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Identification of natural dyes in historical textiles from Romanian collections by LC-DAD and LC-MS (single stage and tandem MS)
Journal of Cultural Heritage 13 (2012) 89–97
Case study
Identification of natural dyes in historical textiles from Romanian collections by LC-DAD and LC-MS (single stage and tandem MS) Irina Petroviciu a,∗,e , Ina Vanden Berghe b , Ileana Cretu c , Florin Albu d , Andrei Medvedovici e a
National Museum of Romanian History, 030026 Bucharest, Romania Royal Institute for Cultural Heritage, 1000 Brussels, Belgium National Museum of Art of Romania, 010063 Bucharest, Romania d Bioanalytical Laboratory, S.C. LaborMed Pharma S.A., 032266 Bucharest, Romania e Department of Analytical Chemistry, Faculty of Chemistry, University of Bucharest, 050663 Bucharest, Romania b c
a r t i c l e
i n f o
Article history: Received 7 February 2011 Accepted 5 May 2011 Available online 14 June 2011 Keywords: Natural dyes Liquid chromatography Diode Array Detection Mass spectrometry (single stage MS and tandem MS) Romanian historic textiles (religious embroideries and brocaded velvets)
a b s t r a c t In this study, the dyes present in five 17th- to 18th-century textiles from the National Museum of Art of Romania, three religious embroideries and two brocaded velvets, are characterized and discussed, together with earlier results on textiles from Romanian collections obtained by the same research group. Dye analyses were performed using two methods: the well-established liquid chromatography-diode array detection (LC–DAD) and a recently developed liquid chromatography-mass spectrometry (LC–MS) analytical protocol. The examination of very small historical samples by both techniques allows a better insight in the advantages and limitations of the two approaches to real analyses to be obtained. LC–MS data interpretation is based entirely on the results accumulated for dye standards. Electrospray ionization (ESI) was used in the negative ion mode and an ion trap served as mass analyzer. Both single stage (MS) and tandem (MS/MS) mass spectrometric approaches were considered. The dyes and natural sources identified by both analytical techniques are discussed in the historical context of the textiles, with respect to earlier results collected for similar Romanian objects. The study showed that the dye sources found in the 17th- and 18th-century Romanian velvets and embroideries were produced using a wide variety of dye sources, suggesting influences from Europe as well as from Asia Minor. Dye sources imported from New World have been also detected. The range of biological sources is in very good correspondence with earlier results obtained from textiles in the Romanian Collections. LC-MS (single stage and tandem MS) approaches have been demonstrated to be valuable tools for dye identification in small-scaled samples from historical textile objects only if sufficient knowledge on the dyes and their biological sources is first accumulated within experiments performed on standard dyes and standard dyed fibers. © 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction and research aims Dyes obtained from naturally occurring biological sources have been used in textile dyeing since antiquity. The identification of their use in historic pieces may provide useful information about where, when and how these objects were created and may also contribute to their conservation. Several studies have been dedicated to the identification of dyes from biological sources in various types of textile preserved in Western European collections in the last 50 years [1–6]. In contrast, objects in Eastern European museums and monasteries – including
∗ Corresponding author. E-mail addresses: [email protected], [email protected] (I. Petroviciu), [email protected] (I. Vanden Berghe), [email protected] (I. Cretu), florin [email protected] (F. Albu), [email protected] (A. Medvedovici). 1296-2074/$ – see front matter © 2011 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.culher.2011.05.004
studies on dyes in textiles from Romanian collections – have only recently been considered as subjects for characterization of the dyes present [7–11]. In the present work, dye analysis on three religious embroideries and two brocaded velvets, dating from the 17th to 18th centuries, from the National Museum of Art of Romania is presented and discussed, together with earlier results on textiles from Romanian collections obtained by the same research group. Data resulting from the liquid chromatography–diode array detection (LC–DAD) method of analysis are compared with those produced using a newly developed liquid chromatography–mass spectrometry (LC–MS) analytical protocol, based on the progressive use of single stage (MS) and tandem (MS/MS) mass spectrometry. The comparison of the resulting pattern of dye constituents obtained through application of the two alternative techniques offers a very good insight into the advantages and limitations of the two approaches to real historical problems, where the reality of degradation of the textiles and the very limited sample sizes available
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must be faced. This results in a mutual validation of the analytical protocols developed for the identification of dyes in cultural heritage textiles. The biological sources of dyes identified in the five Romanian textiles using both techniques are evaluated in terms of period, provenance and technique, and compared with earlier results obtained from similar objects [8–10]. 2. Experimental 2.1. Historical samples Fibers about 0.5 cm long were sampled from three religious embroideries and two brocaded velvets dated to the 17th and 18th centuries from the collection of the National Museum of Art, Romania. Sample withdrawal was possible due to the fact that all the objects had recently passed through the textile restoration workshop for conservation. 2.2. References For each analytical technique, dedicated libraries of references were used. These databases were built up in the laboratories of three of the research group partners and consist of UV-visible (UV–vis), single stage and tandem MS data for the dye components discussed [12,13]. 2.3. Sample preparation Individual samples (about 0.5 mg) of dyed laboratory standard or historic fibers were extracted with hydrochloric acid (37%)/methanol/water (2:1:1, v/v/v) and prepared according to the procedures described in detail in [12] for LC–DAD and [13] for LC–MS analysis. For green-coloured samples, where the presence of indigoid dyes must be checked, an additional extraction step was included. For this, after removing the coloured solution resulting from hydrochloric acid extraction, 200 L dimethyl formamide (DMF) was added to the coloured fiber and the mixture was heated at 140 ◦ C for 10 minutes. The solution was then centrifuged at 12,000 rpm for 5 minutes and the supernatant liquid was transferred to an injection vial. 2.4. Instrumentation 2.4.1. LC-DAD Waters LC–DAD equipment was used, data acquisition and treatment being made by the Empower Pro 2002 software. Separation was achieved on a LiChrosorb RP-18 column, 125 mm L × 4 mm i.d. × 5 m d.p. The mobile phase consisted of a mixture of methanol (solvent A), methanol in water (1/9, v/v) (solvent B) and an aqueous (5%, v/v) solution of phosphoric acid (solvent C). Gradient elution was applied according to the profile given below, which includes the re-equilibration step. Time
Solvent A (%)
Solvent B (%)
Solvent C (%)
0 3 29 30 35
23 23 90 23 23
67 67 0 67 67
10 10 10 10 10
The flow rate was set at 1.2 mL/min. The volume injected was 20 L from which 5 L will pass through the column for analysis. The other 15 l is sent to the waste. Detection was made within a 200–800 nm wavelength range, with a spectral resolution of 1.2 nm. Chromatograms were integrated systematically at 254 nm and also at one or more other wavelengths at which the optimum response
of the dye constituent is observed. Results are presented as the relative composition of dyes at the wavelength(s) of integration. 2.4.2. LC-MSD LC–MS and LC–MS/MS experiments were achieved on a system constructed from Agilent Series 1100 modules. Detection was made through a MS/MS ion trap detector using an electrospray ionisation (ESI) ion source, operated in negative ion mode. The control of the chromatographic system and data acquisition were achieved with the Agilent ChemStation software LC 3D version 10.02, incorporating the MSD trap control, version 5.2, from Brucker Daltronics. Separation was achieved on a Zorbax C18 column, 150 mm L × 4.6 mm i.d. × 5 m d.p., thermostated at 40 ◦ C. The mobile phase consisted of a mixture of aqueous 0.2% (v/v) formic acid (solvent A) and methanol/acetonitrile (1:1 v/v, solvent B). Gradient elution was applied according to the profile given below, which includes the re-equilibration step. Time 0 5 10 16 18 18.01 22
Solvent B (%) 15 25 55 100 100 15 15
The flow rate was set at 0.8 mL/min. The injected volume was 5 L (from a total of about 200 L resulting from the sample preparation). Several injections from the same solution may be performed, as described in the Results section below. The DAD detector was placed in series between the column and the MS ion source. UV–vis spectra were acquired over the 200–800 nm range with a resolution of 2 nm. MS detection was made in the negative ion mode with the following ESI operational parameters: drying gas temperature 350 ◦ C; drying gas flow rate 12 L/min; nebulising gas pressure 65 psi; capillary high voltage 2484 V. The ion trap used a maximum accumulation time of 300 ms and a total charge accumulation (ICC) of 30000. The multiplier voltage was set at 2000 V and the dynode potential at 7 kV. When working in the MS/MS mode, the spectral width was 4 a.m.u. and the collisional induced dissociation amplitude 1.6 V. Automated Mass Spectral Deconvolution and Identification System (AMDIS) software was used as a complementary identification tool. Chromatograms obtained with full scan single stage mass spectrometric detection were exported in the Agilent MS Engine format (.ms) and analyzed with the AMDIS software [13]. 3. Results and discussions Samples described in Table 1 were analyzed by LC–DAD and LC–MS. Fig. 1 shows the 17th-century Epimanikia (right hand sleeve, after cleaning) described in Table 1. Table 2 summarizes DAD and MS data obtained for the samples analyzed. For MS analysis, the detection of dyes was made according to a dedicated analytical protocol, described in detail in an earlier publication [13]. It includes chromatographic separation and single MS full scan (FS) detection, followed by data processing through ion extraction chromatograms (IEC) according to m/z values of the molecular ions of dyes in the database. Results are correlated with UV–vis spectral data. For minor compounds the sample was re-injected using single stage MS detection in the selected ion monitoring/multiple ion monitoring modes (SIM/MID) as well as by using tandem MS, product ion scan/single reaction monitoring (SRM)/multiple reaction monitoring (MRM), for unambiguous confirmation of dye components. The confirmation procedure for the dye constituents (single MS-FS, single MS-SIM or tandem MS) may
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Table 1 Characterization of samples discussed in the present study. Textile object (period)
Function of the threads
Color
Sample code
Bedernita, Religious embroidery (1746)
Lining Sewing thread Embroidery thread Thread sewing the lining
Green Pink yellow Green Kaki
A1 A12 A13 A14
Bedernita (lining), religious embroidery (1746)
Embroidery thread Sewing thread Sewing thread
Kaki Green Brown
B15 B17 B20
Epimanikia, Religious embroidery (17th c.)
