Analytical strategies for dyestuffs - Eu-ARTECH

FOURTH YEAR ANNUAL REPORT – DELIVERABLE N. 1 

N1. Task 2a- Analytical strategies for natural dyestuffs in cultural 

heritage objects 

(ICN) 

Eu-ARTECH 

Access, Research and Technology for the conservation 

of the European Cultural Heritage 

Integrating Activity 

implemented as 

Integrated Infrastructure Initiative 

Contract number: RII3-CT-2004-506171 

Project Co-ordinator: Prof. Brunetto Giovanni Brunetti 

Reporting period: from June 1 st 2004 to May 31 st 2005 

Project funded by the European Community 

under the “Structuring the European Research Area” 

Specific ProgrammeResearch Infrastructures action

N1. Task 2a - Analytical strategies for natural dyestuffs in 

cultural heritage objects 

(Resp. ICN) 

The results obtained for each dyestuff on wool and silk and as a pigment are discussed briefly 

under the appropriate heading. Some general points should also be noted. First of all, it is 

important to be aware that the results obtainable by chromatographic methods of analysis, 

used by most of the participating laboratories, and by 3-dimensional fluorescence 

spectroscopy, used by only two (UNI-PG and KIK/IRPA) are very different in kind. 

Chromatographic methods require a small sample to be taken from the object under study. 3dimensional 

fluorescence spectroscopy has the advantage that it is non-invasive: no sampling 

is necessary and a degree of distinction between dyestuffs, or classes of dyestuff, can be 

made, but the detailed qualitative and quantitative information on the composition of the 

colouring matters present on the object, which chromatographic methods can provide, cannot 

be obtained by this technique. 

HPLC-PDA 

As high-performance liquid chromatography (HPLC) is a separation technique, the dyestuff 

constituents present in the sample of textile, paint or other material can be detected 

individually. This allows a more or less precise identification of the dyestuff to be made, 

based on the UV-visible (or other) characteristics of the individual components and their 

retention times: that is, on a characteristic pattern of constituents observable for that colouring 

matter. Identification can thus be made, even though the identities of some of the constituents 

of the colouring matter may be unknown, and quantitative estimates of the amounts of each 

constituent present can be obtained. 

Each of the participating laboratories has chosen chromatographic conditions – that is, 

eluents, gradients, column type and so on – in response to the requirements of the works of 

art, and thus the samples, usually studied there and the questions generally posed. The 

conditions commonly used for dyestuff analysis were originally applied to the examination of 

dyes on textiles, in particular anthraquinone dyes, like those from cochineal and madder, 1 but 

have subsequently been applied to the examination of other dyestuff classes and other types of 

art object. Improvements or modifications have been made according to the needs of the 

institution and as other column types or eluents have been tried and other aspects of the 

method varied, often following a published method. Thus, for example, the column used in all 

cases contains a reversed phase C18 packing of ODS2 type, but the precise packing and 

column type chosen, the particle size, column diameter, and thus the flow rate and time taken 

to complete the gradient used by each laboratory is different (for details see the protocols for 

each laboratory). Both KIK/IRPA and NMS use a water/ methanol/ phosphoric acid gradient; 

KIK/IRPA uses a 125 x 4 mm column (5 µm particle size packing), while that used by NMS 

1 J. Wouters, ‘High performance liquid chromatography of anthraquinones: analysis of plant and insect extracts 

and dyed textiles’, Studies in Conservation 30 (1985), pp. 119–28.

is 150 x 4.6 mm (same particle size packing, different make). 2 ICN follows a similar method 

except that a 150 x 2 mm column (3.2 µm particle size packing) is preferred. 3 GCI uses the 

same basic conditions, replacing the phosphoric acid with formic acid as phosphoric acid 

cannot be used for LC–MS; they use a 100 x 2 mm column (3 µm particle size packing). 

OADC follows a different published method in which a binary water/ acetonitrile eluent 

system is used, adding 0.1% trifluoroacetic acid to each of the eluents; they use a 250 x 3 mm 

column (5 µm particle size packing). 4 NGL uses a similar eluent system to OADC (with an 

addition of 5% methanol to the acetonitrile), but with a 150 x 0.5 mm column (5 µm particle 

size packing) and thus a slower flow rate and considerably longer run time than the other 

laboratories. 5 

Comparison of the results obtained by the participating laboratories shows that these different 

chromatographic conditions may affect the quality of chromatic separation of the components, 

but have only a minor influence on the analytical results themselves; the method of dyestuff 

extraction, however, has a significant effect. Most of the partners (and most laboratories 

working on the examination of natural dyes in the conservation field) use acid hydrolysis 

(HCl/methanol/water, 2:1:1) for the extraction of red and yellow dyestuffs, a procedure first 

developed in the 1980s. 6 The extraction is quite efficient and gives clear-cut, easily 

quantifiable results, but because O-glycosides and carboxylic acids are hydrolysed, 

information on the possible presence of such constituents on the textile or in the pigment may 

be lost. For example, pseudopurpurin (1,2,4-trihydroxyanthraquinone-3-carboxylic acid) and 

purpurin (1,2,4-trihydroxyanthraquinone) may both be present in madder dye, but as 

pseudopurpurin is converted to purpurin on acid hydrolysis, information on its possible 

presence is lost. 

