*3.3. Historical Textile Samples*

The aforementioned spectral features and behaviors of both of brazilwood's main colorant components, and the marker component, urolithin C, were used to guide the interpretation of the SERS spectra obtained from micro-samples of historic brazilwooddyed linen, silk and wool textiles.

In order to provide a validation of the results obtained through SERS, the historical textile samples had previously been investigated by chromatographic methods (HPLC-DAD) and were all known to contain urolithin C. In some samples additional colorants, namely flavonoids, indigoids and tannins, could be identified. All HPLC results are summarized in Table 3 and are described in more detail below in relation to the SERS results. **Table 3.** Description of historical samples with main components identified by HPLC-DAD and SERS (\* = minor species not detected by DAD but confirmed by tandem mass spectrometric detection).


In the chromatogram (insert Figure 4a) of the extract of the brown wool sample S#5, shown at 275 nm, the peak for urolithin C could be detected together with very low quantities of sappanol, a molecular marker of redwood-type dyes [14,31]. A component of tannin dyes but also a known degradation marker of both flavonoid compounds and protein-based threads, 4-hydroxybenzoic acid, was also detected [32].

S#5 was investigated by colloidal SERS directly on the sample, which was followed with hydrofluoric acid pre-treatment, the resulting spectra shown in Figure 4a(i,ii), respectively. The well-resolved spectrum of S#5 with direct colloidal SERS can be seen in Figure 4 ai where the bands associated with urolithin C (1530, 1340, 1167, 987 and 654 cm<sup>−</sup>1) can be observed. Following HF pre-treatment—and unlike the other samples described below there is no improvement in the quality of the SERS response (Figure 4a(ii)), although weak bands associated with urolithin C can still be observed at 1530 and 660 cm−1. It is likely that in the spectra for this sample the 4-hydroxybenzoic acid identified by HPLC analysis can be observed. Although there is a partial overlap with the marker component, the bands at 1618, 1390, 1017 and 830 cm−<sup>1</sup> correspond to those reported in the literature for this molecule [33]. The results obtained from this sample confirm that urolithin C can be detected by SERS in samples where little or no brazilwood colorant components survive, even in samples containing high proportions of 4-hydroxybenzoic acid as is commonly observed in historical textile samples.

In the chromatogram of the extract of the orange silk sample S#11 (insert Figure 4b) shown at 275 nm, ellagic acid is the most intense peak, followed by urolithin C, fisetin and 4-hydroxybenzoic acid. This suggests that S#11 had been dyed with brazilwood and young fustic, consistent with previous studies [34] performed on silk core fibers of golden metal threads. Ellagic acid could have been added to the dye bath to improve the final color, used as a mordant or may have been co-extracted from the bark used when dyeing with young fustic.

In Figure 4b(i,ii), a noisy spectrum was obtained for the direct colloidal SERS where the bands related to the presence of the urolithin C marker component, namely 1530 and 660 cm−1, can be observed. Following acid pre-treatment of this sample, a much more defined spectrum is obtained (Figure 4b(ii)) and the signals identifying the urolithin C constituent can be better appreciated (1530, ~1330 and ~660 cm<sup>−</sup>1). The presence of strong bands in the lower wavenumber range attributed to the ring *ν*(CC) perhaps indicate a more planar orientation in relation to the colloidal surface. Further bands with reasonable intensity at 1605, 1470 and 1287 cm−<sup>1</sup> can be ascribed to the co-presence of ellagic acid in this sample [35], as highlighted by the HPLC analysis. The bands at 1567, 1370, 885 and 548 cm−<sup>1</sup> collectively provide information regarding the presence of flavonoid materials but do not permit determination of the specific dye source [25]. No bands exclusively pertaining to 4-hydroxybenzoic acid could be identified. This result highlights the selective nature of SERS when multi-components of varying solubilities and concentrations can interact with the Ag colloid, and where some components are likely preferentially adsorbed over others. In this case, implementing a further SERS extraction step with methanol/HCl could be of some benefit.

