Identification of Natural Dyes

in Historical Coptic Textiles

from the National Archaeological Museum of Spain
By Estrella Sanz Rodrguez, Angela Arteaga Rodrguez, Mara Antonia Garca
Rodrguez, Marin del Egido and Carmen Cmara

The aim of this work was the identification of natural dyes employed in the manufacture of eight
fragments of the collection of Coptic textiles from the Spanish National Archaeological Museum, using
HPLC-DAD. Two extraction methods, the classical methanol/hydrochloric acid extraction and a mild
extraction using 5% formic acid in methanol, were evaluated using several reference fibres dyed with a
selection of red, yellow, blue, purple and black dyestuffs. In both cases, an additional step, extracting with
methanol/dimethylform-amide, was employed. The results showed that the number of compounds detected
is higher when the mixture with 5% formic acid was used, contributing to give more information
about the source of the dye, although the extraction efficiency was lower in the most cases. The
latter method was selected and applied for subsequent dye extraction from the samples. The dyestuffs
identified in the fragments under study are in agreement with dyestuff commonly reported for Coptic
textiles.
Introduction
The identification of natural dyes present in historical textiles can contribute to answer different questions
linked with the how, when and where a textile was made [1]. Moreover, this analysis can evidence past
restoration processes and provides key information for the application of an appropriate treatment in current
interventions of restoration or conservation.
In all parts of the world, natural dyes have been used since the most ancient times until the end of 19th
century when they were replaced by synthetic dyes. The ancient dyestuffs were organic materials obtained
from plants, insects, shellfish and lichens [2]. The classification of dyestuffs can be based on their application
method (direct, vat and mordant dyes), according to their origin (natural or synthetic, animal or vegetal),
their colour (red, yellow, blue and purple dyes) and in relation with the chemical constitution (chromophore
structure) of the dyestuff molecule. The different chemical classes of chromophores present in natural dyes
yield the following general classification: anthraquinoid, flavonoid, indigoid dyes and tannins. There are other
chromophores existing, which are not included in this classification because they are less common [3-5]. This
classification is useful for the analyst in order to choose the right sample extraction procedure to recover the
components from natural dyes [6]. The extraction step is crucial, within the whole analytical method,
because identification of dyestuffs will be done upon the extracted components. The standard procedure for
extracting natural dyes from textiles involved heating in 6M methanolic hydrochloric acid solution. This
extraction method was introduced three decades ago [7, 8] and actually is still being applied as evidenced in
recent works [1, 9-11]. The process has the advantage of providing a high extraction efficiency for the
majority of dyes, particularly anthraquinoid and flavonoid types, excepting indigoids, which are poorly
extracted because they remain practically insoluble. Moreover, the majority of yellow dyes and some red and
orange dyes are composed of glycosides, which, when heated in strong HCl, are hydrolyzing glycoside
linkages, causing that only the aglycone chromophore can be detected. As a result, most information about
the original dye components and their plant source is lost [12, 13]. Other limitations of this rather
aggressive process are the degradation of several labile compounds and the chemical transformation of
different chromophores [14]. Recently, several investigations have been carried out to overcome these
problems. The most noticeable with respect to improving the solubility of indigoids dyes have been those