One laboratory (NGL) uses the methylating agent boron trifluoride/methanol at a dilution of 

4% to extract the dyestuff. This method was devised to obtain a more efficient extraction of 

dyestuff from a lake pigment bound in paint medium than was usually possible with acid 

hydrolysis and was first used for thin-layer chromatography of anthraquinone and soluble 

redwood dyes in the mid-1970s, although not published 

until later. 7 The paint medium is broken up by the reagent, which also breaks up the lake 

pigment, releasing the dyestuff into the solution. The method has disadvantages: some 

methylation of susceptible groups (present in carboxylic acids, for example) takes place and 

the reaction is not quantitative (side reactions may occur). Extraction of the dye from textiles 

may be less efficient than the acid hydrolysis method. The chromatograms obtained are more 

complex than those obtained using acid hydrolysis and it is less easy to make quantitative 

estimates of constituents present. On the other hand, decarboxylation does not occur and, once 

allowance for any methylation has been made, the pattern of constituents observed reflects the 

actual composition of the colouring matter on the textile or in the pigment to a large extent, as 

information on glycosides and carboxylic acids is retained. As an added benefit, some 

2 J. Wouters and N. Rosario-Chirinos, ‘Dye analysis of pre-Columbian Peruvian textiles with high-performance 

liquid chromatography and diode-array detection’, Journal of the American Institute for Conservation 31 (1992), 

pp. 237–55. 

3 M.R. van Bommel, ‘The analysis of dyes with HPLC coupled to photodiode array and fluorescence detection’, 

Dyes in History and Archaeology 20 (2001), pp. 30–38. 

4 S.M. Halpine, ‘An improved dye and lake pigment analysis method for high-performance liquid 

chromatography and diode-array detector’, Studies in Conservation 41 (1996), pp. 76–94. 

5 J. Kirby, M. Spring and C. Higgitt, ‘The technology of eighteenth- and nineteenth-century red lake pigments’ 

National Gallery Technical Bulletin 28 (2007), pp. 69–87, see note 70, p. 86. 

6 Wouters 1985 (cited in note 1). 

7 J. Kirby and R. White, ‘The identification of red lake pigment dyestuffs and a discussion of their use’, National 

Gallery Technical Bulletin 17, 1996, pp. 56–80.

information on the technology of dyeing the textile or preparing the lake pigment may thus be 

gained. Pseudopurpurin, for example, is partly or fully converted to a methylated form, but it 

is clearly identifiable separately to any purpurin that that the dyestuff sample may contain. In 

practice it has been found that the two extraction methods complement each other very well. 

Other extraction methods that have been applied to samples of dyed textiles or pigments 

include the use of the complexing agent EDTA (ethylenediaminetetraacetate, disodium salt) 

with or without the aid of hydrochloric acid or other solvents, 8 formic acid 9 and hydrofluoric 

acid, 10 but only a very small-scale study of these was made (by ICN) as part of the pilot 

project on the weld dye (q.v.). Acetic acid is sometimes used for samples of dyed textile when 

the presence of an acid-sensitive dye is suspected (ICN). 

A comparison of the results obtained by each laboratory for any one dyestuff will also show 

that, allowing for the fact that some of the dyestuff constituents have not yet been identified at 

all, some laboratories were able to identify more constituents of the dyestuff than others. This 

reflects the fact that not all laboratories have access to detailed analytical information on the 

constituents (by mass spectrometric analysis, for example) or to samples of them as pure 

chemicals: many are not commercially available and those that can be purchased are often 

very expensive. From this point of view, the inter-laboratory comparison exercise has been 

very valuable for the laboratories taking part, as it helps each laboratory to identify some 

components on which it has no information by comparison of its results with those obtained 

by the other partners. 

All the laboratories use photo-diode array (PDA) detection; this enables a UV-visible 

spectrum of each constituent to be produced so that constituents can be identified by this 

means and by retention time. Some laboratories (OADC, GCI and NMS with the University 

of Edinburgh) are able to examine the components separated by HPLC by mass spectrometry, 

giving molecular information and the possibility of confirmation or identification of 

constituents. 

