles confirmed the presence of arsenic (As) and sulfur (S), suggesting the presence of the arsenic sulfide pigment orpiment (Figure 8), and this was confirmed by micro-XRD. A few particles with similar characteristics and elemental profile were also found to be present in the overlying semi-translucent yellow layer (3). In UV illumination (Figure 6b), the substantial translucent yellow glaze layer seemed, in addition to the few inclusions of orpiment, to also contain other particles. There was also a thin fragmentary layer on the surface which fluoresced a milky white in UV; this was most probably varnish (4).

**Figure 6.** (**a**) Paint cross-section taken from the yellow border design in dark field-reflected light. Stratigraphy from the bottom upwards: 1. Red iron earth paint layer; 2. orpiment 3. yellow glaze layer with a few particles of orpiment present, particularly in the lower portion where layers 2 and 3 are intermixed. (**b**) Paint cross-section in 5a photographed in ultraviolet light (excitation filter 340–380 nm). 1. Red iron earth paint layer; 2. orpiment; 3. in UV illumination a particulate structure is visible in the yellow glaze layer; 4. trace of early/original varnish which remains on the surface of the sample. © The National Gallery, London.

**Figure 7.** Back-scattered electron (BSE) image of the paint cross-section in Figure 6, clearly showing the particles present within the glaze layer.

**Figure 8.** (**Left**) Back-scattered electron image of the paint cross-section with X-ray map for arsenic (As) in red, indicating the presence of particles of orpiment in the yellow paint layer and also a few within the overlying glaze. (**Right**) SEM-EDS spot spectrum confirming the presence of arsenic (As) and sulfur (S). This spectrum is representative of a large number of particles that were analysed in the sample.

To gain a clearer idea of the structure and composition of the particles visible under UV illumination in the translucent glaze layer and in the BSE image, SEM-EDS analysis was undertaken. This indicated the presence of sulfur, potassium and calcium, suggesting a complex potassium sulfate salt within the layer (Figure 9). ATR-FTIR analysis indicated that the inorganic component was homogeneous in composition and excluded the presence of either calcium sulfate or indeed chalk (data not shown). It also confirmed that the egg tempera binder was present in both the upper and lower layers, suggesting that the upper yellow layer was a paint or glaze layer rather than a degradation crust or discoloured varnish. Thus, it seems likely that the inorganic material within the upper layer is the substrate for a yellow organic "lake" pigment, which would have been used to produce a yellow glaze paint over the yellow foliate pattern painted in orpiment.

**Figure 9.** Image of the cross-section as shown in 6b with an SEM-EDS spot spectrum from a representative particle within the glaze layer showing the presence of sulfur (S), potassium (K) and calcium (Ca). **Below** (**from left to right**) are back-scattered electron images with X-ray maps (in red) for sulfur, potassium and calcium.

MA-XRF mapping provided further evidence for the painting technique used for the foliate perimeter border. The arsenic map (Figure 10) clearly shows the location of the yellow, orpiment-rich (As2S3) paint of the foliate pattern painted over the red iron oxide background. The foliate pattern is also visible in the potassium map (present in syngenite), which shows where the yellow lake glaze was applied over the orpiment paint along the entire length of the border design. The foliate design is visible as a negative in the iron (Fe-KA) map because the signal for iron is masked by the arsenic from the orpiment-containing yellow paint on top.

**Figure 10.** MA-XRF scanning: (**top**) iron distribution map (Fe-KA) showing the red iron earth paint of the background; (**centre**) arsenic distribution map (As-KA), showing the location of the arsenic sulfide pigment orpiment; (**below**) potassium distribution map (K-KA), showing the location of potassium present in syngenite from the yellow glaze layer along the whole length of the border design on the upper edge of the painting. © The National Gallery, London.

#### *3.2. Identifcation of Syngenite (Micro-XRD Analysis)*

In order to precisely identify the complex sulfate indicated by SEM-EDS and FTIR analysis, micro-XRD was undertaken. The major phase present in all the sub-samples from an unmounted fragment of the glaze layer was confirmed to be syngenite, K2Ca(SO4)2·H2O. The XRD pattern for one of these sub-samples (IS3-5) is shown in Figure 11. The major and minor phases identified in all sub-samples are summarised in Table 1.

**Figure 11.** Micro-XRD pattern of an unmounted fragment (sub-sample IS3-5) from the glaze layer, confirming the presence of syngenite as the major phase.

**Table 1.** Phases identified from micro-XRD patterns of the unmounted sub-samples from the yellow glaze layer.

There is no doubt about the identification of the major phase syngenite in all fragments from the sample. The minor phases are harder to confirm, but the results suggest small quantities of weddellite (calcium oxalate) in three of the five fragments from IS3. The presence of small amounts of oxalate is not unusual in very aged paint layers, particularly at or near the surface. A trace of anhydrite was also suggested in one sample, while an unassigned peak with low intensity at 21.5◦ in four of the fragments could be explained with calomel (Hg2Cl2) or with polyhalite K2Ca2Mg(SO4)2·2H2O, which has a similar composition to syngenite.

#### **4. Discussion**

#### *4.1. Organic Lake Pigments: Recipes and Possible Dye Sources*

By the mid-13th century, the method of making red lake pigments by extracting the dye from the raw material using alkali and precipitating the pigment by the addition of potash alum had been known for several hundred years [6] (for lac and kermes, see nos. 174A–6, pp. 52–53, for example or for madder, nos. 188–9, p. 54). The deep-crimson lac lake in particular has been identified quite frequently in 13th- and 14th-century works [7] (pp. 89–95), and the way this translucent pigment has been used to paint the shadows in the folds of the Virgin's skirt is very characteristic. Yellow organic pigments seem to have been used as glazing pigments less frequently; in fact, they are rarely identified in paintings of this period, perhaps partly because they are not lightfast, and analytical evidence is thus lacking. They have, however, been identified in 14th-century Italian (Tuscan) paintings used in mixtures: for example, with blue pigments, such as azurite, to give greens, and with rather muted yellow ochres to give a brighter, more vibrant yellow [8]. In the present case, HPLC analysis was undertaken on a small paint sample taken from the foliate design in an attempt to identify any remaining dyestuff in the glaze layer, but no result was obtained. This is probably due to the very tiny sample size and perhaps also because yellow dye components are notoriously light-fugitive [9].

Yellow lake pigments are not well-documented: there are very few recipes for them dated before the late 14th century. Those that are known indicate that the pigments were made by precipitation or adsorption onto a suitable white substrate, which could include a local white earth or limestone, gesso, marble chips, lead white or even white flour, sometimes with the addition of alum to the dye solution. The earliest pigments based on organic dyes were made in this way and examples are recorded in Classical Roman sources: in *De architectura* (c. 30–20 BCE), Vitruvius describes how to make a yellow ochrecoloured pigment by heating dried violets in water and grinding the coloured solution with chalk [10]. Pigments were also made from flower petals directly, expressing the juice and mixing it with a suitable white substance. This, too, was a very old method, described in the first book of the so-called *De coloribus et artibus romanorum*, attributed to Eraclius (or Heraclius). This text consists of three books, the first two of which are in verse form. These probably date from around the 10th century and the author is believed to have been Italian. In a recipe from the first book, fresh flowers were ground on a smooth stone with raw gypsum, giving a pigment that could be stored dry. The third book, which is a prose text probably dating from the 13th century and by a French author, contains similar instructions to those described by Vitruvius centuries earlier [11] (vol. 1, no. II, pp. 184–185, no. LV (245) pp. 250–251. These methods were often used to make pigments from blue flowers, such as cornflowers (adding lime if a green was required) and are repeated in later sources. It is worth noting that these pigments probably were intended for use in aqueous media on parchment or paper.

It was only towards the end of the 14th century that recipes incorporating alum as an ingredient, together with a white material as substrate, began to appear, although in practice, pigments of this sort were probably used considerably before this. The 15thcentury *Segreti per colori*, better known as the Bolognese manuscript from its publication by Mrs Merrifield in 1849, contains instructions for a bright yellow for use on paper made with saffron and lead white and a more familiar yellow, named *arzica*, made using a concentrated solution of the dye from weld (*Reseda luteola* L.) to which alum and either finely ground marble or lead white were added [11] (vol. 2, nos. 184, 194, pp. 480–483). Similar instructions occur in other 15th-century Italian sources. Alkali is rarely mentioned as an ingredient, although one example is a recipe for a yellow described as 'finer than orpiment or German giallolino', made using a strong extract of ripe buckthorn berries (*Rhamnus cathartica* L.) to which is added a solution of strong alkali mixed with alum. This gives a green solution, which is mixed with a fine white earth, repeating this several times to build up the colour [11] (vol. 2, no. 105, pp. 428–429). It is slightly surprising that this recipe should give a yellow as the ripe berries are the source of the pigment sap

green; unripe berries are generally used for yellow pigments. Yellow pigments made from buckthorn berries are notably warmer in colour than those given by weld, which can be cool and acidic-looking.

The absence of alum from the yellow lake pigment found over the orpiment used to paint the decoration on the red border suggests that the yellow pigment was made in the simplest way, mixing a concentrated solution of yellow dye extracted from a plant source with the white material forming the substrate, repeating this process to build up the colour as required. If the pigment was a warm yellow colour, buckthorn could be a source of the dye, although other sources such as sawwort or weld are also possible.

Although there is no evidence at all for the use of alkali in making such a pigment at a date as early as the 13th century (required if it is assumed the syngenite substrate was unexpectedly synthesised during the lake-making process), there is no reason against this being done either; the use of alkali to extract flavonoid dyes such as these does result in more intense colours, and this might have been observed by dyers [12]. However, the possible identification of safflower red in the trace of red glaze on the border of the Virgin's veil in a damaged 13th-century painting of the Virgin and Child in the Istituto Santa Caterina in Arezzo suggested that safflower red was worth studying: this is unusual in that alkali must be used for extraction of the dye [1].

As mentioned above (Section 2.3.3), safflower contains two dyes, a red and a yellow, neither of which is lightfast; the water-soluble yellow dye must be washed out before the red can be extracted. The use of safflower in Europe as early as the 13th or 14th centuries is not well-documented, least of all in painting; the tentative identification of the pigment used as a glaze over gold in the 13th-century painting in Arezzo by Daveri and co-workers is a rare example [1] (pp. 401–402), [13]. By contrast, it was quite regularly used in 14th-century Middle Eastern manuscript production for illumination over gold, exactly as seen in the Arezzo painting, and was also used to dye paper [14,15]. It seems likely that, like several other dyes, the extraction and use of the safflower red dye in Italy had its origins in Arabic practice spreading across Mediterranean regions and then further north; certainly early descriptions of the dye and its extraction are found in 10th- and 11th-century Arabic sources [16] (see p. 195, attributed to Abu M¯ us¯ a J ¯ abir ibn ¯ H. ayyan, died ¯ around 806–816 CE), [17] (pp. 30–31, 44–45; al-Mucizz ibn Bad¯ ¯ıs, written in about 1025 CE). By the first decade of the 14th century, its use in dyeing was well-understood and is, for example, recorded in Lucca [18]. A few years later in 1316, also in Lucca, dyeing equipment sold to Landino Parpaglioni by the merchant Ranieri Malisardi included two presses of some description, a wide, shallow vessel and a sieve, all for working with safflower [18] (p. 170). That safflower was a regular item of trade is also indicated by the description of how to test its quality by the Florentine merchant Francesco Balducci Pegolotti in *La pratica della mercatura*, a handbook compiled between about 1320 and 1340. Pegolotti describes how the sample of safflower to be assessed is tested in a way very similar to the manner described in Section 2.3.3 above, washing out the yellow dye and then obtaining the red by mixing the washed petals with dry wine lees, pouring water through the mixture, and finally acidifying with strong vinegar. If a good vermilion red colour is produced, with very little effervescence, the sample is of good quality; if the solution becomes white, with a lot of effervescence, the safflower is of bad quality [19].

