The results are organised showing first the results of the technical photography, then discussing the organic dyes, followed by XRF mapping and compression testing in relation to the identified dyes. The organic dyes section is subdivided according to the colours of the fibres. A complete summary of all the results is reported in Table 1.
3.1 Technical Photography
The technical photography revealed some UV fluorescence effects. Figure 3 shows the visible light photograph and the UV induced visible fluorescence image of all the samples analysed. The white camelid samples (PAR-011, MNAAHP-006a and MNAAHP-003) are the only ones showing UV induced visible fluorescence. All three were shown to be undyed by chromatographic analysis (see Paragraph 3.2.1 and Table 1). The natural fluorescence of wool is a known phenomenon [32]. None of the other images collected during technical photography showed recognisable photochemical effects.
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 featureless (no peaks) 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. HPLC-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 anthraquinone red compounds, are reported in Figure 4a.
All the HPLC-DAD chromatograms (Figure 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 the literature [33]. HRMS allowed us to unequivocally identify and confirm the presence of several anthraquinones thanks both to the exact mass values acquisition (as deprotonated molecule, [M-H]-) and the comparison with the tandem mass spectra of known compounds [17,33]: pseudopurpurin (m/z = 299.019), munjistin (m/z = 283.002), lucidin (m/z = 269.043), xanthopurpurin (m/z = 239.031), purpurin (m/z = 255.029), rubiadin (m/z = 253.051) and nordamnacanhtal (m/z = 267.035; in traces). A still unidentified peak, detected at 11.5 min, featured both the UV-Vis and product-ion mass spectrum typical of anthraquinones (Figure S2a-c, Supplementary Materials). The HPLC-ESI-Q-ToF extracted ion chromatogram (EIC) of sample MNAAHP-009 is shown as representative of the composition of all the read threads (Figure 4b).
The reported composition suggests that the dye source employed belonged to a Relbunium vegetal species [34]. 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 [17,18,24] and available as reference materials 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 distinct 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) (Figure 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 well-known molecular markers of various vegetal species [35], but in the Peruvian area the most common indigo-producing species are Indigofera suffruticosa and Cybistax antisyphilitica [34]. The profiles of the extracts from reference textiles dyed with these two botanical sources have not been investigated yet, thus they are both likely candidates for the blue Paracas samples [29,36,37]. The indigotin/indirubin ratio (in this case, the ratio between the DAD peak area of indigotin and indirubin (AIng/AInr) integrated at the maximum of absorbance in the 550-650 nm range) 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 [38], but can highlight differences or similarities amongst the three blue samples analysed. In samples PAR-024 and MNAAHP-010 the chromatographic peak area of indigotin is three times that of indirubin (AIng/AInr 3.2 and 3.1, respectively), while 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.
Finally, in general, all the samples show a relevant amount of indirubin, which is seldom detected in European Indigofera species [35]. This is in accordance with previous evidences collected from the analysis of some pre-Hispanic Andean cotton blue fabrics [39]. A plausible explanation for this difference may consist in a South American vat dyeing technology that favoured the formation and uptake of indirubin by the yarn.
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 S3a-b (Supplementary Materials). The two green samples (PAR-037 and PAR-036) both contain indigotin and indirubin. Indigotin was also detected in the yellow sample 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 but resemble those of synthetic blue dyes (Figure S3d, Supplementary Materials). No further information on these components was obtained by mass spectrometric detection in negative, nor in positive ionization mode. The presence of indigotin and other blue components in this sample might be due to a contamination of the yellow fibre from adjacent threads. This textile is supported on a modern blue cloth in storage and the most likely explanation is that some fibres from the support fabric were accidentally sampled along with the yellow thread MNAAHP-008.
With regard to the yellow components, the HPLC-DAD chromatograms relative to all the green and yellow threads 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 S3c, Supplementary Materials). High Resolution MS allowed us to identify these yellow flavonoids as quercetin (m/z = 301.033), 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 [17,18].
Besides blue indigoids (indigotin and indirubin, both m/z = 261.064) and yellow flavonoids, red anthraquinones were also detected (pseudopurpurin, m/z = 299.019, xanthopurpurin, m/z = 239.031, purpurin, m/z = 255.029), as shown in the EIC chromatograms (Figure 6a), whose profile suggests that the source of red is Relbunium. Evidence 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 [21], but no previous report is available on any yellow-blue-red recipes for obtaining green.
With regard to the other green sample PAR-037, the yellow component was not detected by either HPLC-DAD or by HPLC-HRMS, neither in positive nor in negative detection mode. Since indigotin and indirubin were detected in this sample (see above), different hypotheses may explain the final green colour. XRF analysis showed that this thread contains copper, which may be responsible for the green hue. Alternatively, the yellow compounds could have faded so severely that our techniques are unable to detect them (see for instance the reactivity of flavonoids as shown in [40]); or the fibre itself could possess a brownish-yellow colour, which combined with blue produces the final green (the pigmentation of South American camelids has already been described in [41]); or a specific recipe may have been used to yield a greenish hue with indigo-producing plants (as shown in [42]). It is interesting to note that the two green threads belong to the same textile, 1935.32.173, where an embroidered green ground now shows patches of different hues that were presumably once evenly coloured (Figure 1). PAR-036 was collected from the turquoise patches and PAR-037 from the more dominant olive-green ground.