Embroidery support (edge) Embroidery support (edge) Embroidery support (edge) Lining Embroidery thread Embroidery thread Embroidery thread
Green Pink Pink yellow Yellow Orange Red Green
D23a D23b D23c D24 D26 D27 D29
Sakkos, Brocaded velvet (17th c.)
Embroidery thread Embroidery thread Silk core metallic embroidery thread Lining Lining
Pink yellow Red Pink yellow Green Red
F37a F37b F38 F39 F40
Robe of Princess, brocaded velvet (16–17th c.)
Weft
Green
E34
be directly correlated with the occurrence of the specific analyte in the sample (Table 2). When the semi-quantitative evaluation made using diode array detection (indicated through normalisation of a peak area to the sum of the peak areas in the chromatogram) gives a result for the constituent of interest greater than 10%, the FS operating mode is usually sufficient for the MS detection. If the semi-quantitative DAD evaluation for the constituent is between 1 and 10%, single stage MS in the SIM mode or MS/MS approaches are necessary for successful MS detection. When DAD detection indicates the occurrence of a compound at a level below 0.5%, it may be necessary to use AMDIS deconvolution software for MS detection. 3.1. Red dyes Red dyes were detected in eight samples, four from religious embroideries and four from brocaded velvets. Anthraquinone dyes were present in all the cases where samples still have a visibly red color (3/8 samples). In one of these samples carminic acid, the main dye component in Mexican cochineal (Dactylopius coccus Costa), Armenian cochineal (Porphyrophora hameli Brandt) and Polish cochineal (Porphyrophora polonica L.), was detected by DAD. Its presence was also confirmed by MS in both cases. Three minor
compounds, dcII (the C-glucoside of flavokermesic acid [14,15]), kermesic and flavokermesic acids are also present in fibers dyed with cochineal. In the present work, only dcII was detected by the presence of its molecular ion (m/z = 475), according to the FS-Ion Extracted Chromatogram (IEC). The presence of kermesic and flavokermesic acids was confirmed by the profiles of their fragments after spectral deconvolution with AMDIS (Fig. 2). Based on the calculation between the ratio of the minor compounds and carminic acid in HPLC-DAD analysis [16–18], the biological source in F37b was established as Mexican cochineal (D. coccus Costa). For two other red samples alizarin and purpurin, the main anthraquinone constituents in madder (Rubia tinctorum L.), were detected by both DAD and MSD. The presence of minor anthraquinone compounds from madder, anthragallol, munjistin, xanthopurpurin and rubiadin was also established. In five cases described as “pink” or “pink yellow” a marker compound for redwood (Caesalpinia spp.) dyeings, called “srw–soluble redwood” according to Wouters [4] or “type C” by Nowik [19] was identified by both DAD and MSD. For the latter, identification was made by FS-single stage MS followed by IEC of m/z = 243 a.m.u., the major ion of “srw” according to the literature [13,20]. Confirmation of this identification was achieved by detection by single stage MS in the SIM mode and by MS/MS.
3.2. Yellow dyes
Fig. 1. Epimanikia described in Table 1. Religious embroidery (17th century) worked in the Byzantine tradition, preserved in the Art Collection Museum, National Museum of Art of Romania, inv. 88371.
Flavonoid yellow dyes were detected in 15 out of a total of 20 analyzed samples. Dyer’s broom (Genista tinctoria L.) was identified as the main biological source in five samples and as a second biological source in two other samples. In all the green samples blue “indigo” dyes (from Isatis tinctoria L., Indigofera spp. or other species, discussed below) were also present. The identification of dyer’s broom was based on the detection of luteolin, genistein and apigenin, by both LC–DAD and MS detection techniques. In almost all cases, chrysoeriol – a minor compound recently identified in both weld and dyer’s broom [13,15,21,22] – was also detected by single stage MS in SIM mode or by MS/MS. Luteolin and apigenin – without the presence of genistein – were identified by both DAD and MSD in five samples, only one being from brocaded velvet, a dyestuff constituent profile suggesting
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Table 2 Results obtained by alternative diode array and mass spectrometric (MS or MS/MS) detection modes. Sample code Visual color
Results
Biological source b
Dye constituents MSD
Main source(s)
Traces
51 lu, 29 ge, 8 ap, 2 chry, 10 in 77 srw, 7 qu, 1 kf, 7 rht, 2 rhz, 6 ea 90 lu, 6 ap, 2 chry, 2 in 32 dat, 5 ge + lu, 4 kf, 1 irht, 1 ap, 23 in, 28 em, 5 chrys
lu(1), ge(1), ap(1), chry(2), in(2) srw(1), qu(2), kf(2), ea(2)
Dyer’s broom and “indigo” Redwood and buckthorn berries
– Tannin plantc
lu(1), ap(1), in(2) lu(1), ge(2), ap(2), dat(1,3), em(1), in(2)
Weld and “indigo”d Bastard hemp, rhubarbe and “indigo”
Dyer’s broom
lu(1), ap(2), dat(1,3), em(1), kf(2), in(2)
Bastard hemp, rhubarb, weld or eq. and “indigo” –
lu(1), ge(1), ap(1), chry(2), in(2), lu(2) lu(1), ge(1), ap(2), chry(3), in(2) srw(1), ca(1,3), ea(2), fk(*), ka(*)
Dyer’s broom and “indigo” Weld (or another flavone-containg plant) Dyer’s broom and “indigo” (Mexican) cochineal, redwood and tannin plant
A1 A 12
Green Yellow pink
A 13 A 14 B 15
Green Kaki (yellow/green) Kaki
B 17 B 20 D 23 a D 23 b
Green Brown Green Pink
D 23 c D 24
Pink yellow Yellow
D 26 D 27 D 29
Orange Red Green
F 37 a F 37 b
Yellow pink Red
F 38
Pink yellow
68 srw, 14 fi, 15 sul, 3 ea 71.5 ca, 1.5 dcII, 1 fk, 0.5 ka, 25.5 ea, [288 nm: 0.8 dcII, 97.7 ca, 1.5 fk + ka] 81 srw, 10 fi, 9 sul, +ea
F 39 F 40 E 34
Green Red Green
39 lu, 46 ge, 12 ap, 1 chry, 2 in 87 al, 12 pu, +ag 57 lu, 5 ap, 1 chry, 37 in
18 dat, 25 lu, 1 ap, 19 in, 32 em, 1 kf, 4 chrys 43 lu, 43 ge, 10 ap, 1 chry, 3 in 92 lu, 8 ap 32 lu, 66 ge, 1 ap, +chry, 1 in 64.5 ca, 12 srw, 23 ea, +fk, 0.5 ka 90 srw, 1 lu, 9 ge 54 lu, 36 ge, 2 ap, 1 chry, 5 ea, 2 qu, +fi, +sul 35 fi, 56 sul, 8 ea, + kf 69 al, +xp, 25 pu, 1 ru, +ag, 46 lu, 4 ap, 2 chry, 43 in, 4 ea, 0.5 fi, 0.5 sul
Redwood srw(1), lu(2), ge(2), ap(3) lu(1), ge(1), ap(2), chry(2), fi(3), sul(3), ea(2,3), qu(3), Dyer’s broom and redwood
– – – Dyer’s broom Young fustic and qu based dye
fi(1,3), sul(1,3), ea(2) pu(1), ru(1), al(1), mu(1), xp(1), ag(2) lu(1), ap(2), chry(2), fi(2), sul(2), in(2) srw(1,2), fi(2), sul(2), ea(2) ca(1), dcII(2), fk(*), ka(*), ea(2)
Young fustic Madder Weld and “indigo”
– Young fustic
Redwood, young fustic (Mexican) Cochineal and tannin plant
–
srw(1,3), fi(2), sul(2), ea(2) lu(1),ge(1), ap(1), chry(2), in(2) al(1), pu(1), ru(1), ag(2) lu(1), ap(2), chry(2), in(2)
Redwood, young fustic
Dyer’s broom and “indigo” Madder Weld and “indigo”
– – –
The following abbreviations were used: al: alizarin; ag: anthragallol; ap: apigenin; ca: carminic acid; chrys: chrysophanic acid; chry: chrysoeriol; dat: datiscetin; ea: ellagic acid; em: emodin; fi: fisetin; fk: flavokermesic acid (also called laccaic acid D); dcII: flavokermesic acid, C-glucoside; ge: genistein; in: indigotin; irht: isorhamnetin; kf: kaempferol; ka: kermesic acid; laA: laccaic acid A; lu: luteolin; mu: munjistin; pu: purpurin; qu: quercetin; rht: rhamnetin; rhz: rhamnazin; ru: rubiadin; srw: soluble redwood; sul: sulfuretin; xp: xanthopurpurin. a Numbers before the abbreviation for a dye represent the relative composition (%) corresponding to peak areas integrated in the chromatogram monitored at 255 nm (unless specified in the column); “ + ” indicates values lower than 0.5%. b Numbers between brackets have the following meaning: (1) detected through single stage MS–FS/IEC; (2) detected through MS–SIM/MID modes; (3) detected through MS/MS (MRM, product ion scan); (*) is used for dyes evidenced through deconvolution by AMDIS software. c The term “tannin plant” is used as a shorter name for “tannin-producing plant” and is not indicative of a particular dye source. d “Indigo” refers to several plants that produce indigo. e The term “rhubarb” should be read as “rhubarb or another emodin-containing plant”.