Pegolotti's description matches the few extant dyeing recipes from this period very closely [20,21]: if he had put a piece of textile into the final solution the red colour would have been precipitated onto it. To make a pigment, the red material could be allowed to settle, collected, and ground with a suitable white substrate. Alternatively, the white material (gesso, for example) could be present in the vessel so that precipitation took place onto it. However, not enough is known about workshop practices at this time to provide a firm indication of how such a pigment would have been made and, without positive identification of any dye components from HPLC analysis, the dye source remains unknown for the presumed organic pigment on the yellow foliate design on the border of *The Virgin and Child Enthroned* by Margarito d'Arezzo.

#### *4.2. Syngenite and Its Possible Use as a Substrate for a Yellow Organic Lake Pigment*

Syngenite has previously been identified on paintings, but it has generally been found only in very small amounts and usually as a degradation product [22–24]. For example, syngenite was identified in an orpiment-containing paint layer, applied in imitation of gold, on a 13th-century painting by Cimabue [22]. The authors suggest that syngenite is present as a secondary mineral following the degradation of the orpiment-containing paint layer. Similarly, syngenite was identified together with schultenite, mimetite and palmierite in areas originally painted with orpiment in a 17th-century Dutch painting [23]. It is also suggested that these secondary minerals formed as a result of the degradation of orpiment.

However, in the sample taken from the Margarito painting under discussion, no evidence of orpiment degradation was found by SEM-EDS analysis and no arsenolite was identified during XRD analysis. Arsenolite is usually the starting point for deterioration of orpiment via light-induced photooxidation and its absence in the samples under investigation is significant. Indeed, the syngenite observed on the painting by Margarito is homogeneously present within an apparently continuous glaze layer over the yellow foliate design of the border (Figures 6–10). This is puzzling and requires explanation. It is our hypothesis that its presence is most likely explained as the substrate of a yellow organic "lake-type" pigment which, when applied over a layer of orpiment (see Figure 6) must have produced a rich and intense yellow or orange-yellow colour (depending on the dye source used), perhaps intended to imitate the appearance of gold.

Assuming the syngenite does indicate the presence of a yellow organic pigment, the question then arises as to whether the syngenite itself was used as a white substrate on which to adsorb a yellow dye (see discussion in Section 4.1) or whether it is an alteration product of some kind. While it is impossible to come to any definitive conclusions in this regard, some possible scenarios are outlined below.

The syngenite may have been found, either in mineral form or precipitated from solution (perhaps accidentally) and used in the lake-making process as a convenient white substrate. Although rare in absolute terms, syngenite can be found in marine and volcanic deposits [25,26], and this would be the simplest and most straightforward explanation. In Italy, for example, deposits are known in the Campania, Piemonte, Lazio and Apulia regions. However, the particles in the cross-section are relatively small and fairly uniform in size and do not have the appearance of having been ground from a mineral source. In addition, although exploratory experiments using syngenite to adsorb a yellow dyestuff (in this case, extracted from weld) were partially successful (Section 2.3.2) they also indicated that syngenite is prone to decomposition and only remains as a pure substrate during the pigment-making process under certain conditions.

Syngenite can be prepared relatively easily by stirring stoichiometric amounts of gypsum with potassium sulfate [3], and much of the modern scientific literature on this topic is focused on trying to avoid its formation during the production of the relatively useful potassium sulfate from industrial waste streams containing phosphogypsum [27]. Although potassium sulfate is unlikely to have had any technological uses in the 13th century, if it was known at all, the extremely low solubility product of syngenite means that it will readily precipitate from a solution containing the correct proportion of calcium, potassium and sulfate ions. It is therefore possible to imagine an alternative scenario, avoiding the need to collect the syngenite, whereby it was unexpectedly synthesised during the manufacture of the organic pigment itself, perhaps from a 'standard' white substrate such as gypsum.

Preliminary experiments exploring the possibility of syngenite production during the lake-making process indicated that it could be formed by the addition of gypsum to a solution containing potassium carbonate—a substance that would have been available in the 13th century, either converted from cream of tartar, or in the form of wood ash alkali (Section 2.2). The use of potassium carbonate (or any other alkali) when preparing a yellow organic pigment is unusual, but would have been necessary when using a dye source such as safflower (see discussion in Section 4.1). A more pressing problem in this case, however, is that calcium carbonate (chalk) is also formed during the reaction and no chalk was observed in the syngenite-containing layer in the cross-section from the Margarito painting. The recipe for the preparation of safflower pigments requires the addition of acid (in the form of citric acid from lemon juice or acetic acid from vinegar) and while this has the potential to remove the chalk, preliminary experiments were unable to produce a syngenite-containing substrate (Section 2.3.3). Another possibility, not yet explored, is the use of a strong alkali, potassium hydroxide, made by putting wood ash ley over quick lime.

It should be acknowledged that when making pigments of this type, the process of adding the substrate to a strongly coloured solution was often repeated to build up the colour and this repetition (and the exact conditions in the dye solutions) are particularly difficult to recreate. Thus, while it is entirely possible that syngenite, either collected or unexpectedly synthesised, was used as the white substrate on which to adsorb a yellow dye, there is currently no specific evidence for this. The alternative possibility, whereby the syngenite is a degradation product, formed within the paint due to degradation of a 'standard' yellow lake substrate such as gypsum, is also worth considering but is similarly speculative and is perhaps the least likely scenario. Firstly, there is no obvious source of potassium ions within the paint layers, nor any indication of how they would have migrated throughout the paint to create such a uniform layer of syngenite. It is possible that a harsh cleaning of the painted surface using alkali may have provided such an environment; however, the relatively low density of syngenite compared to gypsum would presumably result in a significant dimensional change to the paint layer during any such transformation. The paint layer containing the syngenite is still intact, following closely to the yellow orpiment lines of the design and does not appear to have been disrupted in this way.

#### **5. Conclusions**

A multi-analytical approach has provided evidence for the use of several types of organic colourant on *The Virgin and Child Enthroned* by Margarito d'Arezzo, with both indigo and a red lake pigment unequivocally identified. Micro-XRD analysis confirmed syngenite (K2Ca(SO4)2·H2O) was present in a translucent paint layer on top of a yellow scroll design on the border, and it is argued that this is likely to be the substrate for an organic yellow lake pigment. Exploratory experiments to investigate whether the syngenite itself may have been used as a white substrate on which to adsorb a yellow dye or whether it might represent an alteration product of some kind were inconclusive, but documentary evidence confirms that yellow organic pigments were being produced from an early date and several possible dye sources suggested. Since there is very little direct evidence for the use of yellow organic pigments in 13th-century panel paintings, the identification of syngenite as a possible substrate or alteration product is both an unusual and an important observation.

**Author Contributions:** Conceptualization, D.P., H.H. and J.K.; Cross-section preparation and analysis by SEM-EDS, H.H.; micro-XRD, J.N.; MA-XRF scanning and FORS analysis, H.H. and D.P., HPLC and ATR-FTIR analysis, D.P.; Substrate and lake preparation, D.P. and J.K.; Documentary sources research, J.K.; writing—original draft preparation, reviewing and editing, D.P., H.H. and J.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Data Availability Statement:** Data supporting reporting results available on request.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

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## *Article* **Mapping Materials and Dyes on Historic Tapestries Using Hyperspectral Imaging**

**Constantina Vlachou-Mogire 1,\*, Jon Danskin 2, John R. Gilchrist 2 and Kathryn Hallett 1**

**Abstract:** Hyperspectral imaging has emerged as a promising analytical method of artwork due to its potential in combining non-invasive analytical capabilities and imaging allowing the survey of the entire (or of a large area of the) surface of an artwork, which is a highly significant application for historic tapestries. This project deployed a high-resolution ClydeHSI Art Scanner, which was used with both a push-broom visible to very-near infrared (VNIR; 400–1000 nm) and near infrared (NIR; 900–1700 nm) hyperspectral cameras. Initial testing focused on the characterisation and mapping of the different materials used on historic tapestries (wool, silk, metal threads). To facilitate the dye characterisation, a collection of wool and silk samples dyed with recipes based on medieval practices was used. The samples measured using the system and the data collected formed an external reference library including the type of the natural dyes and mordants used during their production. The outcomes of the on-site deployment of this analytical instrumentation for the characterisation and analysis of 16th century tapestries on display at Hampton Court Palace will be discussed.

**Keywords:** Flemish tapestries; textiles; natural dyes; hyperspectral imaging; non-invasive dye analysis; brazilwood; yellow dyes; fading

#### **1. Introduction**

Historic tapestry collections have been considered among the most significant forms of art from the 15th to 18th century in Europe. Historic Royal Palaces (HRP) is the custodian of Hampton Court Palace in London, UK, and its important tapestry collection, developed primarily by Cardinal Wolsey (1475–1530) and Henry VIII (1509–1547), which is part of the Royal Collection [1]. The characterisation of tapestry materials is crucial for assessing the condition and formulating suitable conservation treatment strategies for these nationally significant large-scale textiles.

Tapestry is a 'weft-faced' woven textile where the coloured weft threads carry the pictorial intricate design while contributing to the physical weave structure of the textile. Materials used for the weft are primarily wool and silk dyed in a wide range of colours with natural sources (plants and insects). In the most expensive tapestries, metal threads, comprising gold, silver or silver gilt wrapped around a silk core, were also used.

Micro destructive chromatographic methods have been commonly used for dye analysis as they allow the different molecular components to be separated and then characterised. High-performance liquid chromatography (HPLC) has enabled the identification of a large number of dyes used in textiles [2,3]. However, this requires sampling usually from the reverse of the tapestry, which is not always feasible and the results are limited to the sampled area [4]. The development of non-invasive analytical approaches for the identification of tapestry materials or the analysis of the natural dyestuffs has progressed over the last few years, including Fourier-transform infrared spectroscopy (FTIR) and fibre optics reflectance spectroscopy (FORS) [5–8]. Raman spectroscopy is another method which can

**Citation:** Vlachou-Mogire, C.; Danskin, J.; Gilchrist, J.R.; Hallett, K. Mapping Materials and Dyes on Historic Tapestries Using Hyperspectral Imaging. *Heritage* **2023**, *6*, 3159–3182. https://doi.org/ 10.3390/heritage6030168

Academic Editors: Marei Hacke, Sara Norrehed, Joanne Dyer, Jo Kirby, Art Proaño Gaibor, Ilaria Degano, Zvi Koren, Sandström Edith and Claudia Pelosi

Received: 8 December 2022 Revised: 23 February 2023 Accepted: 13 March 2023 Published: 16 March 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

be deployed for the analysis of dyes in particular surface-enhanced Raman spectroscopy (SERS); however, it requires a small sample (approximately 80 μg); therefore, it is a micro invasive technique [9]. X-ray fluorescence spectroscopy is also useful for analysing metallic mordants [10,11]. However, these non-invasive analytical techniques also provide limited information on the spot analysed.