including an exclusive extraction step for these dyes using pyridine, dimethylformamide (DMF) or
dimethylsulfoxide (DMSO) [15-17] solvents, in which blue and purple dyes are more soluble, and one
proposed by Surowiec et al. [18], which is based on HCl hydrolysis and involving an additional DMF/Methanol
(MeOH) extraction step. The introduction of this additional step offered a notable improvement for the
recovery of indigotin. Regarding preservation of labile compounds and glycosidic linkages, the development of
milder extraction schemes is actually a tendency of general importance. Different approaches have been
proposed herein. More systematic studies focused exclusively on dyed textiles were compared by Valianou et
al. in 2009 [19]. For example, Zhang and Laursen [20] developed a mild extraction method in which HCl was
replaced by 5 % formic acid (HCOOH) in MeOH solution, which is more efficient than the common HCl
scheme when extracting anthraquinone and flavonoid dyes from dyed silk, wool and cotton fibres, further
preserving glycosisdic linkages. Although this method was successfully applied to historical microsamples
extracted from pre-Columbian Andean [21] and Chinese textiles [22], another study, focused on the
extraction of Rubia tinctorum L. components from wool fibres, reported that classical HCl extraction provides
the most satisfying results [23]. In another investigation, dyed wool was treated with aqueous solutions of
ethylenediaminetetraacetic acid (EDTA), oxalic acid, oxalate, citrate and citric acid [24]. It was reported that
none of the five procedures was better than the classical method, although the oxalic acid extraction was
comparable to HCl extraction for alizarin and carminic acid. In their study, Valianou et al. [19] compared five
extraction methods, including the use of HCl, citric acid, oxalic acid, trifluoroacetic acid (TFA) and a
combination of HCOOH and EDTA with respect to: (a) number of compounds extracted, (b) relative quantities
of compounds extracted and (c) values for the signal-to-noise ratio of the compounds extracted. It was
shown that the TFA method provided in this sense the best overall results.
Since dyes are mixtures of organic compounds and a fibre can be dyed employing various dyes, those
chromatographic techniques which are able to separate very complex mixtures are the most appropriate tools
for this type of analysis. High performance liquid chromatography (HPLC) is by far the most commonly used
chromatographic technique for analysis of natural dyes, enabling the separation of dye components from a
small sample amount [25]. A HPLC system can be coupled to different detectors. Because the vast majority
of dye components are strong chromophores, UV-Vis absorbance detectors or, more usually, diode array
detectors (DAD) are commonly applied in analysis of natural dyes in extracts from plant or animal sources
[7, 8, 26-28], from contemporary dyed materials [29, 30] and from archaeological textiles [9, 18-20, 3135]. Employing DAD, the detection can be done at any wavelength in the UV or visible spectrum and a
complete spectrum of any substance eluting from HPLC column can be obtained. As a result, dye molecules
can be characterised in terms of retention time from the HPLC system and their UV-Vis spectrum. However,
DAD detectors have the disadvantage that they are not very specific and similar compounds present similar
spectra. Therefore, identifying the particular components in the often complex dye mixtures requires a more
discriminating technique than UV-Vis spectroscopy. For example, the flavonoid aglycones and their glycosides
often show identical UV-Vis spectra [12]. For this reason, the actual trend goes towards the coupling of mass
spectrometry detectors (MS) to the HPLC system, which offers the mass spectrum of each component
separated, thus allowing to characterise unknown compounds. In fact, over the last years, most of the
research in this field tends towards uniting and complementing all the information obtained by on-line
coupling of these two detectors, DAD and MS [1, 9, 13, 19, 21, 22, 36-40].
Egypt was one of the first countries where dyestuffs were used, and its climatic and cultural conditions are
favorable to conservation of archaeological textiles. The literature about the characterization of natural dyes
in Coptic textiles is relatively extensive. For example, Wouters presented different studies using HPLC-DAD of
extracts from Coptic objects [7, 41, 42]. Later on, between 2003 and 2004, results about the natural dyes
present in Coptic textiles from National Museum in Warsaw were presented employing HPLC-DAD [43], LCDAD-MS [44] and LC-DAD/fluorescence detection/MS [45 ]. Other interesting research article was presented
by A. Verhecken [46], where the objective was to establish a correlation between the age of a textiles from
Egypt, Syria and Israel and the dyestuff used in them. Further work was carried out by R. Hofmann-de
Keijzer et al. [47], where the authors give an overview of dyes and dyeing techniques used in the Late
Antiquity in Egypt presenting their results about an investigation of natural dyes in two Coptic textile
fragments from the Museum fr Angewandte Kunst (Vienna).