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Dye constituents DADa
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Fig. 2. LC–MS and LC–MS/MS of sample F37b (red), where (Mexican) cochineal was identified. Upper image, from top to bottom, the UV–vis chromatogram; MS–FS, IEC for carminic acid (molecular ion m/z = 491 and ion produced by decarboxylation in the source m/z = 447); IEC for dcII (molecular ion m/z = 475 and ion produced by decarboxylation in the source m/z = 431); IEC for ellagic acid. Lower images: detail of ion profiles after AMDIS deconvolution for flavokermesic (molecular ion m/z = 313 and ion produced by decarboxylation in the source m/z = 269) and kermesic (molecular ion m/z = 329 and ion produced by decarboxylation in the source m/z = 285) acids. The figure illustrates the flexibility in use of mass spectrometry which allows the gradual detection of minor compounds.
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Fig. 3. LC–DAD chromatograms for sample B15, integrated at 255 and 350 nm, respectively, with detection of datiscetin, emodin, indigotin, luteolin, apigenin, kaempferol and chrysophanic acid, suggesting the use of bastard hemp, weld (or another flavone-containing plant; indigo/woad and rhubarb (or another emodin containing plant) the compound marked with “?” has similar UV spectrum to datiscetin, but different retention time.
the use of weld (Reseda luteola L.). However, recent studies have demonstrated that other biological sources containing luteolin and apigenin, of which sawwort (Serratula tinctoria L.) is the most well known [14,15], also exist and could have been used as textile dyes. As a consequence, only those samples where chrysoeriol was detected together with luteolin and apigenin are likely to have been dyed using weld; in the other cases the use of another flavone-containing plant may not be excluded. When the presence of weld (or another flavone-containing plant) corresponded to green-coloured fibers, “indigo” dyes were also detected, while in another sample a combination of rhubarb (or another emodincontaining plant), bastard hemp and “indigo” dyes were found to have been used. Fisetin and sulphuretin, the main dye components in young fustic (Cotinus coggygria L.) were detected in three samples within the present study. Young fustic was detected as a single biological source in one case and was identified together with soluble redwood in pink-yellow samples. Traces of young fustic were also detected in two additional samples where fisetin and sulphuretin were detected by single stage MS in SIM mode or by MS/MS product ion scan. Datiscetin, the main dye component in bastard hemp (Datisca cannabina L.) was identified by DAD in two kakicoloured samples, in both cases together mainly with emodin and with chrysophanic acid, kaempferol and isorhamnetin as minor constituents, as well as flavonoids from either dyer’s broom or weld (or another flavone-containing plant) and “indigo” (Fig. 3). Poor chromatographic resolution is obtained between datiscetin and luteolin. Additionally, another compound exhibiting a very similar UV–vis spectrum to datiscetin, but having increased retention, could be observed in the chromatogram. Evidence for the presence of datiscetin (m/z = 285) in both samples was based on the MS information collected from a standard sample of wool dyed with bastard hemp. As no other dye was reported in this source, the identification of datiscetin by MS was also confirmed by MS/MS analysis, based on the fragmentation pattern obtained through product ion scan. In LC–MS separations, as
illustrated in Fig. 4, datiscetin co-elutes with kaempferol; however, comparison of the MS/MS product ion scan spectra allowed identification of both datiscetin and kaempferol in the kaki-coloured sample B15. Rhamnetin was identified by DAD together with quercetin, kaempferol and rhamnazin in an ochre-yellow sample (A12), suggesting the use of berries from a species of buckthorn (Rhamnus spp.). Analyses of two wool references, dyed with either the bark or the fruits (berries) from alder buckthorn (Rhamnus frangula L.) resulted in the detection of mainly emodin and minor amounts of rhamnetin and quercetin for the former, bark-dyedsample, and mainly rhamnetin, isorhamnetin and quercetin for the latter. The components detected in sample A12 by MS thus confirm the use of berries from a Rhamnus species as the source of dye. Emodin, the main dye component in alder buckthorn bark, rhubarb, yellow dock and other biological sources was also detected in two samples. It was not possible to evidence the presence of chrysophanic acid (m/z = 253) by MS, based on the IEC, nor by the identification of the fragment m/z = 239, which would correspond to the fragment induced by de-methylation. A more detailed study on a pure standard of chrysophanic acid (not available at the time of the present experiments) is needed in order to be able to identify it in historical samples. The limited information on these two anthraquinone dyes of vegetable origin, emodin and chrysophanic acid, as textile dyes may be explained by their rare identification in historical textiles. Like emodin, the presence of chrysophanic acid may indicate the use of alder buckthorn bark (Rhamnus frangula L.), or the roots of rhubarb (Rheum sp.) or dock (Rumex sp.) [23–25]. However, no systematic study in which the biological source of the dye has been identified unambiguously as one or other of these plants has so far been reported. 3.3. Blue dyes Although blue-coloured samples were not selected for analysis, indigotin was detected in eight green-coloured samples. For mass spectrometric detection, FS-single stage MS in SIM mode according to the molecular ion of indigotin (m/z = 261) was used.
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Fig. 4. LC–MS and LC–MS/MS data from sample B15, supporting the identification of datiscetin, emodin, luteolin, apigenin, and kaempferol (identification for indigotin is not given; chrysophanic acid, although detected by LC–DAD, was not detected by LC–MS).
Indigotin is the main dye component in woad, Isatis tinctoria L. and in the indigo plant itself, Indigofera spp. but no analytical method has been reported to distinguish between these species [26]. The term “indigo” is thus used to refer to indigotin-producing plants.
3.4. Tannin Ellagic acid was detected in seven samples with both detection techniques. The presence of ellagic acid indicates the use of a tannin-containing plant material either for textile dyeing or for
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Table 3 Biological sources attributed to samples analyzed throughout the present study, compared to other results for samples from religious embroideries and brocaded velvets from Romanian collections previously identified through the LC–DAD method by the same research group [8–10]. Religious embroideries 15th-16th c.