Hyperspectral imaging (HSI) has emerged as a promising analytical method for works of art [12,13]. The systems used in cultural heritage are usually operated in reflectance mode and cover the visible (Vis), near infrared (NIR) and short-wave infrared (SWIR) spectral regions depending on the type of sensor their cameras have [14]. This technique produces high-quality optical spectral signatures in reflectance at each image pixel position, providing the ability to identify the materials used for the artwork and map their spatial distribution over the object surface. The method has been used primarily in paintings [15–17] and manuscripts [18,19]. Other applications include prints [20], ethnographic collections [21,22], murals [23], photographs [14] and archaeological objects [24].

During the deployment of this method, spectral data are acquired at each spatial position across a measurement line and the spectral cameras are scanned to create a data cube that can be formed into x, y and spectral axes. The processing of these large data sets has been an active area of research particularly for the identification of pigments, lakes and other materials used in the construction of paintings such as binders [16–18,25–31]. Another application of automated classification techniques is related to the authentication of artworks [32].

HSI has also been applied to the examination and analysis of textiles [33]. Zhao looked into the use of a portable spectroradiometer with a range from 2500 nm to identify and characterise textile fibres [34], while another interesting application successfully deployed HSI combined with FORS in the visible to very near infrared range (VNIR 400–1000 nm) for the characterisation of twelve dyestuffs and three mordants [35]. Finally, Vermeulen was successfully deployed a range of non-invasive analytical techniques including HSI to characterize organic colorants used in 20th-century traditional textiles from Mexico [36].

HSI allows the survey of the entire (or of a large area of the) surface of an artwork, which is beneficial for the analysis of historic tapestries; however, there are limited studies related to this application. Delaney deployed an hyperspectral camera (NIR 967–1680 nm) combined with FORS to map the different materials (wool, silk and metal threads) on a 16th century tapestry using the spectral regions between 1500 and 1600 nm and 2100 and 2400 nm [37]. De la Codre investigated the materials used in three 18th century Royal tapestry manufactories Abusson, Gobelins and Beauvais using HSI (400–2500 nm). In this research, dyed samples were used as reference materials and two different classification methods were compared for data processing and material identification [38]. Furthermore, the characterisation of yellow dyes in the tapestries and the influence of the composition and degradation on analysis were also studied [39]. This research has also been included as a case study in a paper exploring the applications of a small hyperspectral camera operating in the visible–near infrared range (VNIR) [24].

This collaborative research considers the advantages and potential limitations of HSI applied to the study of historic tapestries deploying a high-resolution ClydeHSI Art Scanner. Initial testing focused on the characterisation and mapping of the different materials used on historic tapestries (wool, silk, metal threads). To facilitate the dye characterisation, a collection of wool and silk samples were used which had been dyed during the Monitoring of Damage in Historic Tapestries (MODHT) EU research project using recipes based on medieval practices [40]. The samples were measured using the system and the data collected formed an external reference library, including the type of the natural dyes and mordants used during their production. Finally, the analytical instrumentation was deployed on site for the characterisation and analysis of several 16th century tapestries on display at Hampton Court Palace.

#### **2. Materials and Methods**

#### *2.1. Historic Tapestries*

The *Story of Abraham* is a set of ten Flemish tapestries depicting scenes from the Old Testament. The tapestries, each measuring approximately eight meters across by five meters high, were constructed from dyed wool and silk yarns, while large areas were decorated with precious gold and silver metal threads. The *Story of Abraham* is considered the most artistically and historically significant set of tapestries in the UK's Royal Collection, and have hung in their original location in the Great Hall at Hampton Court Palace since the early 1800s (Campbell 2007). The tapestries are likely to have been commissioned by King Henry VIII, their design is attributed to Pieter Coecke van Aelst and they were constructed in the workshop of Willem de Kempeneer in Brussels (c.a. 1541-43) [41].

In this study, three tapestries of the *Story of Abraham* set were analysed (Figure 1). Details of the tapestries and the size of the areas scanned are listed in Table 1.

This research builds on the Henry VIII's Tapestries Revealed project, which focused on virtual restoration of the original colour palette of the *Oath and Departure of Eliezer* tapestry. As part of this research, spectral imaging technology was used for the first time to accurately assess the impact of fading, and develop a model which, when projected on the tapestry, produced the recoloured effect [42].

**Figure 1.** The three tapestries from the *Story of Abraham* set analysed in this project: *Oath and Departure of Eliezer* (**a**), *Sacrifice of Isaac* (**b**) and *Eliezer and Rebekah at the Well* (**c**). The white rectangles indicate the areas scanned on each tapestry (Royal Collection Trust/© His Majesty King Charles III 2022).

**Table 1.** Details of the *Abraham* tapestries and the areas of investigation.

#### *2.2. Reference Samples*

Tapestry samples created during the European Commission-funded project Monitoring of Damage in Historic Tapestries (MODHT) were used in this research [40]. The samples were woven from wool and silk yarns representative of historic tapestry materials and dyed using medieval natural colourant formulations that would have been used in 16th century tapestry weaving [43].

Dye analysis of European tapestries showed that a limited number of dyes from natural sources such as plants and insects were used to create a wide palette of colours. This was achieved by deploying different mordants (inorganic salts for bonding the dye to the fibre), adding chemicals, or overdyeing a colour with another one (yellow with blue for green) [4,40,43]. Table 2 summarizes the information on the materials, dyestuff and mordant combinations used for the production of the MODHT model tapestry samples. Details of the actual recipes and the testing of the samples are available in publications developed as outputs of the MODHT project [40,43]. In this paper, the same abbreviation codes are used.

#### *2.3. Hyperspectral Imaging*

The hyperspectral imaging system deployed in this research was a high-resolution ClydeHSI Art Scanner, with both a push-broom visible to very near-infrared (VNIR; 400–1000 nm) and near infrared (NIR 900–1700 nm) hyperspectral cameras. The cameras were mounted on a stage providing the capability for the instrument to scan large areas of 1.5 × 1.5 m. Tapestry fragments from the Historic Tapestry Fragments Collection housed at the HRP Heritage Science Laboratory were initially tested using the scanner to define the operational conditions to ensure the best signal to noise ratio and initial analytical protocol. The instrument was then carried into the Great Hall where, over a period of one day, areas on three Abraham tapestries were analysed (Figure 2). A line scan, push-broom, spectral camera creates a two-dimensional image by scanning its field of view across a measurement line and then moving the camera to create additional lines in the scan direction. This gives a field of view in the across track direction, the measurement line, and an image in the along track which forms a data hypercube. There is a trade-off between field of view and spatial resolution. This means that for high resolution scanning, the field of view is narrow and in order to image large areas in high resolution, multiple image strips need to be measured. These strips are measured with a spatial overlap so that they can be stitched together using feature mapped procedures. Table 3 summarises the number and size of the individual image strips as well as information on the resulting composite images for each tapestry. The initial tapestry analysed was the *Oath and Departure of Eliezer,* where an area of approximately 1 m2 was scanned in seven strips. The largest area was captured on the *Sacrifice of Isaac tapestry,* which was about 2 m2 and consisted of 17 strips. On the final tapestry, *Eliezer and Rebekah at the Well,* due to time limitations, only one strip was captured, measuring 0.1 m2.

**Table 2.** MODHT samples material, dye and mordant information.

**Table 3.** Specifications of the hyperspectral analysis on the tapestries.

**Figure 2.** The MODHT samples representing a wide range of natural dyes used in the manufacture of 16th century historic tapestries.

The tapestries were scanned vertically; the acquisition time for one strip was 15 s with a spatial resolution of 0.25 mm/pixel. Two halogen light sources positioned at 45◦ with respect to the cameras were used for illumination of the scanned area; the light exposure level in the visible spectrum was ca. 2000 lx. This illumination level was measured under static conditions and, as a result, when line scanned measurements are performed, the speed of acquisition means the illumination of a measurement area occurs for a fraction of a second. The estimated light dose of exposure for one second is 0.5 lux hours, a minor contribution to the 150,000 lux hours threshold adopted as an acceptable annual dose in the course of usual display. The system was set up to allow an overlap of 15% between consecutive strips to facilitate the stitching at the post-processing phase and to combine them into one composite hyperspectral image (Figure 3c, Table 3). At the start and end of each scan, a reflective white Polytetrafluoroethylene block was measured to record the instrument spectral/spatial response function to provide the reference for converting the raw data of the composite hyperspectral image signal into reflectance data. The reflection data were calculated from the measurement of the instrument response using a white tile reference and a dark noise reference with the camera shutter closed. Further details on the hyperspectral cameras specifications and the scan parameters are indicated in Table 4.

For the operation of the cameras and spectra acquisition, as well as the processing of the hyperspectral data, the SpectraSENS Clyde HSI software was used. The composite hyperspectral reflectance image was initially processed with an unweighted smoothing average before using principal components analysis (PCA) and spectral angle mapping (SAM) algorithm to achieve the distributions of the material across the tapestry areas analysed and identify the dyes used for their manufacture.

**Figure 3.** The hyperspectral imaging scanner set up in front of the *Sacrifice of Isaac* tapestry in the Great Hall, Hampton Court Palace, UK (**a**) The hyperspectral image strips from the VNIR and NIR cameras (**b**) The composite hyperspectral images in VNIR (top) and NIR (bottom) (**c**).

PCA is a statistical tool that represents data using uncorrelated variables named principal components (PC). PCA is used to reduce variables, detect general patterns in the data and identify outliers. As a result, the hyperspectral cube is reduced in dimension and the clustering is taking place according to a number of components. In unsupervised deployment, the clustering algorithm automatically detects patterns which are useful for initial processing of the data [14,27,32]. PCA was performed independently on the VNIR and NIR data sets to identify spectral outliers and reduce the number of dimensions in the hyperspectral cubes to less than 10 PC dimensions. The resulting false colour images from this processing indicated the mapping of the different dyes across the area of analysis based on their spectral responses.

SAM is a classification algorithm commonly used in the processing of hyperspectral imaging data. This algorithm assesses the angle between each spectrum at each pixel with the spectra of a reference library (endmembers) [25,26,29,35,38]. In this project, an external library was used with spectra acquired from the MODHT tapestry model tapestries where the dyes and mordants are known (Appendix A Table A1). The SAM algorithm then proceeds in the classification of each pixel of the reflectance hyperspectral image and produces an endmember classification map showing the distribution of each dye on the area of analysis. The map is associated with a list of the identified endmembers and shows their distribution on the area under investigation.

#### **3. Results**

#### *3.1. PCA Analysis*

PCA was applied to the VNIR and NIR reflectance hyperspectral cubes of the tapestries. The results produced detailed mapping of the dyes based on their spectral responses where the design of the tapestry was reproduced in high resolution but in false colour. Figure 4 shows the *Oath and Departure of Eliezer* tapestry area analysed with HSI (Figure 4a) and the false colour images following PCA analysis of the VNIR (Figure 4b) and NIR (Figure 4c) hyperspectral cubes.

**Figure 4.** The *Oath and Departure of Eliezer* tapestry area analysed with HSI. (**a**) False colour images following PCA analysis of VNIR (**b**) and NIR (**c**) reflectance hyperspectral cubes.

Following closer interrogation of the PCA images, some interesting observations could be reported. For example, on the section of the border of the *Oath and Departure of Eliezer* tapestry, which seems relatively uniform under visible light, the PCA false colour images highlighted an area of restoration with a bright orange/red colour (Figure 5a). Furthermore, on the architecture, although the visible image has the same beige colour, the mapping of the dyes in the VNIR PCA image suggests the use of different dyes (Figure 5b). Moreover, the PCA NIR image shows that the dye highlighted in purple has most likely been used on the sleeve of the figure as well as the design of the architecture (Figure 5b).

