The results will be organised showing the results of the technical photography, then discussing the organic dyes first, followed by XRF mapping and compression testing in relation to the identified dyes. The organic dyes section will be subdivided in relation to the colour of the fibre. A complete summary of all the results is reported in Table 1.
3.2 Analysis of the organic dyes
3.2.1 White threads
The white threads (samples MNAAHP-003, MNAAHP-006a, PAR-011 and PAR-026) were analysed by HPLC-DAD and the chromatograms extracted at all the channels in which red, yellow, green, blue and dark fibres may absorb (450 nm, max abs 300–400 nm and max abs 550–650 nm) are shown in Figure S1a-c. (Supplementary Materials). All chromatograms (Figure S1a-c) are flat allowing us to conclude that the white cotton yarn (sample PAR-026) and the camelid threads (samples MNAAHP-003, MNAAHP-006a and PAR-011) are undyed. LC-HRMS confirmed the lack of dyes above detection limit in all the white samples.
3.2.2 Red threads
The HPLC-DAD chromatograms of all the red fibres of the set (samples MNAAHP-001, MNAAHP-007, MNAAHP-009, PAR-007, PAR-009, PAR-019, PAR-021 and PAR-025), extracted at 450 nm to enhance the S/N ratio for the peaks due to the anthraquinone red compounds, are reported in Fig. 4a.
All the HPLC-DAD chromatograms (Fig. 4a) show the same qualitative profile despite the different peak intensities given by the different absolute concentration of the extracts. Pseudopurpurin (14.9 min), munjistin (15.3 min) and purpurin (20.5 min) were identified thanks to the comparison with reference materials, analytical standards, and based on UV-Vis spectra reported in literature (21), while the UV-Vis spectrum of the unidentified peak at 11.5 min featured a maximum of absorbance at 490 nm and the typical shape of anthraquinones. The high sensitivity and selectivity of HRMS allowed us to unequivocally identify and confirm the presence of several anthraquinones thanks both to the exact mass values acquisition and the comparison with the tandem mass spectra of known compounds (13, 21): pseudopurpurin (m/z = 299.019), munjistin (m/z = 283.002), lucidin (m/z = 269.043), xantopurpurin (m/z = 239.031), purpurin (m/z = 255.029), rubiadin (m/z = 253.051) and nordamnacanhtal (m/z = 267.035; in traces). The HPLC-ESI-Q-ToF Extract Ion Chromatogram (EIC) of sample MNAAHP-009 is shown as representative (Fig. 4b). Unfortunately, no peaks ascribable to the unidentified anthraquinone detected by HPLC-DAD were found in the Total Ion Chromatogram collected by HPLC-ESI-Q-ToF, possibly to its low-ionization.
The reported composition suggests that the dye source employed belonged to Relbunium vegetal species. The comparison of the obtained profiles with those of some Relbunium species employed for dyeing alpaca and sheep fibres from the Saltzman collection (Relbunium hypocarpium, Relbunium ciliatum, Relbunium-unknown species), already characterized in the literature (13, 18) allowed us to conclude that the most plausible vegetal source for the three samples is Relbunium hypocarpium. The small differences in composition could be due to the use of a mixture of different Relbunium species, different dyeing processes or degradation of the textiles.
3.2.3 Blue threads
The HPLC-DAD profiles of the three blue fibres of the collection (sample MNAAHP-004, MNAAHP-010 and PAR-024) feature two peaks ascribable to indigoid compounds: indigotin (20.4 min) and indirubin (21.6 min) (Fig. 5). The analysis performed with HPLC-HRMS confirmed these attributions and revealed the presence of isatin (m/z = 146.023), indigotin precursor, in MNAAHP-004. These indigoids are the molecular markers for the identification of Indigofera o Isatis vegetal species (22).
The indigotin/indirubin ratio (in this case, the ratio between the DAD peak area of indigotin and indirubin (AIng/AInr) cannot provide a criterium to unambiguously identify the specific indigo plants used, given that the quantity of indigoid dyes depends on extraction procedures and dyeing recipes (23), but can highlight differences or similarities amongst sample sets. First, all the samples analysed show a proportion of indirubin to indigotin higher than that of any European Indigofera species (22). This is in accordance with previous evidences collected from the analysis of some pre-Hispanic Andean cotton blue fabrics (24). A plausible explanation for this difference may consist in a South American vat dyeing technology which favoured the formation and uptake of indirubin by the yarn.
Moreover, while in sample PAR-024 and MNAAHP-010 the concentration of indigotin is three times higher than that indirubin (AIng/AInr 3.2 and 3.1, respectively), in sample MNAAHP-004 it is nearly the same (1.2). This might suggest the use of a different recipe or a different dye source.