Cochineal All (DC) Kermes Lac dye Madder Safflower Redwood Young fustic Weld (or another flavone-containing plant) Dyer’s broom Buckthorn berries Rhubarb (or another emodin-containing plant) Bastard hemp Tannins Logwood Indigoid
6 (3)–16th c. 3 21 29 2 24 24 26 8 0 2 1 39 0 40
Brocaded velvets
17th–18th c.
19th c.
Present
Previous
1 (1) 0 0 1 0 3 1 4 4 1 2 2 1 0 5
3 (1) 0 0 3 1 4 1 3 0 3 2 2 5 1 7
weighting silk [4,24]. In the present study, a high proportion of ellagic acid from a tannin-producing plant source was detected twice in red samples in combination with cochineal while much lower proportions were detected in a yellow pink sample together with redwood and buckthorn berries. Ellagic acid was also detected in all the five cases when fisetin and sulphuretin were identified, indicating the use of the dye from the heartwood of young fustic. This is probably due to the presence of a trace of the tannins present in young fustic (primarily in the leaves and twigs). 3.5. Discussion on the biological sources together with previous results on religious embroideries and brocaded velvets from Romanian collections Attribution of the biological sources of dyes identified and confirmed in the analyzed samples is summarized in Table 2. All the sources detected in the present series of analyses were also identified in one or more groups of samples analyzed before by the same research team. According to the studies performed until now, six sources of red dye have been identified in religious embroideries and brocaded velvets from Romanian collections dating from the 15th to the 19th centuries (Table 3). Half of these are of animal origin (cochineal, kermes and lac) and half derive from plant sources, and from various parts of the plants: roots (madder), petals (safflower) and wood (redwood). Cochineal (both Old and New World) and redwood were detected in all the textile groups studied; lac dye, madder and safflower were only present in embroideries. The combination of lac dye and madder was the main source of red used in the support fabric for embroideries in the 15th and 16th centuries. According to literature, lac was hardly used in Europe for textile dyeing, but only for dyeing leather and as an organic pigment. It was mentioned as imported into the Ottoman Empire as early as the 15th century and, according to literature, it has been detected in Ottoman textiles [27,28]. Its use in religious embroideries would thus suggest an Oriental origin for these materials. Kermes was only identified in religious embroideries and brocaded velvets dated to the 15th and 16th centuries, which is in accordance with literature mentioning that, due to its lower cost and ease of use Mexican cochineal eventually became the only animal source of red dye used in Europe, soon after the discovery of the New World [25].
1 (0) 0 0 0 0 3 3 4 1 2 1 0 2 1 5
15th–16th c.
2 (0) 1 0 0 0 4 3 7 0 0 0 0 6 0 3
17th c. Present
Previous
1 (1) 0 0 1 0 2 2 1 1 0 0 0 0 0 2
0 0 0 0 0 0 0 1 0 2 0 0 0 3 0 1
Six sources of yellow flavonoids were identified in the series, weld (or another flavone-containing plant), young fustic and dyer’s broom being the most widely used. Except for the latter that was not present in 15th–16th century embroideries, the others were detected in all the groups considered. Bastard hemp, buckthorn berries and rhubarb (or another emodin-containing plant) were only detected in embroideries, bastard hemp up to the 18th century, buckthorn berries not before the 17th century and rhubarb (or another emodin-containing plant source) in textiles from the 15th to the 18th century. However, both bastard hemp and rhubarb (or another emodin-containing plant) were only detected in Ottoman textiles [28], which also supports the suggestion of an Oriental origin for the materials. As far as the blue dyes are concerned, “indigo” was identified in religious embroideries and brocaded velvets from the 15th to the 19th century, while logwood was only present in 17th- to 19thcentury embroideries. This is no surprise when we remember that this source was only available after the discovery of the New World [29]. 4. Conclusions All the dyes identified on the 17th- and 18th-century Romanian velvets and embroideries are in very good correlation with the existing knowledge on dyes and biological sources used in Europe and Asia Minor during this period [1,2,5,6,24,25,28]. The results may be also correlated in terms of period and fiber function with previous data obtained for similar textiles by the same research team [8–10]. From the whole group of analyses performed up till the present on textiles from Romanian collections, it may be concluded that the combination of lac dye and madder was the main source of red in the 15th- to 16th-century embroideries, while kermes was used when a more precious textile was intended. Mexican cochineal was preferred in later textiles. Lac dye, bastard hemp and rhubarb (or another emodin-containing plant) were only detected in embroideries. Based on this fact and considering that these dye sources were not identified in textiles from Europe, but only in Ottoman pieces, it may be stated that at least part of the materials used in embroideries dating from the 15th to the 18th centuries have an Oriental origin. As far as the techniques applied are concerned, it can be concluded that a good correlation between the results produced
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by LC–DAD, LC–MS and LC–MS/MS was found, not only for the major dye components, but also for the minor accompanying constituents. For some of the minor components co-eluting under the chromatographic conditions of the LC–MS method, spectral deconvolution with AMDIS software was established. The study showed that LC–MS and LC–MS/MS approaches were confirmed as versatile tools for the identification and confirmation of dyes in historic textiles, if consistent work is first accumulated on a collection of standard dyes and dyed fibers for construction of suitable spectral libraries. Acknowledgements The authors would like to thank the National Museum of Art of Romania and the Putna Monastery, Romania for access to their collections. They are also grateful to LaborMed Pharma, who offered an open access to the LC–MS/MS analytical instrumentation and to Ms Marie-Christine Maquoi from the KIK/IRPA laboratory for the expert assistance in LC–DAD dye analysis. Professor Recep Karadag from Marmara University, Istanbul, Turkey, who offered a bastard hemp-dyed fibre, is also acknowledged. The authors are also grateful to Ms. Jo Kirby, National Gallery London Scientific Department (retired), for carefully reading and improving the English text. References [1] J.H. Hofenk de Graaff, W.G. Th Roelofs, On the occurrence of red dyestuffs in textile materials from the period 1450–1600, in: 4th Meeting ICOM Committee for Conservation, Madrid, 1972. [2] J.H. Hofenk de Graaff, T.W.G. Roelofs, The analysis of flavonoids in natural yellow dyestuffs occuring in ancient textiles, in: 5th Meeting ICOM Committee for Conservation, Zagreb, 1978, p. 1–15. [3] J. Wouters, Analyse des colorants des tapisseries brugeoises, in: Bruges et la tapisserie des xvie et xviie siècles, Mouscron, Bruges, 1987, p. 515–526. [4] J. Wouters, Dye analysis of Florentine borders of the 14th to 16th centuries, Dyes in History and Archaeology 14 (1995) 48–58. [5] I. Karapanagiotis, L. Valianou, Y. Sister Daniila, Chryssoulakis, Organic dyes in Byzantine and post-Byzantine icons from Chalkidiki (Greece), Journal of Cultural Heritage 8 (2007) 294–298. [6] M. Van Bommel, J.H. Hofenk de Graaff, Master dyers to the court of Sicily, Dyes in History and Archaeology, 22/23, publication due 2012. [7] I. Petroviciu, J. Wouters, Analysis of natural dyes from Romanian 19th and 20th century ethnographical textiles by DAD-HPLC, Dyes in History and Archaeology 18 (2002) 57–62. [8] I. Petroviciu, J. Wouters, I. Vanden Berghe, I. Cretu, Dyes in some textiles from the Romanian Medieval Art Gallery, Dyes in History and Archaeology 22/23, publication due 2012.