**Figure 4.** SERS spectra obtained (i) with regular colloidal interaction and (ii) following HF pretreatment respectively for samples: (**a**) wool S#5, (**b**) silk S#11; (**c**) linen S#27 and (**d**) silk S#9. The corresponding HPLC-DAD chromatograms of the extracts of the samples @275 nm are inserted with abbreviations: pHBA = 4-hydroxybenzoic acid; EA = ellagic acid; UC = urolithin C; LUT = luteolin; and SUL = sulfuretin and are available enlarged in supporting information S1.

In the chromatogram (insert Figure 4c) of the extract of the linen sample S#27 shown at 275 nm, the peak for urolithin C is the most intense, while several other dye materials are also present. These include minor components typical of brazilwood and logwood dyes (sappanol; hematein; brazilein; hematoxylin; and 4-hydroxybenzoic acid) [14,31]. This sample also contains ellagic acid, suggesting the co-presence of tannins, again either used to modify the color or as a mordant during dyeing [7].

For the linen sample S#27, acquisitions performed with the colloidal SERS directly on the sample and after the hydrofluoric acid pre-treatment gave rise to the spectra shown in Figure 4c(i,ii), respectively. By direct colloidal SERS, the main features—albeit quite noisy of the brazilwood marker urolithin C can be seen, with a band at 1530 cm−<sup>1</sup> accompanied by broad bands at 1589, 660 and 990 cm−<sup>1</sup> (very weak). The band at 1350 cm−<sup>1</sup> possibly indicates the co-presence of brazilin/brazilein or even haematin from logwood, a further neoflavonoid which would not have been taken into consideration had it not been for the HPLC results. Following HF pre-treatment, the bands at 1566, 1357 and 467 cm−<sup>1</sup> are slightly shifted from the characteristic bands reported here for brazilin/brazilein (shown in Figure 2), and the peak at 1377 cm−<sup>1</sup> attributed to brazilein cannot be observed. It is plausible to hypothesize the presence of logwood also given the similarity of the SERS spectral features [25]. A few further weak bands can be seen in Figure 4c(i,ii), namely those in the 1470–1490 cm−<sup>1</sup> range, and a band at 1270 cm−<sup>1</sup> seen only in Figure 4c(i). These bands cannot be specifically assigned, but most likely belong to the additional minor components, 4-hydroxybenzoic acid and ellagic acid, as identified by HPLC. In this sample the urolithin C component is readily available to interact with the colloid without any pretreatment steps and despite the confirmed presence of other coloring components. Once these other components were subsequently liberated from the mordant/textile complex following acid hydrolysis, they can be preferentially observed and distinguished. This underlines the necessity of the two-step process for a fuller sample characterization.

In the chromatogram of the extract of the deeper orange silk sample S#9 (insert Figure 4d) shown at 275 nm, urolithin C is the most intense peak. In addition, sappanol and 4-hydroxybenzoic acid are identified, as are sulfuretin, a sulfuretin-glucoside and fisetin, all ascribable to the use of young fustic [36]. Luteolin was also observed, suggesting the co-presence of a second yellow flavonoid dye. The presence of indigotin suggests that indigo or woad was also used to obtain the desired hue.

The SERS spectra obtained from S#9 can be observed in Figure 4d(i,ii) for direct colloidal SERS and after HF pre-treatment, respectively. Interestingly, in both spectra the main bands of urolithin C (1530, ~1335 and ~660 cm−1) can be observed. In this case, the acid pre-treatment does not enhance the quality of the spectra. Given the delicate nature of the substrate and acid hydrolysis conditions, it is worth nothing that no bands relating to the protein content of the silk matrix are observed by SERS [37]. Furthermore, no bands attributable to brazilein are apparent either. Beyond the marker component, a number of other weaker bands can be observed relating to the flavonoid content in the sample highlighted by HPLC analysis. Specifically, in Figure 4d(i), the bands at 1573 and 1240 cm−<sup>1</sup> relate to luteolin, the band at 844 cm−<sup>1</sup> is suggestive of components with a flavonoid structure, while those at 587 and 620 cm−<sup>1</sup> are features which correlate with the suggested fustic content [26]. HPLC analysis also indicates the presence of indigo, which could be observed instead by conventional Raman pre-SERS (spectrum not shown). The identification of the marker component is apparent in this sample in the presence of flavonoid and indigoid dyes and is evidence of the use of brazilwood even when the brazilwood colorants have not survived.
