Chromatographic Investigation of Dyes Extracted from Coptic Textiles from the National Museum
in Warsaw
Marek Trojanowicz, Jowita Orska-Gawrys, Izabella Surowiec, Bogdan Szostek, Katarzyna
Urbaniak-Walczak, Jerzy Kehl and Marek Wrobel
This work aimed to identify natural dyes in extracts from Coptic fibres. The objects examined
originate from fourth- to twelfth-century AD Egypt Chromatographic separations of all the samples
were carried out using reversed-phase HPLC (high performance liquid chromatography) with UVVis detection and fluorescence detection. Fluorescence detection proved to be better than UV-Vis
diode array detection for the determination of some compounds. Selected samples were analysed
with HPLC-MS (HPLC-mass spectroscopy) which confirmed the LC-UV findings and allowed the
detection and identification of additional compounds. Most of the dyes mentioned in the literature
as having been used by Copts were detected: Armenian cochineal, flavouoid yellow dyes, indigotin,
lac dye, madder and tannins. Some new compositions of significant colours were also discovered,
which had not been reported previously in Coptic textiles. Elemental analysis using SEM-EDS
(scanning electron microscopy energy dispersive spectroscopy) was performed to identify
mordants.
INTRODUCTION
The Early Christian Art Department and the Conservation Department of the National Museum in
Warsaw have prepared a permanent exhibit of Coptic art. The exhibit provides an opportunity to
present treasures of Christian art from the valley of the Nile, in particular a collection of 80 Coptic
textiles dating from the late third/early fourth century AH to the twelfth century AD that belongs to
the National Museum in Warsaw. Small decorative appliques, medallions and belts, as well as
fragments of larger tapestries and a small cone-shaped cap, are part of this collection [1].
Most of the preserved fragments came to Poland at the turn of the nineteenth century, thanks to
many travellers and explorers who visited Egypt looking for ancient relics. Some of the textiles
were purchased from antique dealers, others were bought from private owners or were obtained
through donations. Twenty-nine objects are from the National Museum in Gdansk. They came from
the necropolis in Oasis Fajum and Achmim-Panopolis in northern Egypt and were bought from the
Austrian archaeologist and collector R. Forrer. Twenty textiles are from the National Museum in
Wroclaw. Most of them came from excavations in Fajum, Achmim-Panopolis and Arment, some of
them belonging to the collection of T. Graft. Part of the collection, for example 11 fragments given
to the National Museum in Warsaw by Unesco as a compensation for the loss of objects during
World War II, is of unknown origin. The textile fragments in the investigated collection are in
different states of preservation. Most are badly deteriorated and some are in a critical state. They
show extensive physical and chemical damage and are often discoloured and crumbly.
The aim of the work presented here was the chemical identification of natural substances used for
dyeing Coptic textiles from the collection of the National Museum in Warsaw, as part of a
comprehensive research programme that includes chemical, microbiological and mechanical
investigations conducted on the textiles. Results of this work will be used to establish the most
appropriate conservation conditions and can also be helpful in dating or elucidating the place of
origin of the archaeological objects examined.
Ancient dyestuffs originate from extracts of plants, insects and molluscs. Dyes can be extracted
directly from these natural species or can be obtained after various chemical pre-treatments such as
complexing with metals, hydrolysis or oxidation. Although the literature about the chemical
examination of historical textiles is quite extensive [2-11], limited attention has been paid to Coptic
textiles. The investigation of dyes in textiles from Christian burials in Egypt dating from the fourth
to the sixth century, based on chemical reactions, was pioneered by Pfister in the 1930s [12]. The
first HPLC (high performance liquid chromatography) examination of extracts from four Coptic
objects dating from the third to the eighth century was reported by Wouters [9]. From this author's
later works [10, 13], it can be concluded that natural dyes with different compositions were used at
different periods in time. For example, a mixture of madder and kermes in the proportion 95:5 was
used to dye Egyptian textiles in the Byzantine period, while a mixture of madder and lac dye in the
proportion 50:50 was used in the early Arabic period. The dyes identified so far on Coptic textiles
are listed in Table 1. As can be seen, the main red dyes identified were dyers' madder (Rubia
tinctorum L.) and wild madder (Rubia peregrina L.) with alizarin and purpurin as principal
colourants, kermes (Kermes vermilio Planchon) with kermesic acid and flavokermesic acid,
Armenian cochineal (Porphyrophora hameli Brandt) with carminic acid and lac dye (Kerria lacca
[Kerr, 1782]) with laccaic acids. Weld (Reseda luteola L.) seems to be the most commonly used
yellow dye, with luteolin and apigenin as principal colourants. Indigotin was identified, which
is the principal colourant of the blue dyes woad (Isatis tinctoria L.) and indigo (Indigofera tinctoria
L.). Tyrian purple (Murex trunculus L. and Murex brandaris L.), with brominated indigotins as
principal colourant, could also have been used by Copts, although in rare cases, since in antiquity
this dye was very expensive and, hence, not commonly used. Some dyes exhibit natural
fluorescence or can achieve fluorescence properties after complexation with certain metallic
cations. Fluorescence properties of canmnic acid in aqueous solutions [15] and indigotin on fibres
[16, 17] have been mentioned. Flavonoids, which arc also food constituents and important
anrioxidants, were determined by HPLC with fluorescence detection [18-21] and with a postcolumn derivatization with aluminium and fluorescence detection [22, 23]. Increase in fluorescence
signal after addition of aluminium was also noticed for other red dye compounds such as alizarin,
purpurin,
Table 1 Known composition of dyestuffs used for dyeing of Coptic textiles
brazilein, emodin, as well as for extracts from cochineal and kennes [24].