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[9] I. Petroviciu, J. Wouters, I. Vanden Berghe, I. Cretu, Dye analysis on some 15th Century byzantine embroideries, Dyes in History and Archaeology. 22/23, publication due 2012. [10] I. Petroviciu, I. Vanden Berghe, I. Cretu, J. Wouters, Analysis of Dyestuffs in 15th–17th Century byzantine embroideries from Putna Monastery, Romania, Dyes in History and Archaeology, 24/25, publication due 2012. [11] M. Trojanowicz, J. Orska-Gawrys, I. Surowiec, B. Szostek, Chromatographic investigation of dyes extracted from Coptic texiles from the National Museum in Warsaw, Studies in Conservation 49 (2004) 115–130. [12] J. Wouters, N. Rosario-Chirinos, Dye analysis of pre-combian peruvian textiles with HPLC and DAD, Journal of the American Institute for Conservation 31 (1992) 237–255. [13] I. Petroviciu, F. Albu, A. Medvedovici, LC/MS and LC/MS/MS based protocol for identification of dyes in historic textiles, Microchemical Journal 95 (2010) 247–254. [14] D. Peggie, The development and application of analytical methods for the identification of dyes on historical textiles, PhD thesis, University of Edinburgh (2006). [15] D. Peggie, A. Hulme, Mc. Nab, H.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), Microchemica Acta 162 (2008) 371–380. [16] J. Wouters, A. Verhecken, The Coccid insect dyes: HPLC and computerized analysis of dyed yarns, Studies in Conservation 34 (1989) 189–200. [17] J. Wouters, A. Verhecken, The scale insect dyes (Homoptera:Coccoidea) species recognition by HPLC and Diode-Array analysis of the dyestuffs, Annales de la Société entomologique de France 25 (1989) 393–410. [18] I. Vanden Berghe, Investigation of cochineal dyeing parameters and refinement of the cochineal identification system’, Dyes in History and Archaeology22/23, publication due 2012. [19] 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) 129–144. [20] I. Karapanagiotis, E. Minopoulou, L. Valianou, Y. Sister Daniilia, Chryssoulakis, Investigation of the colourants used in icons of the Cretan school of iconography, Analytica Chimica Acta 647 (2009) 231–242. [21] E. Ferreira, New approaches towards the identification of yellow dyes in 463 ancient textiles, PhD thesis, University of Edinburgh (2001). [22] http://www.organic-colorants.org. [23] H. Schweppe, Handbuch der Naturfarbstoffe, Vorkommen, Verwendung, Nachweis, Nikol Verlagsgesellschaft mbH&Co, KG, Hamburg, 1993, p. 224–228. [24] J.H. Hofenk de Graaff, The colourful past. Origins chemistry and identification of natural dyestuffs, Abegg Stiftung & Archetype Publications Ltd, Riggisberg and London, 2004. [25] D. Cardon, Natural dyes–sources, tradition, technology science, Archetype Publications, London, 2007. [26] J. Wouters, Possible future developments in the analysis of organic dyes, Dyes in History and Archaeology 20 (2005) 23–29. [27] I. Vanden Berghe, M.C. Maquoi, J. Wouters, Dye analysis of Ottoman silk, in: M. van Raemdonck (Ed.), The Ottoman silk textiles from the Royal Museums of Art and History in Brussels, Turhout Brepols, Brussels, 2004, pp. 49–60. [28] N. Enez, H. Böhmer, Ottoman textiles: dye analysis, results and interpretation, Dyes in History and Archaeology 14 (1995) 39–44. [29] F. Brunello, The art of dyeing in the History of Mankind, Vicenza, 1973, p. 175–221.
Case study
Identification of natural dyes in historical textiles from Romanian collections by LC-DAD and LC-MS (single stage and tandem MS) Irina Petroviciu a,∗,e , Ina Vanden Berghe b , Ileana Cretu c , Florin Albu d , Andrei Medvedovici e a
National Museum of Romanian History, 030026 Bucharest, Romania Royal Institute for Cultural Heritage, 1000 Brussels, Belgium National Museum of Art of Romania, 010063 Bucharest, Romania d Bioanalytical Laboratory, S.C. LaborMed Pharma S.A., 032266 Bucharest, Romania e Department of Analytical Chemistry, Faculty of Chemistry, University of Bucharest, 050663 Bucharest, Romania b c
a r t i c l e
i n f o
Article history: Received 7 February 2011 Accepted 5 May 2011 Available online 14 June 2011 Keywords: Natural dyes Liquid chromatography Diode Array Detection Mass spectrometry (single stage MS and tandem MS) Romanian historic textiles (religious embroideries and brocaded velvets)
a b s t r a c t In this study, the dyes present in five 17th- to 18th-century textiles from the National Museum of Art of Romania, three religious embroideries and two brocaded velvets, are characterized and discussed, together with earlier results on textiles from Romanian collections obtained by the same research group. Dye analyses were performed using two methods: the well-established liquid chromatography-diode array detection (LC–DAD) and a recently developed liquid chromatography-mass spectrometry (LC–MS) analytical protocol. The examination of very small historical samples by both techniques allows a better insight in the advantages and limitations of the two approaches to real analyses to be obtained. LC–MS data interpretation is based entirely on the results accumulated for dye standards. Electrospray ionization (ESI) was used in the negative ion mode and an ion trap served as mass analyzer. Both single stage (MS) and tandem (MS/MS) mass spectrometric approaches were considered. The dyes and natural sources identified by both analytical techniques are discussed in the historical context of the textiles, with respect to earlier results collected for similar Romanian objects. The study showed that the dye sources found in the 17th- and 18th-century Romanian velvets and embroideries were produced using a wide variety of dye sources, suggesting influences from Europe as well as from Asia Minor. Dye sources imported from New World have been also detected. The range of biological sources is in very good correspondence with earlier results obtained from textiles in the Romanian Collections. LC-MS (single stage and tandem MS) approaches have been demonstrated to be valuable tools for dye identification in small-scaled samples from historical textile objects only if sufficient knowledge on the dyes and their biological sources is first accumulated within experiments performed on standard dyes and standard dyed fibers. © 2011 Elsevier Masson SAS. All rights reserved.
1. Introduction and research aims Dyes obtained from naturally occurring biological sources have been used in textile dyeing since antiquity. The identification of their use in historic pieces may provide useful information about where, when and how these objects were created and may also contribute to their conservation. Several studies have been dedicated to the identification of dyes from biological sources in various types of textile preserved in Western European collections in the last 50 years [1–6]. In contrast, objects in Eastern European museums and monasteries – including
∗ Corresponding author. E-mail addresses: [email protected], [email protected] (I. Petroviciu), [email protected] (I. Vanden Berghe), [email protected] (I. Cretu), florin [email protected] (F. Albu), [email protected] (A. Medvedovici). 1296-2074/$ – see front matter © 2011 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.culher.2011.05.004
studies on dyes in textiles from Romanian collections – have only recently been considered as subjects for characterization of the dyes present [7–11]. In the present work, dye analysis on three religious embroideries and two brocaded velvets, dating from the 17th to 18th centuries, from the National Museum of Art of Romania is presented and discussed, together with earlier results on textiles from Romanian collections obtained by the same research group. Data resulting from the liquid chromatography–diode array detection (LC–DAD) method of analysis are compared with those produced using a newly developed liquid chromatography–mass spectrometry (LC–MS) analytical protocol, based on the progressive use of single stage (MS) and tandem (MS/MS) mass spectrometry. The comparison of the resulting pattern of dye constituents obtained through application of the two alternative techniques offers a very good insight into the advantages and limitations of the two approaches to real historical problems, where the reality of degradation of the textiles and the very limited sample sizes available