3-D fluorescence spectroscopy 

The non-destructive technique of 3-dimensional fluorescence spectroscopy as applied to the 

characterisation or identification of natural dyes is still relatively new. 11 The fluorescence 

characteristics of the dyestuff are observed at defined excitation and emission wavelengths, 

taking account of both emission and excitation spectra. It is also necessary to take account of 

possible fluorescence from the substrate itself (textile fibre, parchment, paper and so on); it 

was found, for example, that the results obtained from the same dyestuff on silk, on wool and 

8 E.J. Tiedemann and Y. Yang, ‘Fiber-safe extraction of red mordant dyes from hair fibers’, Journal of the 

American Institute for Conservation 34 (1995), pp. 195–206; X. Zhang and R.A. Laursen, ‘Development of mild 

extraction methods for the analysis of natural dyes in textiles of historical interest using LC-diode array detector- 

MS’, Analytical Chemistry 77 (2005), pp. 2022–5. 

9 Zhang and Laursen 2005 (cited in note 8). 

10 J. Sanyova and J. Reisse, ‘Development of a mild method for the extraction of anthraquinones from their 

aluminum complexes in madder lakes prior to HPLC analysis’, Journal of Cultural Heritage 7 (2006), pp. 229– 

35. 

11 S. Shimoyama and Y. Noda, ‘Non-destructive determination of natural dyestuffs used for ancient coloured 

cloths using a three-dimensional fluorescence spectrum technique’, Dyes in History and Archaeology 12 (1993), 

pp. 45–56; C. Clementi, C. Miliani, A. Romani and G. Favaro, ‘In situ fluorimetry: a powerful non-invasive 

diagnostic technique for natural dyes used in artefacts. Part I: Spectral characterization of orcein in solution, on 

silk and wool laboratory-standards and a fragment of Renaissance tapestry’, Spectrochimica Acta Part A 64 

(2006), pp. 906–12; C. Clementi, B. Doherty, P. L. Gentili, C. Miliani, A. Romani, B.G. Brunetti and A. 

Sgamellotti, ‘Vibrational and electronic properties of painting lakes’, Applied Physics A (2008), in press.

as a pigment differed slightly. Initially it was also found more difficult to obtain results from 

pigment powders due to the irregularity of the surfaces; this can be resolved by careful 

selection of filters, which also enhances reproducibility and specificity of the technique. 

Another approach is taken by KIK by using the 3 dimensional excitation-emission plot as a 

first screening plot to select the area(s) of fluorescence. In this phase, it is chosen to use 

always a systematically gradually change of excitation and emission filters depending on the 

excitation wavelength during the multi-scan, in order to minimise the risk of loosing 

fluorescence signals in unknown samples because of different filter selection. In a second 

step, one or more single line scans are done at the most suitable excitation wavelengths as 

detected out of the 3D-plot, using filter selection if necessary to optimise the emission profile. 

The pattern of fluorescence observed is for the dyestuff as a whole; the contributions of 

individual constituents cannot generally be monitored. Thus, as far as the red dyes were 

concerned, it is possible to distinguish between samples containing cochineal dyestuff (from a 

scale insect source) and the vegetable dyes, madder, safflower and sappanwood; it is more 

difficult to determine the source of the yellow dyes (dyer’s broom and weld), both of which 

contain luteolin among other constituents. It is possible to say, however, that the dye is of a 

flavonoid type. It is particularly interesting that it is possible identify the presence of 

chlorophyll A in the textiles dyed with dyer’s broom and weld (where these were examined) 

and the lakes with a calcium-containing substrate, but not in the lakes with a substrate of 

hydrated alumina; chlorophyll A was not identified by any of the laboratories using 

chromatographic systems.

Weld – the pilot project 

Weld was used for the pilot project, thus the dyeing of wool and silk, the preparation of the 

pigments and the subsequent analyses were treated in some depth so that the partner 

laboratories were able to assess and compare different methods of examination before 

extending the survey to the other dyes studied. The recipes used to prepare the textile and 

pigment samples using weld were subsequently used for dyer’s broom; these two plant dyes 

are very similar, both containing the flavonoids luteolin and apigenin as the principal 

colouring matters, and may present problems during analysis. 


Using an excitation wavelength of 366 nm, one emitting species with λmax 550 nm was 

detected by 3-D fluorescence spectroscopy in weld lakes 1 and 2; this emission is, in fact, 

independent of the excitation wavelength and is characteristic of flavonoids, according to the 

Perugia laboratory database. In addition, an emission at around 670 nm, with a weak shoulder 

at about 720 nm indicated the presence of chlorophyll A, identified by comparison with the 

emission, excitation and absorption spectra of chlorophyll A and B standards. Weld lake 3 

showed the flavonoid emission maximum at 525 nm and no chlorophyll could be detected. 

Weld lake 3 contains a form of amorphous hydrated alumina as substrate, whereas lakes 1 and 

2 contain a high proportion of calcium carbonate, with some calcium sulphate and a little 

amorphous hydrated alumina. 

Chlorophyll, with absorption and emission of fluorescence at much the same wavelengths as 

in the lakes, was also detected on the silks (a relatively narrow emission at about 675 nm with 

a shoulder at 725 nm) and to a lesser extent on the wool (about 670 nm; no shoulder visible). 