#### *3.2. SAM Analysis*

The SAM algorithm was applied in the processing of the VNIR reflectance data cubes of the three Abraham tapestries for the identification and mapping of the dyes used in their manufacturing. The non-invasive classification of the dyes was achieved with the use of a spectral reference library based on the HSI analysis of the MODHT model tapestry samples. The spectra range used for this analysis was between 500 and 920 nm in order to reduce poor noise to signal ratios. Furthermore, a high tolerance angle of 1.5 rad was applied to facilitate the characterisation of dyes. Previous research has suggested that SAM analysis using high tolerance angle (2.56 to 1.81 rad) resulted in the identification of four yellow dyes (weld, safflower, chamomile and turmeric), while these were not able to be characterised using their reflectance spectra [35].

**Figure 5.** (**a**) Section of the border of the *Oath and Departure of Eliezer* tapestry area analysed with HSI. Following PCA analysis. the mapping of the dyes revealed a restored area. (**b**) The PCA VNIR mapping indicates the use of different dyes.

#### 3.2.1. MODHT Samples Analysis and Compilation of Endmember Library

Forty-two MODHT samples were analysed with HSI VNIR (400–900 nm) and NIR (900–1700 nm) cameras, and the resulting reflectance spectra were used as the spectral reference library for performing SAM analysis on the HSI data collected from the Abraham tapestries.

Figure 6 shows four MODHT replica tapestry samples representing yellow (YW1), red (RW1), green (GRW1) and blue (BW1) colours (Figure 6a) and their respective reflectance spectra in VNIR and NIR (Figure 6b). Details of the endmember library are summarized in Appendix A Table A1, including the dyestuff, mordants, visible images and reflectance spectra for forty-two MODHT samples. This table also includes the colours attributed to the endmembers in the SAM classification. These digital colours were selected as an approximation of the original colour group of the MODHT sample such as pink, red, light and dark green, light and dark blue, brown, beige and black, while grey indicated metal threads.

**Figure 6.** MODHT replica tapestry samples (**a**) and their respective reflectance spectra in VNIR (top) and NIR (bottom) graphs (**b**).

The classification of the dyes used in this section of the *Oath and Departure of Eliezer* tapestry indicated the use of brazilwood with lye treatment for the pink (RS1c) or dark pink, brown/red (RS1d) silk areas, while there is also a smaller amount of the brazilwood dye RS1a. In wool, the only red dye identified was madder (RW2); however, this was in traces. The blue areas were dyed with woad (BS1a), while a darker shade (BS1b) was produced with a longer dyeing time. The yellow silk areas were dyed with both greenweed (YS3) and weld (S1b); however, for wool, greenweed (YW2) was primarily used, while weld (YW1) had a much lower quantification value. Green in the silk areas was produced with threads dyed first with weld and then with woad (GS1a); however, threads which were dyed first with woad and then with weld (GS2b) were also identified in two different shades, light and dark. On the wool green areas, only threads dyed first with weld and then woad (GW1) were identified. Oak gall was classified in silk and wool areas, while Alder bark and Alum mordanted threads were identified only in wool. Black was limited to wool threads and was produced primarily with BLKW4, which was produced with a combination of iron sulphate and copper sulphate mordant.

**Figure 7.** The *Oath and Departure of Eliezer* tapestry area analysed. (**a**) The SAM analysis with the identified endmember mapping. (**b**) The graphs summarize the silk (top) and wool (bottom) dyes identified after SAM analysis (**c**).

The *Oath and Departure of Eliezer* as part of previous research was recorded in highresolution imaging in both obverse and reverse [42]. Figure 8a shows the detail of the area analysed with HSI, while Figure 8c shows the same area in reverse where the tapestry was protected from light exposure and the original colour palette has been preserved. Visual examination of the reverse image confirmed the use of green and yellow dyed yarns on the architecture. However, most significantly, the use of brazilwood was also indicated in the background of the border following a comparison with the brazilwood dyed sample (Figure 8b). During this research project, samples were exposed to a xenon arc lamp in an accelerated light ageing chamber (Xenotest 150S) with an IR and UV filter system intended to replicate sunlight through window glass and following the conditions set in BS EN ISO 105-B02. The relative humidity was controlled by the apparatus to 65 ± 2%, and the temperature was 20 ± 2 ◦C. Trial samples showed that a total exposure of 60 Mlx h

(megalux hours) was a suitable dose, producing a significant and measurable difference in physical properties, and approximating the light exposure which may have been received by a 400-year-old tapestry, based on archival research and current illumination levels. These accelerating ageing experiments confirmed that brazilwood is one of the most fugitive dyes [42].

The *Oath and Departure of Eliezer* was also used in this research to map the different materials used for the weaving of the tapestry, wool, silk and metal threads. For this purpose, the SAM algorithm was applied in the processing of the NIR reflectance data cube of the tapestry and the endmember library included the undyed silk and wool MODHT samples spectra.1500 to 1600 nm of the wool and silk reflectance spectra can be used in reflectance imaging spectroscopy to map these fibres [37] therefore, the spectral range used for this analysis was between 1497 and 1567 nm. Furthermore, a high tolerance angle of 1.5 rad was applied. The results are shown in Figure 9b, while the percentage of area coverage for each endmember is presented in Figure 9c. This analysis reproduced the design of the tapestry in high resolution, providing a clear understanding of the distribution of the materials. In almost 50% of this area, the weft threads used silk in the head and parts of the dress of the figure, as well as the architecture and the border. Wool yarns covered approximately 20% of this area and they were deployed in specific areas of the design. Finally, about 30% of the area was covered by metal threads; this included part of the figure's dress as well as the background of the architecture and the decorative border.

**Figure 8.** The *Oath and Departure of Eliezer* tapestry obverse (**a**) and reverse (**c**) The brazilwood dyed sample in the middle before (right) and after (left) light ageing (**b**).

#### 3.2.2. Sacrifice of Isaac

The scanned area from the *Sacrifice of Isaac,* measuring about 2 m2 and located at the bottom viewing right of the tapestry, shows a male figure; on the right of the scanned area, a section of the border is also included. The results from the SAM analysis shown in Figure 10b provide an estimate of the original colour palette of the tapestry, which has faded due to long exposure to light. Significant changes are observed in the yellow and green areas. Furthermore, the metal threads used primarily on the male figure's clothing, the border on the right and the basket in the background have tarnished (Figure 10a).

The SAM classification endmembers identified in the area of analysis are presented in Figure 10c graphs which show the dyes on silk on the left and the dyes on wool on the right. These graphs show the percentage of area coverage following the spectral matching to the endmember reference library (Appendix A, Table A1).

**Figure 9.** The *Oath and Departure of Eliezer* tapestry area analysed. (**a**) The NIR SAM analysis with the identified endmembers mapping (**b**) SAM analysis results (**c**).

**Figure 10.** The *Sacrifice of Isaac* tapestry area analysed (**a**). The SAM analysis with the identified endmembers mapping (**b**). The graphs summarize the silk (left) and wool (right) dyes identified after HSI SAM analysis (**c**).

The analysis results indicated that the blue areas were dyed with woad (BS1a), while a darker shade (BS1b) was produced with a longer dyeing time. On the yellow silk areas, greenweed (YS3) was primarily used, while a fewer yarns were dyed with weld (YS1b). However, for the yellow wool yarns, only greenweed (YW2) was used. Green was primarily produced with silk yarns dyed either first with weld and then with woad (GS1a), or threads which were dyed first with woad and then with weld (GS2b) in two different shades, light and dark. For the silk red tones, the use of brazilwood with lye treatment (RS1d and RS1c) was identified, as well as a smaller amount of the brazilwood dye RS1a. Red wool yarns were dyed with madder (RW2 and RW1). Oak gall was identified in silk and wool threads, while Alder and Alum mordanted threads were identified only in wool. Black was identified primarily in wool and was produced with three different recipes (BLKW1, BLKW2 and BLKW4). It is interesting to note that for some of the brown areas on the tapestry, the SAM simulation presents them as black, for example, on the flesh at the two legs of the unseen figure on the left. This might have been due to the use of a black dye with another one to produce the brown colour. However, only the black element was revealed in the analysis.

#### 3.2.3. Eliezer and Rebekah at the Well

The scanned area from the *Eliezer and Rebekah at the Well* tapestry was much smaller than the previous two areas, measuring 0.1 m2. Nevertheless, the analysis of this area provided the opportunity to study how the tapestry weavers deployed dyes in different tones to deliver the design of the tapestry, which, in this case, is a detail of a woman with a blue dress (Figure 11a). The results from the SAM analysis shown in Figure 11b indicated that the pleats on the dress were originally highlighted with green- and yellow-coloured threads to add volume and texture into the design of the fabric. Furthermore, large areas of the shoulder and the sleeve of the figure were constructed with metal threads. It is interesting to note that for the areas across the metal threads, yellow-coloured threads were used to match their golden colour. The SAM classification mapping image provides an estimate of the original colour palette of the tapestry (Figure 11b) as the present condition of the tapestry, following long exposure to light, has suffered from fading, and the metal threads have tarnished and blackened (Figure 11a). Accelerated ageing studies have confirmed that the yellow dyes (mostly weld) are highly fugitive [42]. Therefore, the yellow highlights on the dress are currently neutral, while the green-coloured areas originally made with yellow and blue dyes are primarily blue, as indicated in Figure 11a,b.

The SAM classification endmembers identified in the area of analysis are presented in the Figure 11c graphs, showing the dyes on silk at the top and the dyes on wool at the bottom. The data show the percentage of area coverage following the spectral matching to the endmember reference library (Appendix A, Table A1).

The blue areas were dyed with woad (BS1a), while a darker shade (BS1b) was produced with a longer dyeing time. The yellow silk areas were dyed with both greenweed (YS3) and weld (S1b), whereas for wool, greenweed (YW2) was primarily used, while weld (YW1) was used less frequently. Green in the silk areas was produced with threads dyed first with weld and then with woad (GS1a); however, threads which were dyed first with woad and then with weld (GS2b) were also identified in two different shades, light and dark. On the green wool areas, only threads dyed first with woad and then weld (GRW2) were identified. Oak gall was identified in silk and wool threads, while Alder and Alum mordanted threads were identified only in wool. Black was identified in wool and was produced with three different recipes (BLKW1, BLKW2 and BLKW4). Finally, small areas of threads dyed with brazilwood were identified. As the area analysed did not have red/pink areas, brazilwood might have been used in the flesh. Alternatively, the areas consisting of metal threads also have a red hue in the SAM mapping, which might be related to the use of brazilwood for the dyeing of the silk cores in combination with young fustic [4,9]. In wool, there was also a small amount of cochineal (RW4) and madder (RW2) identified, which might have been used in the blue colour.

**Figure 11.** The *Eliezer and Rebekah at the Well* tapestry area analysed (**a**). The SAM simulation with the identified endmembers mapping. The arrows indicate areas of colour fading (**b**). The graphs summarize the silk (top) and wool (bottom) dyes identified after HSI SAM analysis (**c**).

#### **4. Discussion**

The SAM classification endmembers identified in the areas analysed on the three *Abraham* tapestries are presented in Figure 12, showing the dyes on silk (a) and the dyes on wool (b). The compilation of the results on the dyes identified are also summarized in Table 5.

The classification of the dyes used in the Abraham tapestries indicated the use of similar materials for their production, confirming that they are part of one set and were made in the same workshop.