The Spanish Cultural Heritage Institute (IPCE) receives numerous historical textiles from museums and
excavations for their conservation, technical analysis and identification of their dye content. Over the last
years, the restoration of the collection of Coptic textile belonging to the National Archeological Museum from
Spain, dating from IV AD to X-XI AD, was accomplished. This collection was studied in the framework of the
project Technological and chronological characterization of the Coptic textile productions: antecedents of the
high medieval Spanish textile manufactures [48]. Characterization of natural dyes present in eleven of these
fragments was carried out by thin layer chromatography (TLC) in the IPCE laboratory [49], finding the
common natural dyes used in the Nile Valley, such as madder, indigo (or woad), weld, lac and probably orchil.
Madder, indigo (or woad) and weld cannot be used for dating in the first millennium, but the presence of lac
dye in one textile suggests that this textile was made later than the VII century, according to A. Verhecken
[46]. The objective of the present study was the identification of natural dyes employed in the manufacture
of another eight fragments belonging to this collection using HPLC-DAD. Prior to analysis, two extraction
methods for dye were evaluated, using several reference fibres dyed with a selection of red, yellow, blue,
purple and black dyestuffs obtained from a reference collection of IPCE which contains more than 300 dyed
fibres, and from a personal collection of Ana Roquero.
Experimental
Instrumentation
The chromatographic system used consisted of a model 600E Multisolvent delivery system (Waters
Chromatography, USA) equipped with a Luna C18(2) HPLC column (150 x 2.1 mm id, 5 m particle size) and
a guard cartridge system (Phenomenex, USA).
Samples were injected by a 717 auto sampler (Waters Chromatography, USA). Separated components of
dyestuffs were detected with a 996 DAD detector, scanning from 200 nm to 600 nm at scan rate of 1
scan/second and with a resolution of 1.2 nm (Waters Chromatography, USA). The mobile phase, pumped at
0.5 ml min-1, consisted of 0.1% trifluoroacetic acid (TFA) in water (A) and acetonitrile (B). The gradient
applied was the following: 10% B isocratic to 1 min, to 30% B (linear) at 30 min, to 100% B (linear) at 50
min. The column temperature was maintained at 35 C.
Reagents, reference fibres and samples
High-purity deionized water (Milli-Q Element system, Millipore, USA), trifluoroacetic acid (TFA) from Fluka
(Sigma-Aldrich, Steinheim, Germany) and acetonitrile (ACN), both from J.T. Baker (Deventer, Holland) were
used for preparation of the mobile phase. Gradient grade methanol (MeOH) from J.T. Baker (Deventer,
Holland), formic acid (HCOOH, 98%) and dimethylformamide (DMF) both from Panreac (Barcelona, Espaa)
were employed for sample preparation.
Extraction methods were evaluated using the ten before mentioned reference fibres, dyed with American
cochineal (Dactylopius coccus Costa), Brazil-wood (Caesalpinia sp), madder (Rubia tinctorum L.), weld
(Reseda luteola L.), old fustic (Chlorophora tinctoria), saffron (Crocus sativus L.), indigo (Indigofera sp.),
Tyrian purple (Plicopurpura pansa L.), alder bark (Alnus sp.) and sumac (Rhus spp.) on wool, except the
Tyrian purple reference fibre, which was dyed on silk.
Fiber samples were obtained from different colored Coptic textiles from the National Archaeological Museum
in Spain. Figures 1-8 show the photographs of these fragments. A total number of 29 sub-samples were
taken.

Sampling in Coptic fragments codes from the National Archaeological Museum. Photos by Jos Baztan.
From left to right: Figure 1: 15083; Figure 2: 1976/130/12; Figure 3: 1976/130/14; Figure 4: 15059; Figure 5: 15076; Figure 6:15064;
Figure 7: 15065; Figure 8: 1976/130/11.