In the case of the flavonoid emission, the fluorescence maxima were at slightly lower values 

for the silk than for the wool: 520 nm for weld on wool, but 501 nm for weld on silk, both at 

an excitation wavelength of 360 nm. The addition of potash to the dye-bath caused a shift of 

the flavonoid emission to longer wavelengths: 552 nm in the case of the wool; the shift is 

rather greater in the case of wool than it is with silk, suggesting that the interaction between 

the dye and the fibre is somewhat different in the two textiles. In the case of the silk dyed in 

the presence of potash, more than one emitting species appeared to be present in the 510–540 

nm wavelength range. The textiles are a markedly different, brighter yellow colour when 

potash is present in the dye-bath. 

Although emission curves differ when using the second method (KIK), because of the 

measurement at other excitation wavelengths and the use of eventually other filters, the 

general outcome is in the same line. The shift towards longer wavelengths is found in the 

potash dye baths as well, while chlorophyll was found in the silks samples, however not in 

wool. 

HPLC-PDA 

All laboratories using acid hydrolysis to extract the dyestuff detected the presence of luteolin 

and apigenin on the textiles and in the lakes, together with many small components with UVvisible 

spectra similar to that of luteolin; the results obtained were very similar. The precise 

ratio of luteolin to apigenin present depended on the sample (95:5 to 86:13). A late-eluting 

component with a UV/visible spectrum very similar to that of luteolin was seen by all the

partners; NMS, with the University of Edinburgh, were able to identify this as a luteolin-7methyl 

ether by MS and NMR. KIK/IRPA reported the presence of a late-eluting component, 

λmax c. 420 nm, not observed in these samples by other participants. 

NGL, using boron trifluoride methanol as the extraction agent, detected several luteolin and 

apigenin glycosides in the pigments, eluting before luteolin, apigenin and the luteolin-7methyl 

ether. The approximate average proportions of glycoside to aglycone were 68:32 (lake 

1), 46:54 (lake 2) and 74:26 (lake 3); a sample taken from a boiled aqueous extract of weld, 

typical of that used for pigment preparation, contained 67% glycoside to 33% aglycone; these 

values will, of course, depend on whether or not any enzymatic breakdown of the glycosides 

takes place (if, for example, the plant matter is allowed to soak for any length of time) and 

various other factors dependent on the recipe used. The most important glycosides present 

were luteolin-7-glucoside and luteolin-3',7-diglucoside; apigenin-7-glycoside and several 

other unidentified luteolin- or apigenin-related glycosides were also detected. It is likely that 

some of the minor peaks observed in chromatograms after HCl hydrolysis are residual traces 

of these glycosides; if the sample is large and insufficient acid is used only partial hydrolysis 

takes place. 

NGL also found that the glycosides were present on the textiles, although there was not much 

luteolin-3',7-diglycoside. In the dyed silk samples, more glycoside was present if potash had 

been used in the dye-bath, about 49% glycoside to 51% aglycone; a marked amount of 

luteolin-7-glycoside was detected, as well as luteolin, apigenin and luteolin-7-methyl ether. In 

the absence of potash, far more aglycone was present on the textile: 28% glycoside by 

comparison with 72% aglycone. The pattern with wool was very similar, although the 

difference was less dramatic: 36% glycoside to 64% aglycone in the presence of potash, 25% 

glycoside to 75% aglycone when potash was absent. This may be explained by suppression of 

enzymatic hydrolysis of the glycosides or some influence on the take-up of dye by the textiles 

in alkaline conditions, but further investigation of the dye-baths is necessary before any 

conclusion can be reached. 

Of the other methods of extraction investigated by ICN, formic acid did not cause as much 

hydrolysis as the hydrochloric acid normally used, so that a mixture of glycosides and 

aglycones could be seen, but the extraction was very inefficient (33% or so). The complexing 

agent EDTA was even less effective, although quite good results were obtained from the weld 

samples at the Metropolitan Museum, New York using this reagent, revealing the presence of 

luteolin-7-glucoside on the textiles. 

Other methods of analysis 

Examination of the substrate for the lake pigments and the mordant for the textile fibres was 

carried out using the non-destructive method of X-ray fluorescence spectroscopy (XRF; UNI- 

PG); this method was able to identify the presence of calcium (Ca) and, in some cases, 

potassium (K), but the lighter element aluminium (Al) was not detected. Fourier transform 

infrared (FTIR) spectrometry and Raman spectroscopy were also carried out by UNI-PG, 

using a fibre optic system between the measuring head and the instrument; however, these 

were found to give more information on the textile itself than on the mordant. UV-VIS 

colorimetry was carried out at UNI-PG and visible colorimetry at NGL; this characterises the 

colour of the textiles or pigments in terms of, for example, the CIE (Commission

Internationale d’Eclairage) L*a*b* colour space system, where L* is lightness, a* a measure 

of redness–greenness and b* a measure of yellowness–blueness. Energy dispersive X-ray 

microanalysis in the scanning electron microscope (SEM–EDX), which requires a small 

sample, was carried out at ICN and NGL; Al, Ca, K and other trace elements present were 

identified. FTIR microscopy could also be carried out on small samples at NGL and ICN, if, 

for example, it was desired to obtain more information on the chemical nature of the substrate 

of the lake pigments.