The use of brazilwood as the main dye for silk red, pink and dark pink yarns is consistent across all three tapestries analysed, particularly brazilwood with lye treatment (RS1c and d) but also with no lye (RS1a and b). In wool, madder (RW2) was used primarily; however, brazilwood (RW1) and cochineal (RW4) were also traced in small quantities. The fugacious nature of brazilwood has been well-documented for a long time and to characterise its aged form on historic textiles has been challenging. It is only recently that the full characterisation took place [44], while a non-invasive methodology using SERS has also been demonstrated [9]. Furthermore, HSI has been used successfully to identify brazilwood lake pigments [31]. The use of brazilwood in The *Oath and Departure of Eliezer* tapestry was also indicated though the visual examination of the reverse side, which was protected from light exposure and the colour of the yarns had not faded (Figure 9). However, future research should focus on the characterisation of the red dyes with HSI and the impact of degradation to the analytical results.

**Figure 12.** The graphs summaries the silk (**a**) and wool (**b**) dyes identified after HSI SAM analysis on the three *Abraham* tapestries: *Eliezer and Rebekah at the Well* (ER), *The Oath and Departure of Eliezer* (ODE) *and Sacrifice of Isaac* (SI).

The blue areas on the Abraham tapestries were dyed with woad (BS1a), which also had a darker shade (BS1b) which was produced with a longer dyeing time.

Although difficulties have been reported in the identification of yellow dyes [39], the data from the HSI analysis of the Abraham tapestries suggested that this method might be used to characterise the yellow yarns. The results indicated that the silk areas were dyed with greenweed (YS3) or weld (S1b), whereas for wool, greenweed (YW2) was primarily used. Further analytical work using an established analytical method such as HPLC could be deployed for validation as these results differed from the outcomes of the yellow yarns analysis in the MODHT project, which indicated that for the tapestries included in that study, greenweed was used mostly for silk and weld for wool [40]. Furthermore, for the MODHT historic samples, this trend appeared to be most evident in the combination dyed yarns for the production of green or orange colours and not exclusively on the yellow yarns. Additionally, there was a disparity between the number of wool and silk yarns analysed, which might have also influenced this outcome [4].

The results indicated consistency across the three tapestries for the production of the dyes in the green yarns. In the silk areas, green was produced with threads dyed first with weld and then with woad (GS1a); however, threads which were dyed first with woad and then with weld (GS2b) were also detected in two different shades, light and dark. On the wool green areas only, threads dyed first with weld and then woad (GW1) were detected.

In all of the tapestries, oak gall was classified in silk and wool areas, while Alder bark and Alum mordanted threads were detected only in wool.

Black was limited to wool threads and was dyed primarily with BLKW4, which was produced with a combination of iron sulphate and copper sulphate mordant.

The SAM analysis of the HSI images provided accurate mapping of the dyes, reproducing the design of the tapestry in high resolution. Furthermore, the mapping of the dyes potentially revealed information on the original colours of the tapestries. However, on the *Sacrifice of Isaac*, the brown areas were presented as black after the SAM simulation, an effect which requires further investigation.

Finally, the SAM NIR analysis successfully mapped the different materials used to produce the tapestry and provided an estimate of the percentage area coverage for each of them. This method could be deployed for assessing the quality of historic tapestries and possibly provide a tool to monitor changes in their condition over time.

**Table 5.** Compilation of SAM analysis endmember matching results for the three *Abraham* tapestries.

#### **5. Conclusions**

This project explored the deployment of HSI in the characterisation and analysis of historic tapestries. A high-resolution ClydeHSI Art Scanner with both a push-broom visible to very near-infrared (VNIR; 400–1000 nm) and near infrared (NIR 900–1700 nm) hyperspectral cameras was used to analyse three of the 16th century Abraham series tapestries in the Great Hall, Hampton Court Palace. The areas analysed on the tapestries ranged from 0.1 m2 to approximately 2 m2 and consisted of wool and silk dyed in a range of colours with metal threads also used in large sections. The tapestries were scanned vertically with an overlap of 15% between consecutive strips to facilitate the stitching at the post-processing phase.

The composite hyperspectral reflectance images were initially processed with an unweighted smoothing average before using principal components analysis (PCA) based on their spectral responses, and the design of the tapestry was reproduced in a highresolution false colour image. Interrogation of these images revealed information on the use of different dyes in faded areas and identified areas of restoration.

Spectral angle mapping (SAM) algorithm was successfully deployed for processing the data further to study the distribution of the materials across the tapestry areas analysed and identify the dyes used for their manufacture. An external library was produced with spectra acquired from the MODHT tapestry model tapestries, (with known dyes and mordants) and used as endmember reference for the spectral matching and characterisation (Appendix A Table A1).

Future work for this ongoing research project will include the development of the mapping of individual dyes and assessing the impact of degradation on the HSI analysis. Furthermore, the deployment of complementary analytical techniques such as HPLC could

provide further evidence on the HSI results with a particular focus on the characterisation of the yellow dyestuffs and the identification of brazilwood. Finally, the deployment of XRF analysis could also confirm the mordants used for the dyeing.

**Author Contributions:** Conceptualisation, C.V.-M.; methodology, C.V.-M., J.R.G. and J.D.; data curation, C.V.-M. writing—original draft preparation, C.V.-M.; writing—review and editing, C.V.-M., K.H. and J.R.G.; visualisation, C.V.-M. All authors have read and agreed to the published version of the manuscript.

**Funding:** The purchase of the Clyde HSI Hyperspectral Imaging Scanner was funded by the UK's ARTS AND HUMANITIES RESEARCH COUNCIL (AHRC) Historic Royal Palaces Heritage Science Laboratory Upgrade award, grant number AH/V012487/1.

**Data Availability Statement:** The data presented in this study can be requested by the corresponding author.

**Acknowledgments:** We would like to acknowledge our colleagues at Historic Royal Palaces for their support during the onsite work, in particular Magda Iwanicka and Gareth James. We are also grateful to Matthew Sheppard and Douglas Gourlay at ClydeHSI for their help and discussion on signal processing.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **Appendix A End Member Library**

**Table A1.** MODHT samples images and associated HSI spectra.

307

#### **References**

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## *Article* **Chromatographic Characterization of Archaeological Molluskan Colorants via the Di-Mono Index and Ternary Diagram**

**Zvi C. Koren**

The Edelstein Center for the Analysis of Ancient Artifacts, Department of Chemical Engineering, Shenkar College of Engineering, Design and Art, 12 Anna Frank St., Ramat Gan 52526, Israel<EMAIL_ADDRESS>
**Abstract:** One of the main research questions regarding archaeological molluscan purple pigments and dyes is whether it is possible to determine which malacological species produced these colorants. For this determination of the zoological provenance of the pigment, a multicomponent analysis must be performed, which can only be obtained from the HPLC technique—the optimal method for identifying all the detectable colorants in a sample. In order to find any trends in the compositions of the dye components from various species of purple-producing sea snails, a statistical formulation is needed. Though principal component analysis (PCA) is a powerful statistical tool that has been used in the analysis of these components, it is based on an algorithm that combines all the componential values and produces new two-dimensional parameters whereby the individualities of the original dye component values are lost. To maintain the integrity of the dye compositions in the purple pigments, a very simple formulation was first published in 2008 and applied to a limited number of samples. This property is known as DMI (short for Di-Mono Index), and for each sample, it is simply the ratio of the peak area of DBI relative to that of MBI, evaluated at the standard wavelength of 288 nm, which has been used for such peak calculations. Currently, considerably more modern and archaeological pigments have been analyzed via HPLC; thus, in the current study, the DMI has been expanded to characterize these purple pigments. Furthermore, a ternary diagram comprising the blue, violet, and red components that can be found in purple colorants is presented for both modern and archaeological purple pigments from the three Muricidae species known in antiquity to produce purple pigments. This triangular diagram is intuitive, retains the integrity of the original dyes, and is presented here for the first time. Both the DMI and the ternary diagram can discern whether a *Hexaplex trunculus* species or perhaps the *Bolinus brandaris* or *Stramonita haemastoma* species were used to produce the pigment. Further, these two representations can also determine whether the IND-rich or the DBI-rich varieties, or both, of *H. trunculus* were used to produce the purple pigment, either as a paint pigment or as a textile dye.

**Keywords:** HPLC; indigoids; indirubinoids; dibromoindigo; molluscan purple pigments and dyes; Muricidae; *Hexaplex trunculus*; Di-Mono Index (DMI); ternary diagram

#### **1. Introduction**

*1.1. Introducing the Sea Snails*

Of all the natural organic and inorganic pigments and dyes—from flora, fauna, and mineralia—used by ancient civilizations [1–3], there is no other prized colorant than "purple" (with all its variegated shades) produced from certain molluscan species. This marinederived imperial colorant was not only heralded as such in antiquity by Greek and Roman writers, e.g., Homer, Aristotle, Vitruvius, and Pliny the Elder, but even historic, not soancient, poets were awed by this molluscan colorant. In the latter case, this wonderment regarding that colorant is uniquely expressed by the famous English poet Robert Browning in his 1855 poem titled "Popularity", who refers to this supreme colorant as the "dye of dyes" [4].

Archaeological Molluskan Colorants via the Di-Mono Index and Ternary Diagram. *Heritage* **2023**, *6*, 2186–2201. https://doi.org/10.3390/ heritage6020116

Chromatographic Characterization of

**Citation:** Koren, Z.C.

Academic Editor: Vittoria Guglielmi

Received: 8 December 2022 Revised: 14 February 2023 Accepted: 16 February 2023 Published: 19 February 2023

**Copyright:** © 2023 by the author. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

It is well-known that in antiquity, there were three related species of mollusks in the Eastern Mediterranean, from which a purple pigment can be produced, and these sea snails are also found today. This Muricidae family—commonly referred to as Murex sea snails—consists of *Hexaplex trunculus* (also known as *Murex trunculus*), *Bolinus brandaris* (=*Murex b.*), and *Stramonita haemastoma* (=*Purpura h.*). The shells of these mollusks are shown in Figure 1. The chemistry associated with the natural biochemical production of the purple pigment and its components has been published in detail [5–7].

**Figure 1.** Three of the most common purple-producing Mediterranean molluscan species of the *Muricidae* family.

Much has been written about the historical accounts related to these mollusks, and the most prominent ancient historian and science reporter to have written about them was the 1st century CE Roman naturalist Pliny the Elder (Gaius Plinius Secundus). Writing in his monumental multi-volume work *Naturalis Historia*, he recounted the types of sea snails used for purple pigment production, their biology, the season in which to gather them from the seabed, as well as other relevant information. In his role of reporting on the natural world and its animal inhabitants—and keeping in mind that he was obviously not a chemist or alchemist—he nevertheless accurately recounted the entire process of dyeing with these molluscan pigments. Yet, there are modern authors who have at times misunderstood Pliny's description of the dyeing process. However, if one combines modern chemical principles with his ancient chronicling, it becomes apparent that he correctly described the primary and auxiliary agents—and their processing—that were needed for the reduction and solubilization of the purple pigment [8].

#### *1.2. HPLC Über Alles*

As for analyzing the purple pigment and identifying all the detectable colorants in it, the optimal method by far is HPLC (high-performance liquid chromatography). The application of the HPLC method for the analyses of molluscan purple pigments was introduced three decades ago by Jan Wouters and André Verhecken, and they successfully separated four-to-five major components of those pigments [9,10]. With that breakthrough, advancements in HPLC analyses have shown that the purple pigment, especially from the *H. trunculus* species, can consist of about 10 colorants [11]. A typical chromatogram, shown

in Figure 2, presents the separation of these components. The dyes constitute the following three chemical groups:

**Figure 2.** HPLC chromatogram of the various indigoids, indirubinoids, and isatinoids (with their representative colors), which may be present in a molluscan purple pigment. Highlighted (in rectangles) are the three main indigoid components present in pigments extracted from *H. trunculus* species.