Flavonoids have recently also been the focus of many mass spectrometric investigations, due to the
ability of the HPLC-MS techniques to identify and selectively quantify them in complex matrices of
plant and food extracts [25—28]. The collision induced dissociation (CID) spectra of selected
flavonoids obtained with negative ion electrospray (ESI) and atmospheric pressure chemical
ionization (APCI) and their chromatographic separations have been examined [29, 30]. The
fragmentation mechanisms of flavonoids have been described in detail for positive, protonated
molecular ions [31, 32] and for negative, deprotonated molecular ions [33]. Laccaic acids derived
from lac-lakes have been examined by positive ion electrospray mass spectrometry |34J. Also,
tannins from oak have been investigated with HPLC-MS [35],
In the present study, HPLC with three methods of detection — diode array UV-Vis (DAD),
fluorescence (FLD) and mass spectroscopy (MS) — was used to identify individual chemical
components of an-thraquinone, indigoid and flavonoid dyes in extracts from fibres of different
colours taken from Coptic textiles. The different detection modes are compared and new
compositions of some colours are reported. FTIR (Fourier transform infrared) microscopy was also
tested as a non-destructive method for the identification of dyes in archaeological objects.
Elemental analysis using SEM-EDS (scanning electron microscopy—energy dispersive
spctroscopy) technique was performed to identify mordants on threads from Coptic textiles.
Elemental analysis of threads can give information about the type of dye used, for example by
identifying bromine in Tyrian purple, and about mordants, which have a significant influence on the
final tone of the colour of the thread. As far as Coptic textiles are concerned, it is known that for
yellow colours weld was used on alum and zinc mordants, for red colours, madder on alum and tin
salts, and for purple and brown colours, madder on an iron mordant [14], Weld with an iron
mordant might also produce an olive-green colour.
RESULTS AND DISCUSSION
Experimental conditions and procedures used in this study are described in the Appendix.
Identification of dyes by chromatography
Investigations based on UV-Vis identification of compounds were carried out in three stages. First,
chromatographic measurements were made on purified dyes and natural dyeing substances
collected from various sources. Then HPLC data were recorded for extracts of dyes from
contemporary dyed fibres, which were dyed with dyestuff extracted from raw material purchased
from Kremer Pigmente. Finally, the extracts from fibres taken from ancient Coptic objects were
analysed under four sets of chromatographic conditions as described in the Appendix. Identification
of dyes extracted from objects was based on retention times and on UV-Vis spectra recorded for
sample extracts and standards.
Fifty-six hydrochloric acid/ethanol/water extracts and 16 pyridine extracts from threads from
Coptic-textiles were investigated. The peak area absorbance values at 255 nm were used to
determine the relative amounts (in percent) of dyes identified in each extract. This wavelength
provides sufficient sensitivity for the detection of all compounds identified. It is also widely used in
the literature as a reference wavelength for comparing results obtained for different extracts from
natural dyes and archaeological threads. It was noticed that the relative sensitivity of diode array
detection, i.e., the relative amounts of dyes identified for each extract, varied depending on the
gradient of eluent used, even if the same extraction method was applied. This was probably due to
differences in the background; spectra measured for blanks (extracts from non-dyed wool) showed
different amplitude and retention depending on the eluent gradient used.
Frequently, fluorescence detection can provide better selectivity and detectability than UV-Vis
detection. Often, a post- or pre-column derivatization is needed to achieve this. Methanolic
solutions of Al(III), Ga(III), In(III) and Zn(II) salts were tested as post-column complex-forming
reagents for enhancing the fluorescence signal of the investigated dyes. Among these, Ga(III)
proved to be the best (see [36] for detailed optimization) and a 10 mM solution of this cation was
used for fluorescence detection of some plant extracts and 56 water/methanol extracts from Coptic
textiles. Table 2 shows the excitation and emission wavelength programme used for HPLC
measurements with fluorimetric detection.
For plant extracts, results obtained were similar to those described earlier in the literature except for
yellow dyes. Luteolin and apigenin are principal colourants of flavonoid dyes; however, they can be
found in many plants and cannot be specific markers for weld only [37], which was frequently
listed in earlier work on Coptic-textiles. Kaempferol has also been reported as another main
component of weld, in addition to luteolin and apigenin [14]. In the present study kaempferol was
not detected by HPLC with DAD and MS, but only with the most sensitive fluorescence detection
[36, 38]. Using the latter, kaempferol, quercetin and rhamnetin were detected in several samples,
which are indicative of other botanical sources, such as Rhamnus species [39|; hence this is
postulated as a source of pigments. Kaempferol, quercetin and rhamnetin have not been detected in
any of the weld standard samples examined with all three detection systems employed.