Dyer’s broom 

The results should be compared with those obtained for weld as both dyes contain luteolin and 

apigenin; the important difference is the presence of the flavonoid genistein and its associated 

glycosides in dyer’s broom. The same recipes were used for dyeing and for lake pigment 

preparation, the only difference being that preparation of the pigment equivalent to weld lake 

2 (with a calcium/aluminium-containing substrate, very similar to that of weld lake 1) was 

omitted, hence there is no dyer’s broom lake 2. 


Dyer’s broom lake 1 showed an emission at 540 nm, a value independent of the excitation 

wavelength; by comparison with the Perugia laboratory database this is an emission 

characteristic of flavonoids. A weak emission at 659 nm with a shoulder at 715 nm is 

characteristic of chlorophyll A. Dyer’s broom lake 3, with a substrate of some variety of 

hydrated alumina, showed a flavonoid emission at 535 nm, but no chlorophyll could be 

detected. A similar pattern was seen with the weld lakes. 

A broad fluorescence emission at 479 nm on silk and 493 nm on wool at the excitation 

wavelength used, 360 nm, is characteristic of flavonoids, according to the Perugia laboratory 

database; as with weld, this emission is independent of the excitation wavelength. A band at 

673 nm (wool), 675 nm (silk) with a shoulder at 720 nm is characteristic of chlorophyll A, 

also as seen in weld; in the case of silk this band was sharp and quite marked. The use of 

potash in the dye-bath caused a shift in the flavonoid emission to 490 nm in the case of silk 

and 542 nm in the case of wool; this shift was greater in the case of wool, as seen with weld, 

and suggests the interaction between the dye and silk is different to that between the dye and 

wool. The chlorophyll emission was not affected by the presence of potash and the fact that 

the emission is higher on silk suggests the affinity of chlorophyll for the two fibre types is 

different; this is also seen with the weld dye. 

By the second FOFS method (KIK), chlorophyll is found again only in the silk samples. 

For both methods, the conclusion towards diagnostic outcome is the same: It is not possible to 

make a reliable distinction between the very similar dyestuffs extracted from dyer’s broom 

and weld by this technique. 

HPLC-PDA 

All the partner laboratories were able to identify the dyestuff from the presence of luteolin and 

genistein in all the lake and textile samples; apigenin was also identified in all samples. As 

several published sources suggest that apigenin is absent from this dyestuff, this is an 

important result. A luteolin-like component eluting after apigenin is likely to be a luteolin 

methyl ether (NMS/University of Edinburgh), as in weld, although not necessarily the same 

one, and there may be more than one. KIK/IRPA reported the presence of a late-eluting 

component, λmax 419 nm, similar to that observed in weld; this was not observed in these

samples by other participants and it is possible that this is due to differences in column 

performance between the systems used by the partners. 

All partners using acid hydrolysis to extract the dye observed small amounts of luteolin- and 

genistein-like constituents, probably traces of the glycosides observed by NGL using boron 

trifluoride/methanol. The glycosides detected by NGL included luteolin-7-glycoside with a 

similar retention time and an identical UV-VIS spectrum to that seen in weld, but with a 

different sugar attached, genistin, the principal glycoside of genistein and an apigenin-7glycoside. 

Genistin and the luteolin-7-glycoside overlap in the analytical system used at NGL, 

but it is easy to distinguish them (and also luteolin and genistein if this is necessary) by 

comparing chromatograms run at 254 nm and 330 nm: genistin and genistein are barely 

visible at 330 nm. In the lake pigments, further glycosides of genistein, luteolin and apigenin 

were detected, with a glycoside content of about 44% in lake 1 and 40% in lake 3. Little, if 

any, glycoside equivalent to the luteolin-3',7-diglucoside present in weld was detected in any 

of the samples; this may be a method to distinguish between the two dyestuffs. More luteolin 

was found to be present on both wool and silk dyed in the presence of potash than in that dyed 

without, but in all cases genistein and luteolin-7-glycoside were substantial components on all 

the textiles. Variable amounts of a large number of other luteolin, genistein and apigenin 

glycosides, or compounds with UV-VIS spectra similar to them, were detected. The most 

important was a genistein-related constituent with a UV-VIS spectrum similar to genistin, but 

with a longer retention time; after luteolin, genistein and luteolin-7-glycoside this was the 

largest constituent on the textiles (more than genistin). This was observed by all the partners 

as it was not destroyed by acid hydrolysis. It was not present (or was a very minor 

constituent) in the lakes, so it is possible that it is an artefact of the dyeing process. 