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must be faced. This results in a mutual validation of the analytical protocols developed for the identification of dyes in cultural heritage textiles. The biological sources of dyes identified in the five Romanian textiles using both techniques are evaluated in terms of period, provenance and technique, and compared with earlier results obtained from similar objects [8–10]. 2. Experimental 2.1. Historical samples Fibers about 0.5 cm long were sampled from three religious embroideries and two brocaded velvets dated to the 17th and 18th centuries from the collection of the National Museum of Art, Romania. Sample withdrawal was possible due to the fact that all the objects had recently passed through the textile restoration workshop for conservation. 2.2. References For each analytical technique, dedicated libraries of references were used. These databases were built up in the laboratories of three of the research group partners and consist of UV-visible (UV–vis), single stage and tandem MS data for the dye components discussed [12,13]. 2.3. Sample preparation Individual samples (about 0.5 mg) of dyed laboratory standard or historic fibers were extracted with hydrochloric acid (37%)/methanol/water (2:1:1, v/v/v) and prepared according to the procedures described in detail in [12] for LC–DAD and [13] for LC–MS analysis. For green-coloured samples, where the presence of indigoid dyes must be checked, an additional extraction step was included. For this, after removing the coloured solution resulting from hydrochloric acid extraction, 200 L dimethyl formamide (DMF) was added to the coloured fiber and the mixture was heated at 140 ◦ C for 10 minutes. The solution was then centrifuged at 12,000 rpm for 5 minutes and the supernatant liquid was transferred to an injection vial. 2.4. Instrumentation 2.4.1. LC-DAD Waters LC–DAD equipment was used, data acquisition and treatment being made by the Empower Pro 2002 software. Separation was achieved on a LiChrosorb RP-18 column, 125 mm L × 4 mm i.d. × 5 m d.p. The mobile phase consisted of a mixture of methanol (solvent A), methanol in water (1/9, v/v) (solvent B) and an aqueous (5%, v/v) solution of phosphoric acid (solvent C). Gradient elution was applied according to the profile given below, which includes the re-equilibration step. Time
Solvent A (%)
Solvent B (%)
Solvent C (%)
0 3 29 30 35
23 23 90 23 23
67 67 0 67 67
10 10 10 10 10
The flow rate was set at 1.2 mL/min. The volume injected was 20 L from which 5 L will pass through the column for analysis. The other 15 l is sent to the waste. Detection was made within a 200–800 nm wavelength range, with a spectral resolution of 1.2 nm. Chromatograms were integrated systematically at 254 nm and also at one or more other wavelengths at which the optimum response
of the dye constituent is observed. Results are presented as the relative composition of dyes at the wavelength(s) of integration. 2.4.2. LC-MSD LC–MS and LC–MS/MS experiments were achieved on a system constructed from Agilent Series 1100 modules. Detection was made through a MS/MS ion trap detector using an electrospray ionisation (ESI) ion source, operated in negative ion mode. The control of the chromatographic system and data acquisition were achieved with the Agilent ChemStation software LC 3D version 10.02, incorporating the MSD trap control, version 5.2, from Brucker Daltronics. Separation was achieved on a Zorbax C18 column, 150 mm L × 4.6 mm i.d. × 5 m d.p., thermostated at 40 ◦ C. The mobile phase consisted of a mixture of aqueous 0.2% (v/v) formic acid (solvent A) and methanol/acetonitrile (1:1 v/v, solvent B). Gradient elution was applied according to the profile given below, which includes the re-equilibration step. Time 0 5 10 16 18 18.01 22
Solvent B (%) 15 25 55 100 100 15 15
The flow rate was set at 0.8 mL/min. The injected volume was 5 L (from a total of about 200 L resulting from the sample preparation). Several injections from the same solution may be performed, as described in the Results section below. The DAD detector was placed in series between the column and the MS ion source. UV–vis spectra were acquired over the 200–800 nm range with a resolution of 2 nm. MS detection was made in the negative ion mode with the following ESI operational parameters: drying gas temperature 350 ◦ C; drying gas flow rate 12 L/min; nebulising gas pressure 65 psi; capillary high voltage 2484 V. The ion trap used a maximum accumulation time of 300 ms and a total charge accumulation (ICC) of 30000. The multiplier voltage was set at 2000 V and the dynode potential at 7 kV. When working in the MS/MS mode, the spectral width was 4 a.m.u. and the collisional induced dissociation amplitude 1.6 V. Automated Mass Spectral Deconvolution and Identification System (AMDIS) software was used as a complementary identification tool. Chromatograms obtained with full scan single stage mass spectrometric detection were exported in the Agilent MS Engine format (.ms) and analyzed with the AMDIS software [13]. 3. Results and discussions Samples described in Table 1 were analyzed by LC–DAD and LC–MS. Fig. 1 shows the 17th-century Epimanikia (right hand sleeve, after cleaning) described in Table 1. Table 2 summarizes DAD and MS data obtained for the samples analyzed. For MS analysis, the detection of dyes was made according to a dedicated analytical protocol, described in detail in an earlier publication [13]. It includes chromatographic separation and single MS full scan (FS) detection, followed by data processing through ion extraction chromatograms (IEC) according to m/z values of the molecular ions of dyes in the database. Results are correlated with UV–vis spectral data. For minor compounds the sample was re-injected using single stage MS detection in the selected ion monitoring/multiple ion monitoring modes (SIM/MID) as well as by using tandem MS, product ion scan/single reaction monitoring (SRM)/multiple reaction monitoring (MRM), for unambiguous confirmation of dye components. The confirmation procedure for the dye constituents (single MS-FS, single MS-SIM or tandem MS) may
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Table 1 Characterization of samples discussed in the present study. Textile object (period)
Function of the threads
Color
Sample code
Bedernita, Religious embroidery (1746)
Lining Sewing thread Embroidery thread Thread sewing the lining
Green Pink yellow Green Kaki
A1 A12 A13 A14
Bedernita (lining), religious embroidery (1746)
Embroidery thread Sewing thread Sewing thread
Kaki Green Brown
B15 B17 B20
Epimanikia, Religious embroidery (17th c.)
Embroidery support (edge) Embroidery support (edge) Embroidery support (edge) Lining Embroidery thread Embroidery thread Embroidery thread
Green Pink Pink yellow Yellow Orange Red Green
D23a D23b D23c D24 D26 D27 D29
Sakkos, Brocaded velvet (17th c.)
Embroidery thread Embroidery thread Silk core metallic embroidery thread Lining Lining
Pink yellow Red Pink yellow Green Red
F37a F37b F38 F39 F40
Robe of Princess, brocaded velvet (16–17th c.)
Weft
Green
E34
be directly correlated with the occurrence of the specific analyte in the sample (Table 2). When the semi-quantitative evaluation made using diode array detection (indicated through normalisation of a peak area to the sum of the peak areas in the chromatogram) gives a result for the constituent of interest greater than 10%, the FS operating mode is usually sufficient for the MS detection. If the semi-quantitative DAD evaluation for the constituent is between 1 and 10%, single stage MS in the SIM mode or MS/MS approaches are necessary for successful MS detection. When DAD detection indicates the occurrence of a compound at a level below 0.5%, it may be necessary to use AMDIS deconvolution software for MS detection. 3.1. Red dyes Red dyes were detected in eight samples, four from religious embroideries and four from brocaded velvets. Anthraquinone dyes were present in all the cases where samples still have a visibly red color (3/8 samples). In one of these samples carminic acid, the main dye component in Mexican cochineal (Dactylopius coccus Costa), Armenian cochineal (Porphyrophora hameli Brandt) and Polish cochineal (Porphyrophora polonica L.), was detected by DAD. Its presence was also confirmed by MS in both cases. Three minor
compounds, dcII (the C-glucoside of flavokermesic acid [14,15]), kermesic and flavokermesic acids are also present in fibers dyed with cochineal. In the present work, only dcII was detected by the presence of its molecular ion (m/z = 475), according to the FS-Ion Extracted Chromatogram (IEC). The presence of kermesic and flavokermesic acids was confirmed by the profiles of their fragments after spectral deconvolution with AMDIS (Fig. 2). Based on the calculation between the ratio of the minor compounds and carminic acid in HPLC-DAD analysis [16–18], the biological source in F37b was established as Mexican cochineal (D. coccus Costa). For two other red samples alizarin and purpurin, the main anthraquinone constituents in madder (Rubia tinctorum L.), were detected by both DAD and MSD. The presence of minor anthraquinone compounds from madder, anthragallol, munjistin, xanthopurpurin and rubiadin was also established. In five cases described as “pink” or “pink yellow” a marker compound for redwood (Caesalpinia spp.) dyeings, called “srw–soluble redwood” according to Wouters [4] or “type C” by Nowik [19] was identified by both DAD and MSD. For the latter, identification was made by FS-single stage MS followed by IEC of m/z = 243 a.m.u., the major ion of “srw” according to the literature [13,20]. Confirmation of this identification was achieved by detection by single stage MS in the SIM mode and by MS/MS.
3.2. Yellow dyes
Fig. 1. Epimanikia described in Table 1. Religious embroidery (17th century) worked in the Byzantine tradition, preserved in the Art Collection Museum, National Museum of Art of Romania, inv. 88371.