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 Figure 6b): luteolin 7-O-glucoside (m/z = 447.070), okanin glucoside (m/z = 449.105), coreopsin (m/z = 433.113), okanin (m/z = 287.030), luteolin (m/z = 285.030) and butein (m/z = 271.057) [43,44]. Okanin and butein and their glucosides occur in several species, such as Bidens, native to South America [45], but in this case, the positive matching with the profile of the extracts of the petals of Cosmos sulphureus, performed in the same conditions, allowed us to suggest that the latter is the raw source used for dyeing [46]. The use of Cosmos sulphureus for dyeing in yellow-orange hues is reported in the literature [26,27] but, to the best of our knowledge, this 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 very similar to that 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 [21]. More specifically, the profile is also extremely similar to that provided in [36] 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 [29], and among them Zumbhul [47] 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 S4, Supplementary Materials) are rather featureless (no peaks). 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 HPLC-HRMS 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. In particular, the EIC profiles (the chromatograms of PAR-022 are provided in Figure 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 =269.038) and chrysoeriol (m/z = 299.045) [48–50], 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 additional flavonoid raw material used can only be assigned to the “[LUTE-APIG] group” mentioned above for sample MNAAHP-008. Interestingly, all the textiles that 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 of the raw material used can be inferred. The high content of iron revealed by XRF (see Section 3.3) suggests that it was used as a mordant, to provide the yarn with a grey colour, as generally occurring for iron-tannate dyes.
Finally, the brown samples (PAR-008, PAR-023 and MNAAHP-005) do not contain any dyes above detection limit. Dark hues are often provided by tannin dyes, but none of their most common molecular markers, such as gallic acid and ellagic acid, were detected [51]. Nevertheless, the use of high-molecular weight condensed tannins, hardly detectable by a liquid chromatographic approach, cannot be excluded. An alternative explanation is that the brown colour was provided by the natural pigmentation of the raw cotton fibres.
3.3 Analysis of the inorganic components
In µXRF mapping every pixel corresponds to a spectrum. Separate grayscale maps are generated for each element detected, where the brightest areas correspond to the highest peak areas, while black represents the lowest values or absence of the peak (Figure 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 by crosschecking the results with the detected dyestuffs, in order to assess whether additives or mordants could be identified. Moreover, the results obtained on single threads were compared with those achieved by micro-XRF elemental maps of textile fragments in a previous study [3]. It is important to bear in mind that the most common mordant, alum (an Al salt), could not be detected. Aluminium is at the lower atomic number limit of elements that can be detected by XRF in an air atmosphere. A small peak for aluminium was observed in the accumulated spectra of the maps collected from textile fragments [3]; however, the data maps showed no specific distribution of the relative levels of aluminium between the variously coloured threads. Nevertheless, it appears likely that aluminium mordants were used in the Paracas textiles, given that Peru has natural resources of alum salts [45].
The following observations were made from the comparison amongst the sample 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 in most colours apart from undyed white cotton. The two highest potassium levels were recorded in yellow threads and could indicate the use of a dyeing auxiliary, possibly an alkaliser in the form of wood ash. The organic dyes of the two yellow threads are different (MNAAHP-008 was dyed with a flavonoid dye of the [lut-apig] group, while PAR-005 was likely dyed with Cosmos sulphureus).
- Calcium was present in every thread, highest in two black cotton threads dyed with Indigofera or Cybistax plus a yellow dye, lowest in most red threads and in the undyed cotton samples (marked as dark grey in Table 1).Thus, calcium also appears to have been a dyeing auxiliary and its elevated presence in the threads heavily dyed with indigoids may indicate the use of a calcium source to alkalise the dyeing vat. Where available, burnt shells are commonly used to that end. In XRF maps of textile fragments [3], calcium was generally highest in the darker shades of brown and black.
- Iron was present in every sample but generally was the lowest in undyed and blue threads; 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. Iron 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. In the latter case, the grey colour might have been purposely obtained by using an iron mordant with a flavonoid dye. Alternatively, iron may have been present in the water used for dyeing; implying that a urine vat for the indigoid dyes could explain the lower levels of iron in the blue threads. Iron was also relatively low in the blue areas of mapped textile fragments [3], where generally iron was highest in some of the darkest black and brown shades but also in some quite light grey and yellow shades.
- Copper was barely detectable above the background but yielded a clear map with relevant signals in several red, yellow and one green camelid threads. In particular, three samples of yellow camelid embroidery, possibly dyed with Cosmos sulphureus, 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. This is also consistent XRF mapping of the textile fragments [3] where zinc was mostly detected in light-coloured areas of grey, white and beige. The use or function of zinc in these fibres remains unknown.
3.4. Compression tests
Compression tests results are presented in Figure 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 indigo-producing vegetal source 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 cotton 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.