OADC also examined the samples by HPLC–MS. Luteolin (MW 284.2–284.3, depending on 

sample) was identified in all samples, together with genistein + apigenin (MW around 268.3: 

apparently these could not be separated). Other unidentified constituents were observed in 

lake 3 and the two silk samples.

Indigo 

Indigo dyestuff is used directly as a pigment; it is insoluble in the binding media commonly 

used for painting. There was thus no need to prepare any pigments. 


Using an excitation wavelength of 600 nm, a very broad fluorescence emission at 742 nm for 

indigo on wool, 746 nm for indigo on silk was obtained. The emission intensity was very low, 

but it was found to be independent of the excitation wavelength, suggesting the presence of 

one emitting species. 

HPLC-PDA 

All the laboratories identified indigotin and indirubin on the indigo-dyed wool and silk, but as 

it is not possible at present to distinguish between indigo from species of Indigofera, woad 

(Isatis tinctoria L.), or any other plant source no more detailed identification can be made. 

Indirubin is frequently present in synthetic indigo so even this cannot be ruled out. 

Boron trifluoride/methanol is a very poor solvent for indigo, thus NGL obtained poor results 

for the two analyses, although the identifications of indigotin and indirubin could still be 

made and traces of several UV-absorbing compounds and one or two yellow constituents 

were also detected. ICN used dimethyl formamide to extract the dyestuff and detected a range 

of constituents, including isatin (which could be a precursor or a breakdown product of 

indigotin), several absorbing in the UV region, two unknown yellow constituents and an 

unknown orange constituent. The remaining laboratories used the acid hydrolysis method 

described in their protocols. NMS also detected a range of other constituents, some UVabsorbing, 

others red and blue, but used a large sample volume to achieve this. 

OADC identified indigotin (MW 260.45) and traces of indirubin (MW 260.29) on the silk by 

HPLC–MS; only indigotin could be identified on the wool. 

The difficulties experienced with the analysis of indigo is likely to be due partly to poor 

extraction or to the use of chromatographic conditions not optimised for indigoid dyes. In 

practice the conditions used are frequently modified if indigoid dyes are to be examined (see, 

for example, J. Wouters and A. Verhecken, ‘High-performance liquid chromatography of blue 

and purple indigoid natural dyes’, Journal of the Society of Dyers and Colourists 107 (1991), 

pp. 266−9).

Cochineal 

Dyestuff extracted from the dyed silk prepared for the project was used to prepare the lake 

pigment. Alkaline extraction of the dye from shearings of textile dyed with kermes, the Old 

and New World cochineal insects and madder was a commonly used method to obtain dye for 

pigment preparation in western Europe in the fifteenth to seventeenth centuries, if not later; a 

recipe of this sort was therefore appropriate for the cochineal lake. 


Using an excitation wavelength of 360 nm, a broad emission was obtained at 650 nm from 

cochineal on wool and 640 nm from cochineal on silk; in both cases there was also a weak 

shoulder at 700 nm. In the case of the lake, the fluorescence emission occurred at 644 nm, 

with a weak shoulder at 703 nm, using an excitation wavelength of 450 nm. By the KIK 

method, the emission maxima at the excitation of 330 nm were found at respectively 630 nm 

for silk and 640 nm for wool. 

In all cases the emission wavelength was independent of the excitation wavelength used, 

indicating the presence of one emitting species. 

HPLC-PDA 

All partners were able to identify the dyestuff as a variety of cochineal from the presence of 

carminic acid as principal component, with several lesser components known as dcII, dcIV, 

dcVII and others; also flavokermesic acid and kermesic acid, although not all partners were 

able to detect the kermesic acid. The identification of the dye as Mexican cochineal is based 

on the relative proportions of some of these minor components, particularly dcII, present. This 

has recently been identified as a C-glycoside of flavokermesic acid, while dcIV and dcVII are 

stereo-isomers of carminic acid (D.A. Peggie, ‘The development and application of analytical 

methods for the identification of dyes on historical textiles’, PhD thesis, University of 

Edinburgh, 2006; D.A. Peggie, A.N. Hulme, H. McNab and A. Quye, ‘Towards the 

identification of characteristic minor components from textiles dyed with weld (Reseda 

luteola L.) and those dyed with Mexican cochineal (Dactylopius coccus Costa)’, 

Microchimica Acta, published online 10.12.2007, DOI 10.1007/s00604-007-0866-0.) Details 

of the calculations are given in the analytical reports produced by KIK/IRPA. The amount of 

dcII present on silk is lower than on wool, making the calculation more difficult. 