3.2.4 Green and yellow threads
The yellow fibres (sample MNAAHP-008, PAR-001, PAR-003 and PAR-005) and the green ones (PAR-036 and PAR-037) will be discussed together, since green hues were usually obtained by consecutively applying yellow and blue dyes. The HPLC-DAD chromatograms of the samples are shown at max absorbance of 300–400 nm (yellow) and 500–600 nm (blue) and reported in Figure S2a-b (Supplementary Materials). The two green samples (PAR-037 and PAR-036) both contain indigotin and indirubin. Indigotin was also detected in MNAAHP-008, along with three unknown blue compounds (b1 − 3) in the 19.7–22.6 min time range, whose UV-Vis spectra do not allow a straightforward interpretation (Figure S2d, Supplementary Materials). The presence of indigotin and other blue components in this sample might be due to a contamination of the fibre from adjacent threads. Nevertheless, a different source of blue in addition to Indigofera or Isatis species was used in this case.
With regard to the yellow components, all the HPLC-DAD chromatograms show very small peaks, with the exception of those in the 18.6–21.8 min time range (g1 − 5) in the green sample PAR-036, whose UV-Vis spectra are typical of yellow flavonoid compounds (Figure S2c, Supplementary Materials). High Resolution MS allowed us to identify these yellow flavonoids as quercetin (m/z = 447.070), and methyl-quercetin (m/z = 315.049). The comparison between the profiles of reference alpaca and sheep yellow dyed fibres from the Saltzman collection materials (Baccharis floribunda, Kageneckia lanceolata, Hypericum larcifolium) and that of sample PAR-036 allowed us to exclude that any of these three quercetin containing species was used for the Paracas fibres under study (13, 14).
Besides blue indigoids (indigotin and indirubin, both m/z = 261.064) and yellow flavonoids, red anthraquinones were also detected (pseudopurpurin, m/z = 299.019, xantopurpurin, m/z = 239.03, purpurin, m/z = 255.029), as shown in the EIC chromatograms (Fig. 6a), whose profile suggests that the source of red is Relbunium. Evidences of textiles from Paracas Necrópolis dyed with a mixture of yellow and blue vegetal source to yield green nuances or with red (Relbunium species) and blue for the violet hues have already been mentioned in the literature (17), but no previous report is available on any yellow-blue-red recipes for obtaining green.
With regards to the other green sample PAR-037, the yellow component was neither detected by HPLC-DAD nor by High Resolution Mass spectrometry.
The application of HPLC-HRMS allowed us to characterize in detail the source used in the yellow fibres. Several flavonoids were unequivocally identified in samples PAR-001, PAR-003 and PAR-005 (the EIC chromatograms of yellow sample PAR-001 are presented in Fig. 6b): luteolin 7-O-glucoside (m/z = 447.070), okanin glucoside (m/z = 449.105), okanin (m/z = 287.030), luteolin (m/z = 285.030) and butein (m/z = 271.057) (25, 26). The positive matching with the profile of the extracts of Cosmos sulphureus petals, allowed us to suggest that this is the raw source used for dyeing (25). The use of Cosmos sulphureus for dying in yellowish-orangish hues is reported in the literature (27, 28) but, to be the best of our knowledge, it is the first time that it has been identified in ancient textiles.
Finally, the extract of the yellow sample MNAAHP-008 contains luteolin-7-O-glucoside, apigenin-7-O-glucoside, chrysoeriol-7-O-glucoside, luteolin, apigenin, and chrysoeriol. This profile is typical of Reseda luteola, one of the most used yellow dyes in the Old World, and matches with the so-called “[LUTE-APIG] group” described by Wouters and Rosario-Chirinos (17). More specifically, the profile is extremely similar to that provided in (29) for the extract of the leaves of the South American Salix Humboldtiana Wild. At present, only a tentative attribution can be made, since Antúnez de Mayolo identified fifteen Andean plant species as sources of yellow dyes (30), and among them Zumbhul (31) described three plants as luteolin-containing species: Alnus jorulensis, Baccharis genistelloides, and Bidens andicola, whose chromatographic profiles are not available in the literature yet.
3.2.5 Brown, black and grey threads
The HPLC-DAD profiles of all the brown (sample MNAAHP-002, MNAAHP-005, PAR-008 e PAR-023), black (sample PAR-020 and PAR-022) and grey (MNAAHP-006b) samples (Figure S3, Supplementary Materials) are rather flat. In MNAAHP-002 extract several peaks were detected and assigned to flavonoid compounds: luteolin-7-O-glucoside (10.7 min), apigenin-7-O-glucoside (12.2 min), chrysoeriol-7-O-glucoside (12.6 min), luteolin (14.8 min), apigenin (16.3 min) and chrysoeriol (16.7 min). The analysis by HR-LC-MS confirmed the attribution of these peaks for sample MNAAHP-002, detected the further presence of indigotin and indirubin in the extract, and highlighted the same composition for the black fibres of PAR-022 and PAR-020 and grey MNAAHP-006b. In particular, the EIC profiles (the EICs of PAR-022 are provided in Fig. 7) featured: luteolin-7-O-glucoside (m/z = 447.070), apigenin-7-O-glucoside (m/z = 431.072), chrysoeriol-7-O-glucoside (m/z = 461.103), luteolin (m/z = 285.030), apigenin (m/z = 209.038) and chrysoeriol (m/z = 299.045) (32–34), in addition to indigotin and indirubin (m/z = 261.064).