• The accompanying UV/Vis spectra of these molluscan colorants, as determined by the PDA (photo-diode array) detector of the HPLC, as well as their molecular structures, are shown in Figure 3. The spectra show that each homologous series of dyes has a common visible wavelength at maximum absorption, λmax, and for the isatinoids, the wavelength is around 415 nm, the indigoids' is about 605 nm, and the indirubinoids' is approximately 540 nm. It is important to note that the λmax values shown in the UV/Vis absorption spectra are specific for the solvent system in which the dye is solubilized. However, dyes dissolved in other solvents will nevertheless typically yield wavelengths that are close to these values.

Undoubtedly, the most efficient methods for analyzing and identifying relatively nonvolatile organic components as whole non-dissociated molecules in a multi-component mixture—such as in the case of the purple pigment—is HPLC. Though GC (gas chromatography) has been used in analyzing molluscan pigments [12], the very high oven temperatures needed for gasifying the components may cause their decomposition, and thus the integrity of the whole molecule may be lost in its identification. Even if only a peripheral fragment has broken away from the parent molecule, this may damage the viability of the analysis itself. For example, the brominated indigoids that constitute the purple pigments from *H. trunculus* are indigo with one bromine atom (MBI) and the doubly

brominated indigo (DBI). In order to obtain an accurate chromatic fingerprinting of these pigments, it is vital that all the original colorant molecules in the pigment remain intact during the analysis. If an analytic method decomposes either DBI or MBI, then artificial amounts of their decomposition products would be formed. If that occurs, then the debromination of MBI would yield an artificial increase in indigo (IND); similarly, if DBI were to decompose during an analysis, then its quantity would be falsely lower. (The quantity of MBI may not change drastically because it would be both formed from DBI and decomposed to IND.) This type of analysis would harm the ability to determine the various colorants in the original sample.

**Figure 3.** UV/Vis spectra and molecular structures of the isatinoids, indigoids, and indirubinoids, showing the visible wavelengths at maximum absorption in the methanol/water/H3PO4 eluent system.

It is important to emphasize the superb detection powers of HPLC to easily "see the invisible" even on the nanogram—or lower—level. Though the HPLC technique is a "destructive" method, it can be described as actually being a "nano-destructive" scheme. As an example of the latter, a successful analysis of a single one- or two-centimeter fiber removed from a yarn of an archaeological textile has been performed via HPLC [13]. This single fiber was effectively invisible to the "naked eye", but can only be clearly seen via a microscope. With that analysis, a clear separation of four dye components on a single fiber was carried out, with relatively strong HPLC peak signals in the chromatogram.

Analytic methods based on spectrometric techniques have also been used to study dyes, in general, and the purple pigment. These methods include Raman, mass spectrometry (MS) without first chromatographically separating the dyes, FTIR, and UV/Vis spectrometry (in the old pioneering days of analysis). However, all of these analytical tools are spectrometric, and the outcome of such an analysis would produce one graph—a spectrum—that is a combination of the spectra of all the components in that pigment. While these techniques may identify the main colorant—or two—in a mixture, spectrometry is limited in that it produces an "overlap of information". Chromatography yields a "separation of information", which provides the identification of all the detectable colorants in a pigment or dyeing. This "separation" yields a "chromatic fingerprint" and is essential in trying to identify the biological provenance of the dyestuff, whether from flora or fauna, and from what specific species [14].

#### *1.3. Statistical Characterizations of Molluscan Purple*

The separating powers of multicomponent HPLC analyses may allow us to determine which malacological species produced the various archaeological pigments and dyes. Towards this end, we need a statistical characterization of the pigments that have been properly analyzed via HPLC. One statistical algorithm for such a characterization is principal component analysis (PCA), and it has been used in the analysis of these components [15,16]. However, this tool has the feeling of a "black box" that incorporates the quantities of the various components, condenses and digests them mathematically, and churns them out in a way that the individuality of the components is lost.

There is a very simple statistical formulation for characterizing molluscan pigments, and it incorporates only two dyes. It was first published in 2008 [17] for a very limited number of samples and has now been expanded to include the results of more recent analyses of archaeological purples. This parameter is known as the Di-Mono Index and is presented in this study to identify the various species of purple-producing mollusks.

Another representation of a statistical characterization of the purple pigments, which maintains the individuality of as many dyes as possible, is the ternary (or triangular) diagram. This graphical depiction best characterizes the dyes so as to allow maximum distinctions among all the species. This intuitive representation would allow for the identification of the zoological provenance of these pigments. This ternary diagram for the molluscan pigments is presented here for the first time.

#### **2. Materials and Methods**

#### *2.1. HPLC: Sample Preparation and Analysis*

The sample preparation and the chromatographic elution system used has been previously published [17] and is summarized here. Extraction of the pigment or dye was performed in a glass vial (and not in a plastic one, as the pigment can adhere to the plastic when heated to a high temperature). The duration of the extraction was 5 min via 200–400 μL DMSO at 100–150 ◦C, depending on the size of the sample, and in subdued light to prevent the artificial photo-debromination of the components. In order to perform a fully quantitative extraction, no residual color must remain on the sample; thus, additional fresh quantities of DMSO may have been needed for the extraction, and all the extracted solutions were combined.

The resulting hot extracted mixture was immediately filtered in a 0.2- or 0.45-μ microspin polypropylene centrifuge tube with nylon filter for 3 min, and immediately injected into the HPLC. In these analyses, the reverse-phase Waters HPLC system consisted of a 3.0 × 150 mm C18 symmetry column as the stationary phase, and the ternary eluent system consisted of methanol, water, and 5% H3PO4 as the mobile phase.

#### *2.2. Materials Analyzed*

The samples analyzed, and their results, consisted of modern and archaeological purple pigments and textile dyes, performed for this study and by other researchers, and are described in Tables 1–3 below in the Results and Discussion section. For the current study, a number of Roman Period archaeological textiles found in the Judean Desert, Israel, have been analyzed and reported in Table 3.

#### **3. Comparison Problems**

#### *3.1. Problems in Comparing Results from Different Researchers*

In order to build as full a picture as possible regarding the characterization of archaeological molluscan colorants, the analytical results of all laboratories need to be combined. However, there are inherent problems in comparing and integrating results from different researchers. Firstly, for reasons described above, only HPLC results will be considered, as this is the only method through which the multi-components in the sample can be determined. Comparing analytical results from different researchers who used different extraction methods and conditions is risky, and it is important to recognize the potential problems that may be encountered, which are detailed below.

#### 3.1.1. Extraction in Minimal Light

Extraction of the dye or pigment should be performed under subdued lighting conditions so that there is just enough light to see the experimental setup. Performing an extraction in a well-lit laboratory room will, at least partially, photo-debrominate the dissolved colorants; thus, the results of dye analyses will be artificially skewed to less DBI and more IND, as mentioned above regarding the limitations of GC analyses. (The quantity of MBI may change only slightly as it is both formed from the debromination of DBI and decomposed when irradiated.) Analysts have not, in general, designated whether their sample extractions were performed in subdued light and this can be a problem when using their results.

#### 3.1.2. Maximizing Extraction via the Optimal Solvent, Temperature, and Time

Different analysts have used different solvents for extracting the pigments [18]. Firstly, in any system, the solubility of a solute will vary when attempting to dissolve it in different solvents. The components in the purple pigment are no exception. The various solubilities of the purple's components have been studied and published [15]. A further factor in the ability of a solvent to dissolve a sample is the temperature of the mixture and the duration of the extraction process. Thus, these three factors (choice of solvent, temperature, and time) may produce different results for the composition of the colorant when analyzing the same sample, if at least one of these three factors is different among the researchers.

Examples of the effective solvents that have been used for the extraction–dissolution of the indigoids and related compounds in the pigment, and the corresponding temperatures and times, include the following:

Though the indigoids have good solubilities in all of these hot solvents, the most effective solvent in dissolving the most stubborn of the indigoids—DBI—is DMSO at high temperatures of 100–150 ◦C, depending on the quantity of the sample. Even when the extraction is performed in subdued light, as mentioned above, no debromination occurs even at 150 ◦C as analyzed using HPLC.

It is important to mention that the acidic-methanolic system for extracting mordant dyes is poor in extracting purple's components. What is a good extracting solvent system for mordant dyes is not necessarily effective for vat dyes. In the typical aluminum–dye complex that is formed in naturally dyed textiles, the acid is needed in order to break the metal–dye bond, and the organic solvent (methanol) is then needed for dissolving the dye that was set free. However, the solubilities of the indigoids in that acid–methanol system are very low, where the solubilities decrease from IND to MBI to DBI. Thus, when using that methanol–acid system, the artificial compositions will show relatively high values for IND, lower than actual for MBI, and much lower for DBI. Additionally, if any indirubin and related brominated derivatives are present, and though their solubilities in methanol are relatively low but higher than the indigoids, then the reported indirubinoid content would also be artificially skewed higher. Thus, the results obtained from the acid–methanol extracting system were not incorporated in the current study.

#### 3.1.3. Immediate Filtration and Injection into the HPLC

As soon as the indigoids are extracted into the solvent, the hot mixture should be immediately filtered and injected into the HPLC, so as to preserve the indigoids in the solution. The automation that is found in modern HPLC/UPLC instruments, with the sample tray able to contain tens of sample vials to be tested, should not be employed for analyzing indigoids. Experiments conducted in this laboratory have shown that if, e.g., DBI is dissolved in hot DMSO and left for extended periods of time, then that dye begins to precipitate out of the solution; thus, an analysis of the solution will not produce the correct compositions. Additionally, even in the dark, the indigoids will eventually decompose, at least in part, to yellowish isatinoids. This precipitation phenomenon is probably due to the occurrence of an association between the solute particles as they can form intermolecular hydrogen bonds among themselves and thus produce larger, heavier, molecules—aggregates or agglomerates. These associated complexes and the low room temperature can cause the indigoids to precipitate out of the solution.

#### 3.1.4. HPLC Elution Methods

Different stationary and mobile phases, as well as elution methods, have been utilized to chromatographically separate the indigoids in the purple pigment. The nature of the eluents used affects the solubilities of the components in the column's stationary phase. Thus, as the absorption of light of the HPLC detector is measured for each eluting component, different eluents may produce different absorbances as the solutions are different. Even if two researchers use the same mobile phase solvents, but their timedependent gradient elution methods are different, the compositions of the mobile phases may be different. Thus, using different stationary and mobile phases and different changes of the volumes of the eluents with time may yield different results for the compositions of the purple colorant among the various researchers.

#### 3.1.5. HPLC Detection Wavelength

When comparing the results of the HPLC analyses on a set of dyes, the same wavelength of detection of the dye component must be used throughout since different wavelengths will yield different absorptions of light in the UV or visible regions. The wavelength chosen is the one at which all of the investigated dyes have significant light absorptions; thus, these absorptions may be used for comparison purposes. The current standard of absorbance measurements for indigoids is 288 nm, which was first used by Wouters and Verhecken [9,10] and has been adopted by others. If the measured ability of each component to absorb light at a given wavelength is known—and this is known as absorptivity (a) or extinction coefficient (ε)—then the actual mass of each dye component can be ascertained. However, not all of the researchers of this purple pigment have used the same wavelength in their measurements; thus, initially, these values cannot be used for comparing one set of measurements at one wavelength with another set of results at a different wavelength. However, this apparent problem may be overcome by knowing the ratio of absorbances at the different wavelengths and then re-calculating the absorbance at the desired wavelength.