With the boron trifluoride/methanol method of extraction all peaks were methylated, leading 

to a very much more complex chromatogram; some of the minor methylated peaks also 

overlap. Using this method, unmethylated dcII is often undetectable and methylated dcII can 

be swamped by the methylated carminic acid peak. This was the case here; NGL found that, 

using the normal sample injection volume, dcII was undetectable in the textiles and the lake 

pigment; a more concentrated sample solution would facilitate detection. 

On the cochineal-dyed wool, OADC identified dcII (MW 364.5), carminic acid (MW 490.4), 

components with MW 411.6 (related to dcIV?) and 334.6 and 490.6 (related to dcVII?) and

flavokermesic acid (312.3) by HPLC–MS. Only carminic acid could be identified on the silk, 

and only traces of flavokermesic acid in the lake, thus the lake was not identifiable by this 

method.

Madder 

The wool used for the project was dyed for the ‘Monitoring the Damage to Historic 

Tapestries’ (MODHT) project under the European Commission Fifth Framework Programme 

(Energy, Environment and Sustainable Development Programme Key Action 4, ‘The City of 

Tomorrow and Cultural Heritage’, subsection 4.2.1 ‘Improved damage assessment of cultural 

heritage,’; Contract No. EV4K-CT-2001-00048, 2002–2005). A sixteenth-century recipe was 

used. Dyestuff extracted from this wool was used to prepare lake pigments 2i and 2ii, 

following fifteenth-century recipes; alkaline extraction of the dye from shearings of textile 

dyed with madder, kermes and the Old and New World cochineal insects was a commonly 

used method to obtain dye for pigment preparation in western Europe in the fifteenth to 

seventeenth centuries, if not later. The powdered madder used for dyeing was also made 

available to the ARTECH partners and this was used for madder lake 1, following a 

nineteenth-century recipe. 


In the case of the madder-dyed wool, the fluorescence spectrum showed a maximum at 615 

nm, with a shoulder at 640 nm (excitation wavelength 450 nm). The shoulder is made more 

obvious if an excitation wavelength in the visible region is used. For madder lake 2i, an 

emission with a maximum at 601 nm and a shelf at 570 nm was obtained, while for madder 

lake 2ii, the fluorescence maximum occurred at 611 nm, using an excitation wavelength of 

450 nm in both cases. 

HPLC-PDA 

The madder dyestuff contains many constituents and what is present on the textile and in the 

lake pigments, both qualitatively and quantitatively, is very much dependent on the conditions 

of dyeing and pigment making respectively. Some partners did not have access to standard 

samples of commonly observed madder dye constituents, such as anthragallol, 

pseudopurpurin, munjistin, xanthopurpurin and rubiadin; some constituents are as yet 

unidentified as no standards are available. A detailed mass-spectrometric examination of the 

dyestuff would be of value and the benefits of a comparison exercise are clearly particularly 

great in the case of this dyestuff. 

All partners were able to identify alizarin and purpurin on the wool and in the lake pigments, 

and also in the powdered madder root provided. Minor components in the madder powder 

included anthragallol, xanthopurpurin and rubiadin. NGL, using boron trifluoride/methanol 

extraction, found that the madder anthraquinones were present in the root in glycoside form as 

well as the free aglycones; thus a large amount of ruberythric acid (the principal glycoside of 

alizarin) was present, also small amounts of pseudopurpurin glycoside and other glycosides. 

Pseudopurpurin itself was present in the root powder as well as purpurin. 

The partners using acid hydrolysis reported a much higher proportion of purpurin than alizarin 

on the wool; boron trifluoride/methanol extraction (NGL) revealed that a higher proportion of

pseudopurpurin than purpurin was in fact present, but confirmed that there was more of the 

two together than alizarin. Most partners reported the presence of an unidentified constituent 

eluting after rubiadin, often seen in historical samples. It is now thought that this may be 

nordamnacanthal, but this has to be confirmed. The same range of constituents were reported 

in the lake pigments. Some partners reported that madder lake 2i contained a marked amount 

of xanthopurpurin (KIK/IRPA, NGL); it also contained an unusual constituent eluting 

between alizarin and purpurin with a UV-VIS spectrum similar to that of pseudopurpurin, but 

showing absorptions at longer wavelengths. It is thought that this has been produced as a 

result of prolonged alkaline treatment during the preparation of the pigment; it may be 

pseudopurpurin-related. 

Examination of the substrates of madder lakes 1 and 2ii by SEM–EDX and FTIR microscopy 

(NGL) showed that the substrate of lake 1 contained much sulphate, as would be expected 

from the recipe used to prepare it (q.v.). Lake 2ii was found to contain organic sulphur from 

the wool protein (keratin), also to be expected from the recipe used. Examples of both types 

have frequently been identified in paintings of the appropriate date (nineteenth century for 

lake 1, fifteenth–sixteenth century for lake 2ii). 