Flavonoid yellow dyes were detected in 15 out of a total of 20 analyzed samples. Dyer’s broom (Genista tinctoria L.) was identified as the main biological source in five samples and as a second biological source in two other samples. In all the green samples blue “indigo” dyes (from Isatis tinctoria L., Indigofera spp. or other species, discussed below) were also present. The identification of dyer’s broom was based on the detection of luteolin, genistein and apigenin, by both LC–DAD and MS detection techniques. In almost all cases, chrysoeriol – a minor compound recently identified in both weld and dyer’s broom [13,15,21,22] – was also detected by single stage MS in SIM mode or by MS/MS. Luteolin and apigenin – without the presence of genistein – were identified by both DAD and MSD in five samples, only one being from brocaded velvet, a dyestuff constituent profile suggesting
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Table 2 Results obtained by alternative diode array and mass spectrometric (MS or MS/MS) detection modes. Sample code Visual color
Results
Biological source b
Dye constituents MSD
Main source(s)
Traces
51 lu, 29 ge, 8 ap, 2 chry, 10 in 77 srw, 7 qu, 1 kf, 7 rht, 2 rhz, 6 ea 90 lu, 6 ap, 2 chry, 2 in 32 dat, 5 ge + lu, 4 kf, 1 irht, 1 ap, 23 in, 28 em, 5 chrys
lu(1), ge(1), ap(1), chry(2), in(2) srw(1), qu(2), kf(2), ea(2)
Dyer’s broom and “indigo” Redwood and buckthorn berries
– Tannin plantc
lu(1), ap(1), in(2) lu(1), ge(2), ap(2), dat(1,3), em(1), in(2)
Weld and “indigo”d Bastard hemp, rhubarbe and “indigo”
Dyer’s broom
lu(1), ap(2), dat(1,3), em(1), kf(2), in(2)
Bastard hemp, rhubarb, weld or eq. and “indigo” –
lu(1), ge(1), ap(1), chry(2), in(2), lu(2) lu(1), ge(1), ap(2), chry(3), in(2) srw(1), ca(1,3), ea(2), fk(*), ka(*)
Dyer’s broom and “indigo” Weld (or another flavone-containg plant) Dyer’s broom and “indigo” (Mexican) cochineal, redwood and tannin plant
A1 A 12
Green Yellow pink
A 13 A 14 B 15
Green Kaki (yellow/green) Kaki
B 17 B 20 D 23 a D 23 b
Green Brown Green Pink
D 23 c D 24
Pink yellow Yellow
D 26 D 27 D 29
Orange Red Green
F 37 a F 37 b
Yellow pink Red
F 38
Pink yellow
68 srw, 14 fi, 15 sul, 3 ea 71.5 ca, 1.5 dcII, 1 fk, 0.5 ka, 25.5 ea, [288 nm: 0.8 dcII, 97.7 ca, 1.5 fk + ka] 81 srw, 10 fi, 9 sul, +ea
F 39 F 40 E 34
Green Red Green
39 lu, 46 ge, 12 ap, 1 chry, 2 in 87 al, 12 pu, +ag 57 lu, 5 ap, 1 chry, 37 in
18 dat, 25 lu, 1 ap, 19 in, 32 em, 1 kf, 4 chrys 43 lu, 43 ge, 10 ap, 1 chry, 3 in 92 lu, 8 ap 32 lu, 66 ge, 1 ap, +chry, 1 in 64.5 ca, 12 srw, 23 ea, +fk, 0.5 ka 90 srw, 1 lu, 9 ge 54 lu, 36 ge, 2 ap, 1 chry, 5 ea, 2 qu, +fi, +sul 35 fi, 56 sul, 8 ea, + kf 69 al, +xp, 25 pu, 1 ru, +ag, 46 lu, 4 ap, 2 chry, 43 in, 4 ea, 0.5 fi, 0.5 sul
Redwood srw(1), lu(2), ge(2), ap(3) lu(1), ge(1), ap(2), chry(2), fi(3), sul(3), ea(2,3), qu(3), Dyer’s broom and redwood
– – – Dyer’s broom Young fustic and qu based dye
fi(1,3), sul(1,3), ea(2) pu(1), ru(1), al(1), mu(1), xp(1), ag(2) lu(1), ap(2), chry(2), fi(2), sul(2), in(2) srw(1,2), fi(2), sul(2), ea(2) ca(1), dcII(2), fk(*), ka(*), ea(2)
Young fustic Madder Weld and “indigo”
– Young fustic
Redwood, young fustic (Mexican) Cochineal and tannin plant
–
srw(1,3), fi(2), sul(2), ea(2) lu(1),ge(1), ap(1), chry(2), in(2) al(1), pu(1), ru(1), ag(2) lu(1), ap(2), chry(2), in(2)
Redwood, young fustic
Dyer’s broom and “indigo” Madder Weld and “indigo”
– – –
The following abbreviations were used: al: alizarin; ag: anthragallol; ap: apigenin; ca: carminic acid; chrys: chrysophanic acid; chry: chrysoeriol; dat: datiscetin; ea: ellagic acid; em: emodin; fi: fisetin; fk: flavokermesic acid (also called laccaic acid D); dcII: flavokermesic acid, C-glucoside; ge: genistein; in: indigotin; irht: isorhamnetin; kf: kaempferol; ka: kermesic acid; laA: laccaic acid A; lu: luteolin; mu: munjistin; pu: purpurin; qu: quercetin; rht: rhamnetin; rhz: rhamnazin; ru: rubiadin; srw: soluble redwood; sul: sulfuretin; xp: xanthopurpurin. a Numbers before the abbreviation for a dye represent the relative composition (%) corresponding to peak areas integrated in the chromatogram monitored at 255 nm (unless specified in the column); “ + ” indicates values lower than 0.5%. b Numbers between brackets have the following meaning: (1) detected through single stage MS–FS/IEC; (2) detected through MS–SIM/MID modes; (3) detected through MS/MS (MRM, product ion scan); (*) is used for dyes evidenced through deconvolution by AMDIS software. c The term “tannin plant” is used as a shorter name for “tannin-producing plant” and is not indicative of a particular dye source. d “Indigo” refers to several plants that produce indigo. e The term “rhubarb” should be read as “rhubarb or another emodin-containing plant”.
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Dye constituents DADa
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Fig. 2. LC–MS and LC–MS/MS of sample F37b (red), where (Mexican) cochineal was identified. Upper image, from top to bottom, the UV–vis chromatogram; MS–FS, IEC for carminic acid (molecular ion m/z = 491 and ion produced by decarboxylation in the source m/z = 447); IEC for dcII (molecular ion m/z = 475 and ion produced by decarboxylation in the source m/z = 431); IEC for ellagic acid. Lower images: detail of ion profiles after AMDIS deconvolution for flavokermesic (molecular ion m/z = 313 and ion produced by decarboxylation in the source m/z = 269) and kermesic (molecular ion m/z = 329 and ion produced by decarboxylation in the source m/z = 285) acids. The figure illustrates the flexibility in use of mass spectrometry which allows the gradual detection of minor compounds.
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Fig. 3. LC–DAD chromatograms for sample B15, integrated at 255 and 350 nm, respectively, with detection of datiscetin, emodin, indigotin, luteolin, apigenin, kaempferol and chrysophanic acid, suggesting the use of bastard hemp, weld (or another flavone-containing plant; indigo/woad and rhubarb (or another emodin containing plant) the compound marked with “?” has similar UV spectrum to datiscetin, but different retention time.
the use of weld (Reseda luteola L.). However, recent studies have demonstrated that other biological sources containing luteolin and apigenin, of which sawwort (Serratula tinctoria L.) is the most well known [14,15], also exist and could have been used as textile dyes. As a consequence, only those samples where chrysoeriol was detected together with luteolin and apigenin are likely to have been dyed using weld; in the other cases the use of another flavone-containing plant may not be excluded. When the presence of weld (or another flavone-containing plant) corresponded to green-coloured fibers, “indigo” dyes were also detected, while in another sample a combination of rhubarb (or another emodincontaining plant), bastard hemp and “indigo” dyes were found to have been used. Fisetin and sulphuretin, the main dye components in young fustic (Cotinus coggygria L.) were detected in three samples within the present study. Young fustic was detected as a single biological source in one case and was identified together with soluble redwood in pink-yellow samples. Traces of young fustic were also detected in two additional samples where fisetin and sulphuretin were detected by single stage MS in SIM mode or by MS/MS product ion scan. Datiscetin, the main dye component in bastard hemp (Datisca cannabina L.) was identified by DAD in two kakicoloured samples, in both cases together mainly with emodin and with chrysophanic acid, kaempferol and isorhamnetin as minor constituents, as well as flavonoids from either dyer’s broom or weld (or another flavone-containing plant) and “indigo” (Fig. 3). Poor chromatographic resolution is obtained between datiscetin and luteolin. Additionally, another compound exhibiting a very similar UV–vis spectrum to datiscetin, but having increased retention, could be observed in the chromatogram. Evidence for the presence of datiscetin (m/z = 285) in both samples was based on the MS information collected from a standard sample of wool dyed with bastard hemp. As no other dye was reported in this source, the identification of datiscetin by MS was also confirmed by MS/MS analysis, based on the fragmentation pattern obtained through product ion scan. In LC–MS separations, as
illustrated in Fig. 4, datiscetin co-elutes with kaempferol; however, comparison of the MS/MS product ion scan spectra allowed identification of both datiscetin and kaempferol in the kaki-coloured sample B15. Rhamnetin was identified by DAD together with quercetin, kaempferol and rhamnazin in an ochre-yellow sample (A12), suggesting the use of berries from a species of buckthorn (Rhamnus spp.). Analyses of two wool references, dyed with either the bark or the fruits (berries) from alder buckthorn (Rhamnus frangula L.) resulted in the detection of mainly emodin and minor amounts of rhamnetin and quercetin for the former, bark-dyedsample, and mainly rhamnetin, isorhamnetin and quercetin for the latter. The components detected in sample A12 by MS thus confirm the use of berries from a Rhamnus species as the source of dye. Emodin, the main dye component in alder buckthorn bark, rhubarb, yellow dock and other biological sources was also detected in two samples. It was not possible to evidence the presence of chrysophanic acid (m/z = 253) by MS, based on the IEC, nor by the identification of the fragment m/z = 239, which would correspond to the fragment induced by de-methylation. A more detailed study on a pure standard of chrysophanic acid (not available at the time of the present experiments) is needed in order to be able to identify it in historical samples. The limited information on these two anthraquinone dyes of vegetable origin, emodin and chrysophanic acid, as textile dyes may be explained by their rare identification in historical textiles. Like emodin, the presence of chrysophanic acid may indicate the use of alder buckthorn bark (Rhamnus frangula L.), or the roots of rhubarb (Rheum sp.) or dock (Rumex sp.) [23–25]. However, no systematic study in which the biological source of the dye has been identified unambiguously as one or other of these plants has so far been reported. 3.3. Blue dyes Although blue-coloured samples were not selected for analysis, indigotin was detected in eight green-coloured samples. For mass spectrometric detection, FS-single stage MS in SIM mode according to the molecular ion of indigotin (m/z = 261) was used.