Fluorescence detection with Ga(III) solution as the post-column reagent proved to be more sensitive
than UV-Vis for the detection of purpurin, rhamnetin,
Table 2 Optimized detection conditions used in HPLC with fluorescence detection
quercetin, gallic acid, kaempferol and munjistin. DAD, however, was more sensitive for the
determination of carminic acid, ellagic acid, luteolin, alizarin, apigenin, lawsone and indigoid dyes.
Examples ot chromatograms of extracts from a yellow Coptic thread are shown in Figure 1.
The chromatographic and mass spectrometric behaviour was investigated for selected dye
compounds of flavonoid-, anthraquinone- and indigo-type. Most ot the compounds investigated can
be ionized with the positive and negative ion electrospray ionization. Formic (methanoic) acid was
added to the mobile phase, although it can potentially affect the sensitivity of LC-MS analysis by
causing the suppression of the electro-spray ionization process in negative ions. The formic acid
was added, however, only to the aqueous component of the mobile phase (no formic acid was used
in acetonitrile) and its effective concentration in the mobile phase decreases as the gradient
progresses towards more organic composition. Formic acid was needed in the mobile phase in order
to obtain good peak shapes for flavonoids, and its presence only in the aqueous component of
mobile phases allows good initial focusing and minimizes its content when analytes elute and are
introduced into the MS. To evaluate the effect of the formic acid presence on ionization efficiency,
some of the samples were run using only the water/ acetonitrile mobile phase, using the mass
spectrometer set up in the MS-MS mode (referred to as MRM). No apparent gain of sensitivity was
observed and peak shape was definitely inferior for most analytes observed. Difficulties with
ionization by electrospray were experienced for indigo and brominated indigotins, but these could
be ionized by atmospheric pressure chemical ionization (APCI).
Mass spectrometric detection, using different scanning modes of a triple quadrupole mass
spectrometer, combined with the UV detection, was demonstrated to be a powerful approach for the
detection and identification of dyes in extracts from archaeological textiles. This approach is
extremely useful in cases where a limited amount of precious sample is available and the maximum
amount of information is to be gained from the samples. MS detection additionally provides
selectivity that is hard to obtain with UV detection. This is advantageous for complex sample
matrices and for resolution of overlapping chromatographic peaks. Figure
Figure 1 Chromatograms of a brown Coptic thread (wool, fourth century AD). Chromatographic
conditions are described in the Appendix (DAD2). The programme of wavelength change for
fluorometric detection is shown in Table 2.
2 illustrates the selectivity and sensitivity obtained with mass spectrometric detection. In this case,
the mass spectrometer is set up to monitor signals for characteristic parent-daughter ion transitions
for pre-selected groups of compounds (MRM mode), allowing their selective and sensitive
detection and providing additional clues for the compound identity, besides the retention time
match. The UV detection was not sufficient to detect these compounds for this sample.
Full scan MS with different ionization modes, combined with simultaneous UV detection, is often
sufficient to identify the main components. Figure 3 presents data obtained in full scan mode (100700 m/z) with negative ion electrospray ionization for an extract from green Coptic thread.
Individual components can easily be identified by plotting traces for molecular ions of investigated
compounds and matching the retention time of the peak with that of the standard. In addition, the
data from the full scan mode can be used to flag the parent ions of unknowns showing up either on
the UV trace or MS trace and subject them to further structure elucidation by collecting their
daughter-ion spectra.
Using this approach, xaiithopurpurin was identified in several samples and monochloroalizarin
together with dichloroalizarin in one [37]. Although chlorine was not detected by EDS, formation of
chlorinated alizarins during extraction with hydrochloric acid is practically impossible, since
chlorination of phenols is usually done with gaseous chlorine as a catalyst. In the Beilstein chemical
reference database, the only synthesis of monochloroalizarin found was achieved via sultonated
derivatives [40, 41], while synthesis of dichloroalizarin was even more complicated [42]. There is,
as yet, no explanation for the presence of chlorinated alizarins, but the results obtained were
reproducible.
In the case of the samples investigated, the detection capabilities of the LC-MS system were
comparable with those observed for the UV-Vis and fluorescence detection, and especially
advantageous for luteolin, apigenin and indirubin.
Comparison of results for UV-Vis, fluorescence and MS detection for some of the extracts from
Coptic-threads is shown in Table 3. It should be pointed out. however, that the relative amounts of
different
Figure 2 Detection of low levels of (A) purpurin, (B) alizarin, (C) indirubin, (D) rhamnetin, (E)
apigenin and (F) luteolin in a sample of a brown Coptic thread (wool, fourth century AD) using the
mass spectrometer in MS-MS (MRM) mode. Corresponding UV trace at (G) 350 nm and (H) 278
nm.