The presence of indigoids accounted for the dark coloration of the threads. Due to the lack of reference materials with comparable profiles, the raw material used can only be assigned to the “[LUTE-APIG] group” mentioned above for sample MNAAHP-008. Interestingly, all the textiles which contained “[LUTE-APIG] group” molecular markers, also contained indigotin and indirubin, thus suggesting that this dye source was preferably used in combination with an indigoid dye.
In the grey sample MNAAHP-006b only luteolin was detected, thus no clear attribution can be inferred.
Finally, the brown samples (PAR-008, PAR-023 and MNAAHP-005) do not contain any dyes above detection limit; thus, their coloration is likely provided by the natural colour of the raw cotton fibres.
3.3 Analysis of the inorganic components
In µXRF mapping every pixel corresponds to a spectrum; from the data cube is thus possible to visualize a separate grayscale map for each element detected, where the brightest areas correspond to the highest peak areas, while the black represents the lowest values or absence of the peak, Fig. 8.
In order to be able to tabulate and compare data from the generated maps, the peak intensities were visually sorted into three or four categories from brightest (white) to darkest (black) with one or two shades of grey in between, see Table 1. The data were interpreted crosschecking the results with the detected dyestuffs, in order to assess whether additives, auxiliaries or mordants could be identified, bearing in mind that the most common mordant, alum (an Al salt), could not be detected. Moreover, we aimed at comparing the results obtained on single threads with those achieved by micro-XRF elemental maps of textile fragments in a previous study (3).
The following observations can be drawn from the comparison amongst the samples set:
as expected, sulphur was only detected in the camelid fibres (white or grey categories), as it is due to the sulphur containing proteins in keratin;
chlorine also appeared to be generally higher in proteinaceous fibres, although the highest chlorine levels in the entire map are associated to one cellulosic black thread (PAR-020). It is important to note that the chlorine peak appears as a shoulder on the sulphur peak, so its presence in the proteinaceous fibres (rich in sulphur) may be an artefact;
potassium was also relatively high on most colours apart from undyed white cotton; the two highest potassium levels were recorded on yellow threads and could indicate the use of a dyeing auxiliary, even if the organic yellow dyes are different (MNAAHP-008 was dyed with Indigofera, a flavonoid dye of the [lut-apig] group and an unknown blue, while PAR-005 with Cosmos sulphureus);
calcium amount was highest in two black cotton threads that were shown to be dyed with Indigofera plus a yellow dye, but was present in every thread with the lowest levels recorded for most red threads and the undyed cotton (marked as dark grey in Table 1);
iron was present in every sample but generally was the lowest in undyed and blue threads; it was highest in the red cotton thread PAR-025 dyed with Relbunium and in a grey camelid thread (MNAAHP-006b) that contained an unknown luteolin-based yellow dye. This may imply that iron was a mordant for the red and yellow dyes, also when used with indigo for green, brown and black threads;
copper was barely detectable above the background but nevertheless yielded a clear map with relevant signals in several red, yellow and one green camelid threads. In particular, three samples of yellow camelid embroidery, dyed with Cosmos sulphureous, all contained relatively high amounts of copper: this may suggest that copper was used as a mordant. This distribution is very consistent with the data obtained for the mapping of the whole textile fragment 1935.32.0211a, as presented in (3);
zinc was barely detectable, but the highest amounts can be clearly pinpointed on the two undyed white camelid threads, the two blue camelid threads and the grey camelid thread with an unknown luteolin-based yellow dye.
3.4. Compression tests
Compression test results are presented in Fig. 9. Compression tests for the cotton showed that the white fibres are mostly in reasonable condition while the brown and black fibres tend to have a wider distribution of conditions, with a higher number of weak or very fragile brown samples compared to the white samples. Of the eleven brown cotton threads tested, four were also analysed by XRF and HPLC-DAD-HRMS. Three of those, one from each category: flexible, weak, fragile, were most likely undyed, while the fourth one (weak) was dyed with a combination of Indigofera and a yellow dye.
Similarly, the undyed white or grey camelid yarns tend to still be flexible/strong whilst the red and possibly the black fibres tend to be in the worst conditions.
Samples from ammonia treated cotton and camelid textiles occur in every category suggesting that the treatment did not have a lasting, or any, effect, except perhaps for red camelid samples. Interestingly, the only red camelid samples that are not classified as very fragile are those that underwent the ammonia treatment. These samples come from different textiles, and do not vary in terms of organic dye or inorganic content. In detail, the dyes in seven of the nine red threads were analysed and all were identified as Relbunium species, and the inorganic components in the red dyed camelid samples were consistent. One sample was relatively high in copper (very fragile), a second one relatively high in iron (weak), while almost all were relatively low in calcium compared to the other colours. Notwithstanding this, the number of observed specimens is too low and the condition assessment method too subjective to conclusively state that the ammonia treatment had any preservative effect.