When measuring the absorbance of each indigoid, it may be best not to first measure its absorbance at the UV wavelength of choice (288 nm), but rather at the visible wavelength at which the dye in question has its maximum absorbance, λmax. Afterwards, knowing the ratio of absorbances at the standard UV wavelength and the visible wavelength, a calculation of the absorbance at 288 nm can be performed. This type of re-calculation was previously performed [17] and was also conducted in the current study. The reasoning for measuring at the visible wavelength first is that an archaeological sample has impurities from the ground in which it was excavated, and some of these non-colorants may absorb UV light at or approximately the standard UV wavelength. Thus, directly measuring the peak area at the time of the eluting dye at the standard UV wavelength, may partially mask its actual peak, thereby reducing its peak purity and possibly incorporating into it the absorbance of not only the dye, but also of the impurities, which would produce a false measurement.

#### **4. Results and Discussion**

#### *4.1. Modern and Archaeological Purples*

Considering all of the limitations and concerns about combining the analytical HPLC results from various analysts, as discussed above, it is, nevertheless, expected that all the analytical results would not deviate far from their true values in order to obtain an overall pattern for the dye components. The results of different researchers were also incorporated into the PCA studies mentioned above.

The following tables show the integrated peak area values as percentages for the five main indigoid and indirubinoid components that can be found in modern and archaeological pigments produced from the purple-producing sea snails. These data are all based on an effective extraction system and the HPLC methodology discussed above. A form of these tables was initially determined by Karapanagiotis et al. [15,16,21] and has now been expanded in the current work to include more samples and information.

The following sample descriptions appear in the tables below: the geographical region where the pigment or dye was produced; a description of the manner in which the colorant was obtained; the solvent used for sample preparation as well as the temperature and heating duration; the HPLC-produced integrated peak areas (as %) of the five main components evaluated at 288 nm; and the original literature reference that discusses these samples in detail. For completeness purposes, each colorant's DMI value, which is discussed below, is also shown in the tables.

Table 1 outlines the information regarding purple pigments produced from modern *H. trunculus* sea snails from different researchers, and each sample was given a "T" number in the tables that were previously established, as mentioned above [15,16,21]. Similarly, Table 2 shows the relevant data regarding modern *B. brandaris* and *S. haemastoma* pigments, and these samples are given "B" and "H" numbers, respectively.

Table 3 lists the various archaeological materials—pigments and textile dyes—that have been analyzed. These historic items span parts of the globe from the Aegean Sea to the Mediterranean and the Middle East to Siberia and England, and are dated from the 17th century BCE up to about only a century ago.

**Table 1.** Relative dye components (calculated at 288 nm as % peak areas via the HPLC-PDA detector) of modern *Hexaplex trunculus* colorants.

\* A question mark appears if the temperature and /or duration of heating were not reported in the publication. \*\* A blue color for the DMI value is indicative of an IND-rich *H. trunculus* pigment, and a red color represents a DBI-rich *H. trunculus* pigment; see text for the detailed discussions.

**Table 2.** Relative dye components (calculated at 288 nm as % peak areas via the HPLC-PDA detector) of modern *Bolinus brandaris* and *Stramonita haemastoma* colorants.

**Table 3.** Relative dye components (calculated at 288 nm as % peak areas via the HPLC-PDA detector) of archaeological colorants. All extractions performed in DMSO.

\* DMSO extraction at 80 ◦C for 15 min. \*\* DMSO extraction at 100 ◦C, 5 min. \*\*\* DMSO extraction at 150 ◦C, 5 min.

#### *4.2. Di-Mono Index*

In order to classify all the malacological pigments, and based on that categorization, be able to determine the zoological provenance of the purple pigment, a simple predictive index was developed. This indicator is known as the Di-Mono Index (DMI), and for each sample, it is the ratio of the peak area of DBI relative to that of MBI, measured at the standard 288 nm wavelength:

#### DMI = A(DBI)/A(MBI) @ 288 nm

The reason that IND, which is found in *H. trunculus* pigments, was not chosen for this property is because one cannot rule out the fact that the indigo may not have solely originated from a molluscan source but that a plant-based indigo was added. It is especially important to emphasize that there have been dyeings that were found to be double-dyed with a molluscan source and either madder, a red-scale insect, and, perhaps, even plant indigo.

In the three tables, the DMI values are also presented. From the values of Table 1, it could be discerned that there is a difference among *H. trunculus* snails, and that some pigments have very low DMI values and others have higher numbers. On the other hand, Table 2 shows that the non-*H. trunculus* snails have very high values.

Figure 4 shows the DMI values for modern pigments produced from *H. trunculus* obtained from different geographical regions (listed in Table 1). It is apparent that for *H. trunculus* snails, the IND-rich (i.e., DBI-poor) bluish or violet pigments have a DMI of up to about 0.6, whereas the DBI-rich reddish pigments have DMI values from about 0.8 to 9.

**Figure 4.** DMI values for modern *H. trunculus* from different geographical regions.

The DMI values for purple pigments from the non-*trunculus* Mediterranean snails—*B. brandaris* and *S. haemastoma*—are shown in Figure 5.

**Figure 5.** DMI values for modern *B. brandaris* (B) and *S. haemastoma* (H) from different geographical regions.

The DMI values shown in Figure 5 are very high, significantly higher than even the DBI-rich *H. trunculus* snails. However, as the *B. brandaris* and *S. haemastoma* share these very high DMI values, it is not possible to differentiate between the pigments from these two species. These elevated values can be simply explained by the special nature of the MBI component, whose properties have been studied [26]. The very high DMI values for non-*trunculus* snail pigments are due to their very low and even negligible quantities of MBI in their pigments, whereas, in general, all *trunculus*-based pigments—whether reddishor bluish-purple—have significant amounts of MBI [27]. From Figure 5 and Table 2, it is observed that when using the optimal extracting solvent (DMSO) for these analyses, the pigments from non-*trunculus* snails have DMI values greater than 40, which is nearly five times the highest value for the DBI-rich *trunculus* pigment with the highest DMI value.

Except for one special case of a historic century-old dyeing from Leeds (UK), which was dyed with non-*trunculus* snails off the African coast [25], all the archaeological samples reported in Table 3 have DMI values of less than 6. This is indicative of *H. trunculus* pigments as depicted in Figure 4. Thus, based on the DMI property alone, it could be inferred that the archaeological samples were produced from *H. trunculus* snails that are either IND-rich (with DMI < 0.6) or DBI-rich (DMI between 0.8–9). It is, of course, possible that both types of *H. trunculus* were mixed, or even that some non-*trunculus* snails were used as red-color additives. However, most probably, the *H. trunculus* was always used.

#### *4.3. Ternary Diagram*

The ternary diagram (also known as a triangular diagram) incorporating the results of the HPLC analyses on modern and archaeological samples is depicted in Figure 6.

The ternary diagram is an aesthetic visual method of presenting multicomponent data in a graphical manner. Ternary or triangular diagrams were first used in the mid-18th century [28] and have become a standard method of data display today for samples that contain at least three components or condensed into three. Numerous examples are found of the use of ternary diagrams in many areas of science related to archaeological or modern samples in, e.g., metallurgy and geology [29–31], physical chemistry and material science [32–34], and botany and zoology [35–37].

Though it is represented in two-dimensional space, the ternary diagram maintains the identities of all the main original dyes and their colors, blue (IND), violet (MBI), and reds.

In the triangular chart, each apex represents 100% of the corresponding dye, and the opposite base designates 0%. In the diagram, IND and MBI are denoted as such, and the third coordinate, labelled "reds", includes the sum of all the main dye components that yield reddish colors, specifically, DBI, DBIR, and INR. Thus, the apex at the top represents 100% IND, the apex at the bottom left designates 100% MBI, and the apex at the bottom right represents 100% "reds". All the various colors from the five main components that could be present in molluscan purple pigments are represented in this diagram, which maintains the individual original identities of the dyes.

The ternary diagram shows that the IND-rich *H. trunculus* pigments are clustered in the middle-to-upper left of the chart within a semi-oval (also known mathematically as a semi-stadium). This positioning is as expected for these indigo-rich and DBI-poor pigments. The chart also indicates that the DMI values for this semi-oval group is ≤0.6, as discussed in the tables above. The other variety, the DBI-rich *H. trunculus* species, are grouped at the bottom right within a shoe-shape arrangement, with DMI values ranging from 0.8–9. The other Muricidae sea snails, *B. brandaris* and *S. haemastoma*, are compressed to occupy a miniscule area at the extreme bottom right, as expected, for these pigments that are even more red than the DBI-rich *H. trunculus* species. Their DMI values, as also previously noted, are ≥ 14, and the optimal DMSO extractions > 40.

**Figure 6.** Ternary diagram showing the relative dye compositions (as % peak areas in the HPLC chromatogram at 288 nm) of pigments produced from modern and archaeological Muricidae sea snail species. The "reds" axis and apex represent the sum of all the main reddish components (DBI, DBIR, INR). The open red circles at the extreme bottom right represent modern *B. brandaris* and *S. haemastoma* purples; the closed circles represent modern *H. trunculus* pigments (blue for IND-rich and red for DBI-rich pigments). The squares and Xs represent various archaeological samples.

The ternary diagram shown in Figure 6 also includes the various archaeological purple pigments and dyes. With very few exceptions, all the archaeological colorants appear within the semi-oval or shoe-shaped areas, and no archaeological pigment is present in the small shape to the right of the tip of the "shoe", which is inhabited by the *B. brandaris* and *S. haemastoma* pigments. This indicates that all archaeological molluscan purples were probably produced from *H. trunculus* species, either the IND-rich or DBI-rich varieties. The red-only non-*H. trunculus* snails may have been added to the DBI-rich *H. trunculus* pigments to produce even redder dyeings. It is important to mention that the archaeological pigments represented by the points present at the borders between the semi-oval and the shoe shape could have been produced from a combination of the two varieties of *H. trunculus* snails.

It is important to note how the two statistical formulations presented in this paper complement each other to determine the zoological provenance of a molluscan pigment. For example, at the tip of the "shoe" in the ternary diagram lies pigment T9 (a DBI-rich *trunculus*), which borders B2 (*brandaris*); thus, it seems that they are nearly clustered together. However, their DMI values are significantly different: the *trunculus* pigment's DMI value is about 9 and the *brandaris* is 14.

#### **5. Conclusions**

The presence of any brominated dye in a colorant automatically indicates that the source of this pigment or dye is malacological, since only sea snails can produce that kind of colorant. HPLC analyses of various modern and archaeological molluscan pigments have shown that *H. trunculus* snails are unique, different from all other purple-producing snails from all of the world's waters. The pigments from all purple-producing sea snails contain DBI—this is the common dye of all molluscan purple pigments. However, all non-*H. trunculus* mollusks produce mainly DBI; thus, their purple pigments are always reddish. The singularity of *H. trunculus* is that its pigments contain all three indigoids (as well as indirubinoids): DBI (as all do), MBI, and IND. This major point about the exclusivity of *H. trunculus* snails is that they alone contain a significant amount of MBI, which is what distinctly separates them from all other species, and is observed in their DMI values.