Figure 3 Selected ion traces obtained using the mass spectrometer in full scan mode for a green
Coptic thread (wool, sixth century AD): 285 m/ z, luteolin (B); 269 m/z, apigenin; 239 m/z, alizarin
(D). (A) Corresponding UV trace at 278 nm showing apigenin at 21.33 minutes and alizarin at
25.15 minutes.
compounds obtained using the DAD 1 and DAD2 gradient programmes could be different.
Additionally, some species detected by fluorescence and/or MS were not detected by DAD, which
most likely means that they were present in very low amounts. Also, data obtained with very
sensitive detection techniques such as fluorescence and MS should be treated with some reservation
since these techniques can detect small variations in composition even within samples taken from
fibres dyed the same colour on the same object. Identification of apigenin, luteolin, quercetin,
rhamnetin, kaempferol, alizarin, purpurin, munjistin. carminic acid, ellagic acid, gallic acid,
indigotin and indirubin was based on reference data obtained for pure compounds or, in the case of
laccaic acids, on data from the literature. Xanthopurpurin, monochloroalizarin and dichloroalizarin
were identified based on structure elucidation by mass spectrometry.
When compared to data in Table 1, some new dye components of significant colours were found in
extracts from Coptic textiles in the present study. Table 4 lists compounds that were used for
identification, although ratios with the main colouring compounds were not evaluated. In all these
samples, luteolin, apigenin. ellagic acid, alizarin and purpurin were identified with the diode array
detector based on their retention times and UV-Vis spectra. Absorption maxima of compounds
identified on Coptic textiles with the diode array detector are listed in Table 5. Identification of
unknowns was achieved when their UV-Vis spectra matched the shapes and maxima (within ±3
nm) of reference spectra. Rhamnetin, kaempferol and quercetin were detected with the fluorimetric
detector on the basis of their retention times only.
In the extract from the red silk fibre, carminic acid, laccaic acid A, laccaic acid B. purpurin,
alizarin, apigenin, luteolin, ellagic acid and gallic acid were found with the diode array detector,
carminic acid, quercetin and kaempferol with the fluorescence detector and alizarin, purpurin,
carminic acid, luteolin. apigenin and xanthopuipurin with the MS detector. In this case the presence
of Armenian cochineal, lac dye, madder, a flavonoid yellow dye (probably weld), woad or indigo
Table 3 Components found in extracts from Coptic threads
Table 3 Continued
and tannins was concluded. This is the first report of the simultaneous presence of carminic acid
and laccaic acids in extracts from Coptic textiles.
Identification of dyes by infrared spectroscopy
The microscopic infrared (µFTIR) investigation of five dye standards, five samples of
contemporary dyed threads and their extracts as well as three Coptic threads and their extracts was
carried out to examine the
applicability of this technique to the identification of the type of dye used for dyeing archaeological
threads. The spectra of dyed threads were compared with the spectra of standards and with a
spectrum of undyed wool. Spectra of all of the investigated dyed fibres exhibited strong absorptions
in two regions, 3500—2500 cm-1 and 1700—1000 cm-1, and showed similarities with the spectrum
of undyed wool. No bands attributable to dye standards were found in the spectra of dyed fibres and
in the spectra of dry residues from thread extracts. It can be
Table 4 Identification of additional components of significant colours of Coptic threads
Table 5 Absorption maxima of selected dye components
concluded that the amount of dye on the fibre is not sufficient to be detected by µFTIR in the
presence of strong signals from wool.
X-ray spectroscopic determination of inorganic mordants
Calcium, oxygen, aluminium, silicon and magnesium were found in all samples in point analysis
mode; carbon was also detected in all samples, since the samples were carbon-coated prior to
analysis, and therefore is not included in the results reported for some of the samples in Table 6.
These elements, together with phosphorus (found in 36 samples), potassium (18 samples), sodium
(24 samples) and chlorine (nine samples) can be associated with contaminants from the
archaeological sites and it is not possible to conclude if they are chemical elements of the mordants.
Sulphur was detected in all samples, which is not surprising since this element is found in animal
fibres. Its occurrence in two flax fibres is very interesting and probably indicates the use of a
mordant. Iron was found in 26 samples and its presence can indicate the use of a mordant during the
dyeing procedure or that the sample was buried in an iron-rich soil. Zinc, reported earlier by other
authors as being used with weld to obtain yellow colours, was found in seven samples. In all of
them, a flavonoid yellow dye or at least kaempferol was identified, suggesting that weld could
indeed have been used. Copper and chromium were found in one brown sample in which madder, a
flavonoid yellow dye (probably weld) and indigotin were identified by HPLC analysis. Titanium
was found in one black thread dyed with madder, a flavonoid yellow dye (probably weld), indigotin
and tannins. These elements are not mentioned in the literature as having been used by Copts as
mordants. Tin was not detected in any of the samples investigated, although it is said that its salts
could have been used in ancient Egypt as mordants [14]. Results of point analysis for some of the
samples are shown in Table 6 and an example of an energy dispersive spectrum obtained for a
Coptic thread is shown in Figure 4. The analysis for each sample was repeated three times, and
reproducibility of measurements was rather poor (±20%). Elevated amounts of oxygen, silicon,
sulphur, calcium and aluminium were observed in line analysis, and of sulphur, aluminium and
calcium in area analysis. It has to be admitted, however, that elemental analysis of archaeological
textile objects rarely provides truly conclusive results about the mordant used in dyeing processes,
in spite of careful cleaning of samples prior to analysis. It is very difficult to say if any given
element identified is from the mordant or if it is from an impurity associated with the dying process
or from the burial environment. Additional problems in mordant identification occur because of the
small amount of mordants on archaeological threads and their disappearance as a result of washing,
textile use and material deterioration.