Extraction procedures
Extraction methods were applied according to the following general schemes:
Method 1: HCl extraction + MeOH/DMF extraction
Samples were placed in a conic vial and were treated with 250 l of a mixture of H 2O : MeOH : 37%HCl
(1:1:2, v/v/v) for 10 minutes at 100 C. The solvent was then evaporated under a N2 current. A volume of
250 l of the mixture MeOH:DMF (1:1, v/v) was added to the dry residue and the mixture was heated for 5
minutes at around 100 C. Then, the solution was transferred to 0.2 m nylon filters Spin-X (micro centrifuge
filter) and centrifuged at 6000 rpm for 10 minutes. The filtrate was evaporated to dryness under a N2 current
and the residue was dissolved in 50 l of MeOH:DMF (1:1, v/v) solution. After shaking it in vortex for 1
minute, the extract was injected to the HPLC-DAD system.
Method 2: HCOOH extraction + MeOH/DMF extraction
Samples were placed in a conic vial and treated for 30 minutes at 45-50 C with 250 l of a mixture of
MeOH:HCOOH (95:5, v/v). The solvent was then evaporated under a N2 current. 250 l of a solution of
MeOH:DMF (1:1, v/v) were added to the dry residue and the mixture was heated for 5 minutes at around
100 C. Then, the solution was transferred to 0.2 m nylon filters Spin-X (micro centrifuge filter) and
centrifuged at 6000 rpm for 10 minutes. The filtrate was evaporated to dryness under N 2 and the residue was
dissolved in 50 l of MeOH:DMF (1:1, v/v) solution. After shaking it in vortex for 1 minute, the extract was
injected to the HPLC-DAD system.
Results and discussion
Comparison between the two evaluated extraction methods
The two extraction methods applied are based on classical methanolic hydrochloric acid extraction, with an
additional MeOH/DMF extraction step as proposed by Surowiec et al. [18] and on the mild extraction
proposed by Zhang and Laursen [20], where 5% formic acid in methanol was used. Surowiec et al.confirmed
the greatest improvement in extraction efficiency for indigotin when using the additional step involving
MeOH/DMF and Zhang and Laursen observed the preservation of flavonoid glycosides. To the best of our
knowledge, no study has been performed comparing the method proposed by Surowiec et al.with others,
where the acidic hydrochloric extraction has been replaced by a mild extraction. The objective was to join the
advantages of both in one extraction method, because the sample amount available for an analysis is always
very small, therefore it is crucial to obtain the maximum information in one analytical run.

Figure 9. Comparison between the two extraction methods evaluated. N upon bar= n detected compounds.

The results obtained in this comparison are summarized in Figure 9. As expected, the indigoid dyes, indigo
and Tyrian purple, were extracted in a similar way because they are mainly affected by the MeOH/DMF
extraction, which is identical in both methods. Four reference fibres, dyed with brazilwood, old fustic, alder
bark and particularly, saffron, were extracted more efficiently employing the mild extraction, or Method 2. As
expected, when saffron was extracted using Method 1, no peaks were detected because crocin and crocetin,
its principal components, are decomposed by hydrochloric acid to compounds non-detectable by HPLC-DAD.
Regarding the rest of reference fibres, for those dyed with American cochineal, madder, weld and sumac,
Method 1 was able to extract more efficiently the dyestuff. The difference for weld was not very high (with
Method 2 a 95 % of what has been achieved with Method 1), for American cochineal, madder and sumac, the
sum area of detected compound employing Method 2 was 40 %, 70 % and 1.5 %, respectively, compared to
results using Method 1. This indicates that anthraquinone dyes and ellagic acid are poorer extracted with
formic acid. For madder, this effect was also observed by other authors [23]. However, the total number of
compounds detected was higher when Method 2 was employed, with the exception of only two dyes:
American cochineal and sumac. These results were attributed to the milder conditions, the ones with which
the glycosidic linkages were preserved, and, thus, the number of detected compounds increases. To set an
example, the number of compounds increases from four to eight, from seven to eleven or from four to twelve
for weld, madder and alder bark respectively. If the extraction efficiency is acceptable, the criteria to choice
the best method would be the extraction of a maximum number of compounds, which will offer more
valuable information about the origin of the dye.
Consequently, Method 2 was selected as optimum and applied for subsequent dye extraction from the Coptic
textile samples.
Application of the optimum method for dye extraction from the Coptic textile samples
The results obtained applying the optimum extraction method to dyestuffs from the Coptic textiles under
study are summarised in Table 1.
Indigotin was identified in five blue samples, eight green samples and five purple samples. These results
indicate that the dye employed for these samples was in all cases indigo (Indigofera sp.) or woad (Isatis
tinctoria L.), whose main component is indigotin, therefore the differentiation between the two species was
impossible.