Using HPLC–MS, OADC identified alizarin (MW 238.29) and purpurin (254.28) on the 

madder-dyed wool, and the same constituents were identified on madder lake 2ii. In the case 

of madder lake 2i, alizarin (MW 238.29) and a constituent thought to be xanthopurpurin (MW 

238.26) were detected. For lake 1, alizarin, purpurin and an unidentified constituent with MW 

282.3 were identified.

Redwood 

The ‘soluble’ redwood dyestuff used was that extracted from sappanwood (Caesalpinia 

sappan L.), a variety that would have been widely used in Europe before 1500. 


One emitting species, independent of the excitation wavelength used and with very low 

emission intensity, was observed on the textiles, λmax 575 nm on silk and 613 nm on wool 

(excitation wavelength 360 nm). Using an excitation wavelength of 400 nm an emission with 

very low intensity and a maximum at 612 nm could be obtained. 

HPLC-PDA 

Brazilein, the principal constituent of ‘soluble’ redwood dyes, is not stable to hydrolysis with 

HCl, but the product of this reaction is seen consistently by all partners using this method of 

extraction as a rather broad peak. OADC, using an acetonitrile gradient rather than the 

methanol used by KIK/IRPA, ICN and NMS, obtained a better peak shape and clearer result. 

Another, as yet identified but far more stable constituent was detected by all partners and this, 

too, is regularly used for identification of a redwood dyestuff (see W. Nowik, ‘The possibility 

of differentiation and identification of red and blue “soluble” dyewoods: determination of 

species used in dyeing and chemistry of their dyestuffs’, Dyes in History and Archaeology 

16/17 (2001), pp. 129–44, component type C). 

NGL obtained very poor results from the textiles as the boron trifluoride/methanol failed to 

extract much dyestuff, even after prolonged treatment, although sufficient was extracted to 

enable identification. Very much better results were obtained from the lake pigment, where a 

characteristic range of brazilin- or brazilein-related constituents (type A in Nowik 2001) were 

observed, but not the brazilein derivative seen by partner laboratories using acid hydrolysis: 

this is not seen using the methylation method of extraction, although another, very broad, 

somewhat similar and probably brazilein-related constituent can be detected. The 

unidentified, stable component observed by all partners (type C in Nowik 2001) was also 

seen.

Safflower 

The red dyestuff carthamin is obtained from the flower petals by alkaline extraction after the 

water-soluble yellow dye has been removed; lemon juice or citric acid is then added to give 

the solution used for dyeing red, or the red dye is allowed to precipitate for use as a pigment. 

In practice the solid red dye was generally mixed with a white extender such as lead white, 

flour or, as in this case, chalk. The yellow dye was used to dye wool only; no silk was dyed 

and there is no record that it was ever used for pigment preparation. 

Safflower yellow wool 


Using an excitation wavelength of 360 nm, a broad fluorescence emission at 554 nm was 

obtained. This was independent of the excitation wavelength used and thus indicative of the 

presence of one emitting species. 

HPLC-PDA 

All partners detected a range of yellow components, none of which could be identified, and 

some found a red component; that found by NMS is late-eluting and was not identified by 

other partners. NGL, using boron trifluoride/methanol extraction, obtained a wider range of 

yellow components, some of which were similar to those obtained by the acid hydrolysis 

extraction method; two early constituents were thought tentatively to be flavonoids. It was 

thought that some of the later components detected were breakdown products; the safflower 

dyes are very sensitive to the boron trifluoride/methanol reagent. Rather similar components 

were also observed in the red dye. No similar constituents have been observed in any other 

dyestuff so they may be characteristic of safflower. No partner was able to say that this dye 

could be identified correctly. 

Safflower red 


For the safflower-dyed wool, the emission spectrum showed a maximum at 568 nm with a 

shelf at 605 nm and a second band at 466 nm (excitation wavelength 360 nm); the intensity of 

this band increased as the excitation wavelength was increased. In the case of safflower red on 

silk, the corresponding values were 622 and 477 nm. For both wool and silk, the position of 

the two bands is independent of the excitation wavelength, thus they are probably due to the 

presence of two emitting species. In the case of the pigment, one emitting species appeared to 

be present with a maximum at 593 nm and a very low emission intensity.

HPLC-PDA 

The dyestuff is unstable to acid hydrolysis and none of the partners using this extraction 

method were able to identify the dye from the presence of carthamin. The dye is not stable to 

boron trifluoride/methanol extraction either, other red products and a range of other 

unidentified components being formed. However, the red derivatives are formed consistently 

and can be used for identification; also carthamin could still be detected, thus NGL were able 

to detect its presence on wool, silk and as a pigment. KIK/IRPA and NGL also observed the 

consistent presence of several unknown UV-absorbing constituents, apparently characteristic 

of the red dyestuff, which can perhaps be used for identification purposes.

Analytical strategies for dyestuffs - Eu-ARTECH

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