CONCLUSIONS
HPLC with different detectors is a powerful method for the identification of dyes. Fluorescence
detection with Ga(III) solution as post-column reagent proved to be more sensitive than UV-Vis
detection for purpurin. rhamnetin, quercetin, gallic acid, kaempferol and munjistin. However, UVVis diode array detection was more sensitive for the determination of carminic and ellagic acids,
luteolin, alizarin, apigenin, lawsone and indigoid dyes. In the case of the samples investigated, the
detection capability of LC-MS was competitive with
Table 6 Examples of elemental composition of Coptic threads obtained by X-ray spectroscopy
those observed for UV-Vis and fluorescence detection and advantageous for luteolin, apigenin and
indirubin. Hence, these three detection methods used with HPLC separation should be considered as
complementary for the determination of dyes in extracts from archaeological samples.
Figure 4 (A) Scanning electron micrograph of a brown Coptic thread (wool, fifth century AD)
showing points analysed and (B) elemental spectrum obtained at point no. 2.
Most of the dyes reported earlier in the literature to be used by Copts were detected in the extracts
investigated. Additional components of some colours were discovered, mainly because of very low
detection limits obtained for flavonols with fluorometric detection. Using LC-MS analysis,
monochloroalizarin and dichloroalizarin were identified in one sample and xanthopurpurin in some
samples of fibres from Coptic textiles.
FTIR microscopy did not provide any information about dyes on archaeological threads because of
the presence of strong signals from wool. High amounts of some elements were detected on the
Coptic threads investigated by SEM-EDS. Tin, whose salts were used as mordants in ancient Egypt,
was not found, but chromium together with copper was identified on one sample. However, results
of elemental analysis can only be properly interpreted when combined with other chemical,
historical and archaeological data.
APPENDIX: EXPERIMENTAL SECTION
Chemicals
The reference substances were either purchased from commercial sources or obtained from
individual researchers. Alizarin, purpurm, carminic acid, ellagic acid, gallic acid and apigenin were
obtained from Fluka (Buch, Switzerland); luteolm, rhamnetin, kaempferol and quercetm were
obtained from ROTH (Karlsruhe, Germany), lawsone from Sigma (Steinheim, Germany), weld,
madder, synthetic indigo and indigo from Indigofera tinctoria, lac dye, cochineal and Tyrian purple
from Kremer Pigmente. Indirubin, 6-monobromo-indigotin and 6,6'-dibromoindigotin were
obtained as gifts from Mr Chris J. Cooksey, Watford, UK, munjistin from Dr N.P. Mischenko from
the Pacific Institute of Bioorganic Chemistry, Vladivostok, Russia, Armenian cochineal from Ms
Ina Vanden Berghe and Dr Jan Wouters of Koninklijk Instituut voor het Kunst-patrimonium,
Brussels, Belgium, kcrmes from Mr Andre Verhecken, Mortsel, Belgium, and from Mr Witold
Nowik of Laboratoire de Recherche des Monuments Historiques, Champs-sur-Mame, France.
Contemporary dyed fibres were prepared in the Conservation Department of the National Museum
in Warsaw. Fibres were dyed with dyestuff extracted from raw material obtained from Kremer. The
dyeing conditions were not optimized to obtain a particular colour, but rather to fix on the threads
enough dye for further analytical determinations. In order to get stable binding of dye, regardless of
the final colour, it was necessary to use a quantity of mordant equivalent to 20% of the weight of
the dry wool; the same quantity of dye was used. Iron (III) sulphate and aluminium potassium
sulphate were used as mordants. The dyeing procedure was carried out in boiling aqueous solutions
of dye and mordant for one hour. Dyeing with vat dyes such as mdigotin and extracts from woad,
indigo and Tyrian purple was carried out by dissolving the dye (20% weight of dry wool) in a
solution containing 5% ammonia and 10% sodium dithionite. The yellow wool threads obtained
turned to blue after exposure to air. Some of the threads dyed with indigo dyes were further dyed
with some of the mordant dyes mentioned above.