Further, it was found that within the *H. trunculus* species, there are two varieties—maybe even subspecies—where one type produces reddish-purple pigments and another produces bluish-purple (violet) pigments. The *H. trunculus* snails producing reddish-purple pigments are DBI-rich, more than IND; however, these pigments are not as reddish as the pigments from non-*H. trunculus* snails, which mostly have DBI in their pigment. The other variety of *H. trunculus* produces bluish-purple or violet pigments because of the overwhelming amount of IND in the pigment, and they are IND-rich. Both types of pigments are termed the generic "purple", which is not a pure color but a mixture of red and blue. A schematic summary of the differences among the sea snails is depicted in Figure 7. While the *H. trunculus* has probably always been used in Mediterranean pigments, the other snails were also employed, not alone but as color additives, if desired, to produce even redder dyeings than those obtained from the DBI-rich *H. trunculus*.

**Figure 7.** Relative compositions of reddish-purple and bluish-purple pigments produced from *H. trunculus*, and reddish-purple pigments from all other mollusks. In addition, a Roman Period Royal Purple textile (the biblical *Argaman* color) from King Herod (1st c. BCE) and 1st c. BCE—1st c. CE violet, bluish-purple yarns (dyed to the biblical *Tekhelet* color) are also shown, both excavated at Masada in the Judean Desert, Israel.

The uniqueness of the current research is that to date, the characterization of the three color components (reddish, violet, and bluish) of molluscan purple pigments by means of a ternary diagram has not been applied. This is one of the major innovative themes of the study. This type of classification should therefore become an accepted standard for characterizing the various color components of such molluscan colorants in order to enable quantitative comparisons among them by using a more visual—and aesthetic—statistical formulation for these samples. The current study has shown that by combining the DMI values with the ternary diagram, the malacological identities of molluscan purple pigments can be determined.

**Funding:** This research received no external funding.

**Acknowledgments:** The author is most grateful for the support given to the research on natural dyes by the Sidney and Mildred Edelstein Foundation.

**Conflicts of Interest:** The author declares no conflict of interest.

#### **References**

*Humanities and Natural Sciences Interwoven for our Understanding of Textiles*; Interdisciplinary Contributions to Archaeology; Ulanowska, A., Grömer, K., Vanden Berghe, I., Öhrman, M., Eds.; Springer: Cham, Switzerland, 2022; Chapter 3; pp. 39–52.

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## *Article* **Thermochromicity in Wool Dyed with 6-Bromoindigo Depends on the Presence and Identity of a Solvent**

**Keith Ramig 1,\*, Timone Eskaros 1, Tazrian Islam 1, Olga Lavinda 1, Sasan Karimi 2, Lou Massa 3 and Christopher Cooksey 4**

**Abstract:** The thermochromic effect of wool dyed with 6-bromoindigo was found to depend on both heat and a solvent. The dyed fabric must be immersed in a solvent while heating for a color change from purple to blue to occur. Ethanol was the most effective solvent in causing the color change. Water was effective as well. 1-Butanol caused a slight color change, while toluene was completely ineffective. These results are interpreted as interaction of the solvent with both the wool surface and adsorbed dye molecular aggregates, causing changes in the size of large red aggregates to smaller blue ones. The mildest conditions for the color change, immersion in water at 60 ◦C, are so easily obtained that it is possible that ancient dyers knew of this as a finishing process for their dyeing, or knew to avoid post-dyeing heat treatment.

**Keywords:** 6-bromoindigo; thermochromic; dyeing; wool; Tyrian purple

#### **1. Introduction**

Shellfish purple (also known as Tyrian purple) is a pigment that is produced from the hypobranchial glands of a variety of shellfish when exposed to sunlight. The precursor in the gland is tyriverdin, which is yellow; as it is converted to the dark-purple 6,6- dibromoindigo (DBI), the gland fluid undergoes a striking variety of color changes [1]. The color changes were first observed in detail by Mr. William Cole in 1685, when he studied the hypobranchial gland fluid from the dog whelk *Nucella lapillus* when spread onto linen or silk. The initial yellow color changes to light green, then deep green, sea-green, Watchet blue, purplish red and finally deep purple red, "beyond which the sun can do no more" [2]. The similar color changes which take place using the Mediterranean species *Hexaplex trunculus* (*Murex trunculus*) or *Bolinus brandaris* (*Murex brandaris*) were illustrated by A. and G. de Negri in 1876 and reproduced by Cooksey and Sinclair [3]. In contrast to this riot of colors, dilute solutions in dimethyl sulfoxide of the major components of shellfish purple, indigo (IND), 6-bromoindigo (MBI) and DBI (Scheme 1), are all blue [4]. Reflectance spectra of wool dyed with IND, MBI and DBI show a wide diversity. Indigo shows a maximum absorption at 650 nm and 6,6- -dibromoindigo at 527 nm. 6-Bromoindigo in low concentration is similar to indigo, but at higher concentration, moves towards the maximum of 6,6- -dibromoindigo [5]. The study of 6,6- -dibromoindigo was extended to cover the dyeing of six fabrics [3]. The shades of purple obtained are shown in Table 1. The result was illustrated in Plate 14.4 of reference [3].

**Citation:** Ramig, K.; Eskaros, T.; Islam, T.; Lavinda, O.; Karimi, S.; Massa, L.; Cooksey, C. Thermochromicity in Wool Dyed with 6-Bromoindigo Depends on the Presence and Identity of a Solvent. *Heritage* **2023**, *6*, 672–680. https:// doi.org/10.3390/heritage6010036

Academic Editors: Marei Hacke, Sara Norrehed, Joanne Dyer, Jo Kirby, Art Proaño Gaibor, Ilaria Degano, Zvi Koren and Sandström Edith

Received: 8 December 2022 Revised: 13 January 2023 Accepted: 13 January 2023 Published: 15 January 2023

**Copyright:** © 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

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**Scheme 1.** Structures of IND, MBI and DBI. Additionally, reduction of the colored oxidized form of MBI by sodium dithionite followed by neutralization by ammonium chloride, giving its reduced colorless *leuco* form, and oxidation of the *leuco* form by aerial oxygen back to the colored oxidized form.

**Table 1.** Shades of purple obtained from dyeing of fabrics with DBI [3].

More extensive studies were later undertaken using 13 fabrics [6,7]. It was first reported by Ziderman in 2003 that wool dyed with MBI, originally blue–violet in color, turns blue upon heating at 60 ◦C in water [8]. These conditions are so simple to obtain that ancient dyers may have used this as a post-dyeing treatment, or they may have avoided such treatment, since it would change a rare and costly purple garment into a more commonplace blue one. There are many potential causes for this thermochromic effect, one of which is a chemical change in MBI: MBI could be thermally decomposing through the loss of a bromine atom, resulting in the production of IND. IND is known to dye wool blue. Although the reduced colorless form—the so-called *leuco* form (Scheme 1)—of MBI is known to lose its bromine atom upon exposure to light [9–11], the oxidized colored form which ends up on the fabric is not known to photochemically or thermally decompose. However, it remained to test for thermal decomposition by extracting the dye from heated MBI-dyed fabric, and analyze the MBI for presence of IND. Another possible cause for the thermochromic effect is a physical change in MBI. The color of a substance can depend on

its crystal structure, and heat can cause a change to this crystal morphology. A complicating factor is that dye molecules may interact with a fabric via several modes of interaction, such as hydrogen bonding, dipole–dipole interactions and van der Waals forces. This makes it difficult to model and, thus, predict the final color of a dyeing. For example, MBI is a blue–violet color in bulk form, but may dye fabrics a wide variety of shades of blue and purple [7].Therefore, it is possible that heat could affect bulk MBI differently than MBI adsorbed onto a fabric.

#### *1.1. Effect of Heat on Both Wool-Adsorbed and Crystalline MBI*

The theory that MBI adsorbed onto wool loses its bromine atom to become IND, and thus causes the wool to turn blue, was put to rest by an extraction experiment. Wool dyed with MBI was heated in boiling water, and the resulting blue fabric was extracted with a hot solvent to remove the dye from the wool. Analysis with high-performance liquid chromatography (HPLC) showed negligible conversion of MBI to IND [12]. Thus, the color change is not due to a chemical change in the dye molecule.

It has been reported that crystalline MBI undergoes a change in both physical form and color upon heating to the vaporization point, and after deposition of the vapor [8]. We reproduced those experiments, and found that MBI does not change color upon heating to its sublimation point, around 200 ◦C. Additionally, the vapor-deposition experiment was repeated. The crystals of MBI obtained did appear to the eye to be of a different crystalline form, but X-ray crystallographic analysis showed them to be identical with crystals obtained by recrystallization from a solvent [12,13]. In addition, a crystal obtained from recrystallization [13] did not change in color or in crystallographic properties upon heating to 77 ◦C [12], a temperature at which MBI-dyed wool rapidly turns blue in water. Thus, it was concluded that MBI, unadsorbed onto wool, does not undergo a heat-induced physical change that would affect its color.

Further experiments and analyses were based on the idea that a physical change to the fabric-adsorbed MBI was occurring. Additionally, IND and DBI were included in the experiments, because they are so closely related in structure to MBI.

#### *1.2. Divergent Behavior of MBI and DBI*

Not only did we successfully reproduce the thermochromic effect seen before for MBIdyed wool, but we also found that many other MBI-dyed fabrics show this same bluing effect upon heating in water [7]. When the thermochromic studies were extended to INDand DBI-dyed fabrics, our expectation was that they might show this same thermochromic effect, due to similarities in chemical structure between the three indigoids. However, it was found that a wide variety of IND-dyed fabrics showed little, if any, color change upon heating in water, while DBI-dyed fabrics turned distinctly more *red* in hue [7]. Figure 1 shows four wool fabric pieces, each dyed with MBI or DBI, plus the effect of heating the fabrics in water.

**Figure 1.** (Left to right) MBI-dyed wool heated in water, MBI-dyed wool with no heat application, DBI-dyed wool with no heat application, DBI-dyed wool heated in water [7].

#### *1.3. Reflectance Spectroscopic Analysis*

The color changes were studied qualitatively using reflectance spectroscopy. As has been seen before [5], we found that the reflectance spectra of MBI-dyed wool and DBI-dyed wool are remarkably similar in their spectral features; each has a reflectance minimum around 530 nm (which equates with perceived red color), and a shoulder on this main minimum around 620 nm (which equates with perceived blue color) (Figure 2) [7]. This means that both types of dyed wool appear to be a mix of blue and red, or similar shades of purple. After boiling in water for 10 min, the reflectance spectra change markedly: MBIdyed wool shows a loss of the "red" 530 nm minimum, and production of one main "blue" minimum at 620 nm. To the eye, the fabric is now true blue. Conversely, the DBI-dyed wool loses its "blue" shoulder at 620 nm, and is left with only a "red" minimum at 520 nm. To the eye, the fabric is now very red. On the other hand, the spectrum of IND-dyed wool does not change much after heating in water.

**Figure 2.** Reflectance spectra of wool dyed with IND, MBI or DBI, showing the effect of heating in water.

#### *1.4. Colorimetric Analysis*

The color changes were analyzed a quantitative basis using colorimetric analysis [14]. The shade of MBI- and DBI-dyed wool is determined by the red and blue hue components. For MBI-dyed wool, the blue and red components are of approximately equal magnitude, while for DBI-dyed wool of a similar dye loading, the red component is significantly greater than the blue. The consequence is that MBI-dyed wool can be considered truly purple in color, while DBI-dyed wool is a more reddish purple. Colorimetric analysis of the dyed fabrics after heating in water shows rearrangement of blue and red hue components: for MBI-dyed wool, the magnitude of the red component decreases markedly, and the blue component drops very little, while for DBI-dyed wool, the red hue component rises and the blue component drops [12].

#### *1.5. Generality of the Thermochromic Effect*