Extraction procedure
Extracts from the contemporary fibres and from archaeological fibres from Coptic textiles were
prepared by an open tube hydrolysis of approximately 0.5—3 mg of the fibre in 400 µl ot a mixture
of 3 M hydrochloric acid and ethanol (1:1 v/v) in a boiling water bath for 15 minutes. The resulting
extracts were filtered using a polypropylene centrifuge filter (VectaSpin Micro, 0.45 µm) and then
evaporated to dryness m a vacuum desiccator. Residues obtained in this procedure were dissolved
in 200 µl of water/methanol solution (1:1 v/ v). Non-dissolved residues were treated with 200 µl of
warm pyridine and filtered again. Hydrolyzate or pyridine extracts were injected into a reversedphase HPLC system.
HPLC and LC instrumentation
The separations for diode array and fluorescence detections were carried out with two HPLC
systems from Shimadzu (Kyoto, Japan) consisting of injection valve with 20 µT loop, column oven
CTO-10AS, photo diode array detector SPD-M10A, spectrofluorometric detector RF-10A, system
controller SL-10A, Shimadzu chromatographic software Class-VP, gradient pump LC-10AT and
phase mixer FCV-10AL in the first system, and two HPLC pumps model LC10 AD and degasser
DGU-14A in the second system. With the first HPLC system, UV-Vis detection was applied; with
the second, UV-Vis and fluorometric detections.
For mass spectrometry detection, two different LC-MS systems were employed. Two different
systems were used because the systems were available at the time the samples were analysed and
the second system (System II) had the in-line DAD detector while the first system (System I) only
had a single wavelength detector. As far as the mass spectrometers used in these systems are
concerned, they have very similar capabilities and sensitivity. Initially, the work was started on a
triple quadrupole mass spectrometer Quattro LC (Micromass, Manchester, UK) equipped with a Zspray API ESI source, interfaced with an HP 1100 HPLC system (Agilent, Palo Alto, Cal., USA)
and in-line variable wavelength UV detector (Agilent) (System I). The work was continued on a
triple quadrupole mass spectrometer (Quattro Micro, Micromass) equipped with Z-spray API ESI
source or APCI source, combined with a 2795 Waters HPLC system (Waters. Milford, Mass., USA)
and in-line model 2996 PDA UV detector (Waters) (System II).
HPLC measurements
For diode array spectrophotometric detection of hydrolysate samples two eluents were used:
gradient of acetonitrile and aqueous solution of 0.1% trifluoroacetic acid at a flow rate of 1.2
ml.min-1 (DAD 1) and gradient of methanol and 25 mM phosphate buffer pH 2.5 at a flow rate of 1
ml.min-1 (DAD 2). For DAD of pyridme extracts gradient of acetonitrile and aqueous solution of
5% tetrahydrofuran at a flow rate of 1 ml.min-1 was used (DAD 3). Separations were carried out .it
40°C on a Phenomenex Luna-C18 (250 x 4.6 mm, 5 µm) column. The volume of injected sample
was 20 µl.
For fluorescence detection, the column effluent after DAD was mixed in a post-column reactor with
a solution of 10 mM gallium(III) nitrate in methanol containing 1% hydrochloric acid. Flow rate of
post-column reagent was 0.2 ml.min-1. For this detection gradient programme DAD 2 was used. The
post-column reactor consisted of 4.0 m (0.25 mm i.d.) stainless steel tubing connected to the HPLC
system with a low dead-volume T-piece from Supelco (Buchs, Switzerland). An HPLC micropump
type 6400 from Knauer (Berlin, Germany) was used to generate the flow of post-column reagent.
For MS detection, chromatographic separation was earned out at ambient temperature with a flow
rate of 0.2 ml.min-1. The mobile phase consisted of 0.3% formic acid in water and acetonitrile
applied in a gradient mode. A Zorbax RX-C18 (2.1 x 150 mm, 5 µm) was used with injection
volume of 50 µl. The triple quadrupole mass detector was connected in series with the HPLC
system after the UV-Vis detector. The chromatographic conditions used in this study are
summarized in Table 7.
Table 7 HPLC conditions used in measuring systems with different detectors
MS measurements
The ESI probe and ion source were operated at 3.5 kV of capillary voltage, cone voltage 30 V,
source temperature 120°C, desolvation temperature 300°C, cone gas flow rate 60 l.hr-1 and
desolvation gas flow rate 500 l.hr-1 for both negative and positive ion generation with System II.
The ESI and ion source of System I were operated at 3.0 kV of capillary voltage for negative ions
and 3.5 kV for positive ions, cone voltage: 30 V, source temperature 120°C, desolvation
temperature 300°C. nebulizer gas flow rate 150 l.hr-1 and drying gas flow rate 700 l.hr-1.
IR measurements
Gallic acid, alizarin, purpurin, luteolin and indigotin were used as standards. Contemporary dyed
threads were dyed with gallic acid (40%) without mordant, gallic acid (40%) on iron mordant
(40%), alizarin and purpurin (20%.) on alum (20%), luteolin (20%) on alum (20%) and indigotin
(20%). Percentage of dry dye or mordant is relative to the amount of dry wool used for dyeing. IR
spectra were collected on a FTIR Perkin Elmer System 2000 spectrometer (Shelton, USA). Two
techniques were used: IR microscopy for threads and microscopic samples of standards in spectral
range 4000-600 cm-1 and transmission IR spectroscopy (KBr pellets) for standards and dry residues
of thread extracts in spectral range 4000-400 cm-1.
SEM-EDS analysis
All samples of Coptic threads were rinsed three times with distilled water and coated with spectrally
pure carbon prior to analysis. Elemental analysis was done using an electron microprobe (Link-Isis)
coupled to a scanning electron microscope (Jeol JSM-630. Peabody, USA). Three techniques were
used. Point analysis was made on 40 samples of Coptic threads. Because of significant differences
in elemental composition within one sample, three point analyses from different areas of the thread
were made for each sample. Line analysis of one sample and area analysis of another were also
made. Line and area analyses were carried out in scanning mode in order to observe a change in
element content along the cross-section of the fibre. All determinations were performed with an
accelerating voltage of 20 keV and a beam current of 10-9 A. Duration of point, line and area
analysis was 200 seconds, 20 minutes and two hours, respectively.
ACKNOWLEDGEMENTS
This work was supported by Grant No. 1H01E00299C/ 4402 from the Polish State Committee for
Scientific Research and CEEPUS Grant H-76. The authors are grateful to the following individuals
for gifts of dyes and dye components: Mr Chris J. Cooksey, Watford, UK; Dr N.P. Mischenko from
the Pacific Institute of Bioorganic Chemistry in Vladivostok, Russia; Ms Ina Vanden Berghe and Dr
Jan Wouters from Koninklijk Instituut voor het Kunstpatrimonium, Brussels, Belgium; Mr Andre
Verhecken, Mortsel, Belgium; Mr Witold Nowik from Laboratoire de Recherche des Monuments
Historiques, Champs-sur-Marne, France; and Prof. Jan Koteja from Agricultural University of
Cracow, Poland. The authors would also like to thank Dr Magdalena Biesaga from the Chemistry
Department of the University of Warsaw, Poland, Prof. Milan Hutta and Dr Radoslav Halko from
the Faculty of Natural Sciences of Comenius University, Bratislava, Slovakia, for their help in the
optimization of the fluorescence detection, Mr Michal Jamroz from the Industrial Chemistry
Research Institute, Warsaw, Poland, for performing uFTIR measurements, and Ms Hanna Rejniak
of the National Museum in Warsaw for technical help in dyeing contemporary fibres. The
information about the state of preservation of the objects examined was kindly provided by Ms
Iwona Pannenko and Ms Elzbieta Rosloniec from the National Museum in Warsaw. The authors
also greatly appreciated valuable reviews of their manuscript, which were very helpful in improving
the paper.
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AUTHORS
MAREK TROJANOWICZ, DSC, graduated from Warsaw University, currently professor of chemistry
in the Department of Chemistry, Warsaw University and in the Institute of Nuclear Chemistry and
Technology in Warsaw. Author of 230 scientific papers and two books in the field of flow analysis,
working also in the field of separation techniques, chemical and biochemical sensors and trace
analysis. Address: Department of Chemistry, Warsaw University, Pasteura I, 02-093 Warsaw,
Poland. Email: trojan@chem.uw.edu.pl
JOWITA ORSKA-GAWRYS, MSC, graduated from Warsaw University, currently research associate in
the Department of Analytical Chemistry of the Institute of Nuclear Chemistry and Technology in
Warsaw, working in the field of the application of HPLC to the analysis of archaeological samples
and radiopharmaceuticals. Address: Institute of Nuclear Chemistry and Technology, Dorodna 16,
03-195 Warsaw, Poland.
IZABELLA SUROWIEC, MSC, graduated from Warsaw University, currently PhD student in the
Department of Chemistry, Warsaw University, working in the field of the application of highperformance separation techniques, spectroscopic methods and mass spectrometry to the chemical
analysis of archaeological objects. Address as for Trojanowicz.
BOGDAN SZOSTEK, PhD, received his MSc degree from Warsaw University in 1991 and PhD from
Southern Illinois University, Carbondale iti 1996, both in analytical chemistry. He is currently a
senior research chemist at DuPont Haskell Laboratory for Health and Environmental Sciences.
Before joining Haskell Laboratory, he did postdoctoral research in the Environmental Research
Division of Argonne National Laboratory (1997) and at DuPont Agricultural Products (1998). His
present research is primarily focused on the development and validation of quantitative analytical
methods as well as qualitative chemical analysis in support of toxicological and ecotoxicological
research utilizing various chromatographie techniques and mass spectrometry. Address: DuPont
Haskell Laboratory for Health and Environmental Sciences, 1090 Elkton Road, Newark, DE
19714, USA.
KATARZYNA URBANIAK-WALCZAK, 1956-2003, PhD, received her MSc degrees from Catholic
Theological University in Warsaw and Westfalian Wilhelms University of Miinster, Germany. She
was awarded a PhD by the Institute of Egyptology of Warsaw University in 1991. From 1994 she
was a lecturer in Coptic language in the Institute of Archaeology of Warsaw University, and at the
same time a custodian of the Early Christian Art Collection at the National Museum in Warsaw.
She was a member of the International Association of Egyptologists and ICOM, and the author of
numerous papers on Coptic art and literature.
JERZY KEHL, PhD, graduated from the Department of Mathematics, Physics and Chemistry of
Warsaw University, obtained his PhD from the Institute of Organic Chemistry of the Polish
Academy of Sciences. Head of the State Laboratory for Conservation of Historical Objects, 197683. Head of the Laboratory of the National Museum in Warsaw, 1982-2002. Since 1985 he has
been an expert in the Polish Ministry of Culture for conservation of metallic objects. Address:
Zablocihska 8/19, 01-697 Warsaw, Poland.
MAREK WROBEL graduated from the Department ot Geology, Warsaw University, and is
currently working with scanning electron microscopy in the GEONAFTA. Address: Geological
Bureau GEONAFTA, Geological Bureau of Polish Oil and Gas Company,
301 Warsaw, Poland.
Jagidloiiska 76, 03-
Resume — Ce travail a pour but d'identifier des colorants naturels extraits de fibres coptes. Les
objets examinés, datant du IV au Xir siècle, proviennent d'Egypte. On a effectué des séparations
chromatographiques de tons les échantillons an moyen de la Chromatographie liquide à haute
performance (HPLC) à phase inversée avec détection UV-visible et détection par fluorescence. La
détection par fluorescence s'est révélée meilleure que la détection à l'aide d'une barctte de diodes
UV-visible pour la détermination de quelques composants. Certains échantillons ont été analysés
par HPLC-spcctrométrie de masse, ce qui a confirmé les résultats obtenus par chromatographieUl'et a permis la détection et l'identification de composés supplémentaires. La plupart des colorants
mentionnés dans la littérature comme ayant été utilisés par les Copies ont été détectés: cochenille
d'Arménie, colorants jaunes flavonoïdes, indigotine, laque, garance et tannins. On a découvert
également quelques nouvelles compositions de couleurs significatives, qui n 'étaient pas
mentionnées auparavant dans les documents sur les textiles coptes. L'analyse élémentaire par
microscopie électronique à balayage couplée à la spectrométrie des rayons X a dispersion
d'énergie a élé effectuée afin d'identifier les mordants.
Zusammenfassung— //; dieser Arbeit wird die Identifizierung natürlicher Farbstoffe in Koptischen
Textilien beschrieben. Die untersuchten Objekte aus dem 4. bis 12. nachchristlichen ¡ahrhuiideit
gehören. Die Untersuchung der Proben erfolgt mit HPLC (Hochdruckflüssigkeitschromatographie)
mit UV-l'is-Detektor und Fluoreszenz-Detektor. Dabei envies sich die Fluorcszcuzdetektiou für
einige Komponenten als besser als die UV- ] is-Detcktion. Ausgewählte Proben wurden mit HPLCMS (HPLC/Massenspcktromctric) untersucht, wodurch die Ergebnisse der LC-UV Untersuchung
bestätigt und weitere Komponenten identifiziert werden konnten. Die meisten in der Literatur
erwähnten Farbmittel der Kopten wurden gefunden: Armenische Cochenille, gelbe
Flavonoidfiarbstoffe, Indigo, Lac-dye, Krapp und Tannine. Darüber hinaus wurden einige spezielle
Farbmittel gefunden, die bisher noch nicht in Koptischen Textilien gefunden werden konnten.
REM/EDX (Rasterelektronenmikroskopie mit energiedispersiver Röutgcumikroaualysc) wurde für
die Analyse der Bcizmittel verwendet.
Resumen — Este estudio se centra en la identificación de colorantes naturales en extrados
obtenidos de fibras de ¡ejidos coptos. Los objetos examinados pueden datarse desde el siglo II ' al
XII en Egipto. Las separaciones cromatográficas de todas las muestras se realizaron usando HPLC
de fase inversa (cromatografía líquida de alta resolución) con detección UV-Vis, y detección
adicional por fluorescencia. La detección por fluorescencia dio mejores resultados que la detección
UV-Vis para el análisis de algunos de los componentes. Varias muestras seleccionadas se
analizaron por medio de HPLC-MS (HPLC-espectromctría de masas), lo cual confirmó los
descubrimientos obtenidos con L(.-LJV, y permitió la detección e identificación de componentes
adicionales. Se detectaron la mayor parte de los colorantes mencionados en la bibliografía como
habituales en los tejidos coptos: cochinilla armenia, colorantes amarillos del tipo de los
flavonoides, indigotina, rojo de laca, rubia y tauiuos. Se descubrieron también nuevas
composiciones cu colores significativos, los cuales no habían sido mencionados anteriormente en
los estudios sobre tejidos coptos. Se llevaron a cabo estudios para la identificación elemental
usando SEM-EDS (microscopía electrónica de barrido-espectroscopia de energía dispersiva) con
el fui de identificar el tipo de mordiente.