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Fig. 4. LC–MS and LC–MS/MS data from sample B15, supporting the identification of datiscetin, emodin, luteolin, apigenin, and kaempferol (identification for indigotin is not given; chrysophanic acid, although detected by LC–DAD, was not detected by LC–MS).
Indigotin is the main dye component in woad, Isatis tinctoria L. and in the indigo plant itself, Indigofera spp. but no analytical method has been reported to distinguish between these species [26]. The term “indigo” is thus used to refer to indigotin-producing plants.
3.4. Tannin Ellagic acid was detected in seven samples with both detection techniques. The presence of ellagic acid indicates the use of a tannin-containing plant material either for textile dyeing or for
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Table 3 Biological sources attributed to samples analyzed throughout the present study, compared to other results for samples from religious embroideries and brocaded velvets from Romanian collections previously identified through the LC–DAD method by the same research group [8–10]. Religious embroideries 15th-16th c.
Cochineal All (DC) Kermes Lac dye Madder Safflower Redwood Young fustic Weld (or another flavone-containing plant) Dyer’s broom Buckthorn berries Rhubarb (or another emodin-containing plant) Bastard hemp Tannins Logwood Indigoid
6 (3)–16th c. 3 21 29 2 24 24 26 8 0 2 1 39 0 40
Brocaded velvets
17th–18th c.
19th c.
Present
Previous
1 (1) 0 0 1 0 3 1 4 4 1 2 2 1 0 5
3 (1) 0 0 3 1 4 1 3 0 3 2 2 5 1 7
weighting silk [4,24]. In the present study, a high proportion of ellagic acid from a tannin-producing plant source was detected twice in red samples in combination with cochineal while much lower proportions were detected in a yellow pink sample together with redwood and buckthorn berries. Ellagic acid was also detected in all the five cases when fisetin and sulphuretin were identified, indicating the use of the dye from the heartwood of young fustic. This is probably due to the presence of a trace of the tannins present in young fustic (primarily in the leaves and twigs). 3.5. Discussion on the biological sources together with previous results on religious embroideries and brocaded velvets from Romanian collections Attribution of the biological sources of dyes identified and confirmed in the analyzed samples is summarized in Table 2. All the sources detected in the present series of analyses were also identified in one or more groups of samples analyzed before by the same research team. According to the studies performed until now, six sources of red dye have been identified in religious embroideries and brocaded velvets from Romanian collections dating from the 15th to the 19th centuries (Table 3). Half of these are of animal origin (cochineal, kermes and lac) and half derive from plant sources, and from various parts of the plants: roots (madder), petals (safflower) and wood (redwood). Cochineal (both Old and New World) and redwood were detected in all the textile groups studied; lac dye, madder and safflower were only present in embroideries. The combination of lac dye and madder was the main source of red used in the support fabric for embroideries in the 15th and 16th centuries. According to literature, lac was hardly used in Europe for textile dyeing, but only for dyeing leather and as an organic pigment. It was mentioned as imported into the Ottoman Empire as early as the 15th century and, according to literature, it has been detected in Ottoman textiles [27,28]. Its use in religious embroideries would thus suggest an Oriental origin for these materials. Kermes was only identified in religious embroideries and brocaded velvets dated to the 15th and 16th centuries, which is in accordance with literature mentioning that, due to its lower cost and ease of use Mexican cochineal eventually became the only animal source of red dye used in Europe, soon after the discovery of the New World [25].
1 (0) 0 0 0 0 3 3 4 1 2 1 0 2 1 5
15th–16th c.
2 (0) 1 0 0 0 4 3 7 0 0 0 0 6 0 3
17th c. Present
Previous
1 (1) 0 0 1 0 2 2 1 1 0 0 0 0 0 2
0 0 0 0 0 0 0 1 0 2 0 0 0 3 0 1
Six sources of yellow flavonoids were identified in the series, weld (or another flavone-containing plant), young fustic and dyer’s broom being the most widely used. Except for the latter that was not present in 15th–16th century embroideries, the others were detected in all the groups considered. Bastard hemp, buckthorn berries and rhubarb (or another emodin-containing plant) were only detected in embroideries, bastard hemp up to the 18th century, buckthorn berries not before the 17th century and rhubarb (or another emodin-containing plant source) in textiles from the 15th to the 18th century. However, both bastard hemp and rhubarb (or another emodin-containing plant) were only detected in Ottoman textiles [28], which also supports the suggestion of an Oriental origin for the materials. As far as the blue dyes are concerned, “indigo” was identified in religious embroideries and brocaded velvets from the 15th to the 19th century, while logwood was only present in 17th- to 19thcentury embroideries. This is no surprise when we remember that this source was only available after the discovery of the New World [29]. 4. Conclusions All the dyes identified on the 17th- and 18th-century Romanian velvets and embroideries are in very good correlation with the existing knowledge on dyes and biological sources used in Europe and Asia Minor during this period [1,2,5,6,24,25,28]. The results may be also correlated in terms of period and fiber function with previous data obtained for similar textiles by the same research team [8–10]. From the whole group of analyses performed up till the present on textiles from Romanian collections, it may be concluded that the combination of lac dye and madder was the main source of red in the 15th- to 16th-century embroideries, while kermes was used when a more precious textile was intended. Mexican cochineal was preferred in later textiles. Lac dye, bastard hemp and rhubarb (or another emodin-containing plant) were only detected in embroideries. Based on this fact and considering that these dye sources were not identified in textiles from Europe, but only in Ottoman pieces, it may be stated that at least part of the materials used in embroideries dating from the 15th to the 18th centuries have an Oriental origin. As far as the techniques applied are concerned, it can be concluded that a good correlation between the results produced
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by LC–DAD, LC–MS and LC–MS/MS was found, not only for the major dye components, but also for the minor accompanying constituents. For some of the minor components co-eluting under the chromatographic conditions of the LC–MS method, spectral deconvolution with AMDIS software was established. The study showed that LC–MS and LC–MS/MS approaches were confirmed as versatile tools for the identification and confirmation of dyes in historic textiles, if consistent work is first accumulated on a collection of standard dyes and dyed fibers for construction of suitable spectral libraries. Acknowledgements The authors would like to thank the National Museum of Art of Romania and the Putna Monastery, Romania for access to their collections. They are also grateful to LaborMed Pharma, who offered an open access to the LC–MS/MS analytical instrumentation and to Ms Marie-Christine Maquoi from the KIK/IRPA laboratory for the expert assistance in LC–DAD dye analysis. Professor Recep Karadag from Marmara University, Istanbul, Turkey, who offered a bastard hemp-dyed fibre, is also acknowledged. The authors are also grateful to Ms. Jo Kirby, National Gallery London Scientific Department (retired), for carefully reading and improving the English text. References [1] J.H. Hofenk de Graaff, W.G. Th Roelofs, On the occurrence of red dyestuffs in textile materials from the period 1450–1600, in: 4th Meeting ICOM Committee for Conservation, Madrid, 1972. [2] J.H. Hofenk de Graaff, T.W.G. Roelofs, The analysis of flavonoids in natural yellow dyestuffs occuring in ancient textiles, in: 5th Meeting ICOM Committee for Conservation, Zagreb, 1978, p. 1–15. [3] J. Wouters, Analyse des colorants des tapisseries brugeoises, in: Bruges et la tapisserie des xvie et xviie siècles, Mouscron, Bruges, 1987, p. 515–526. [4] J. Wouters, Dye analysis of Florentine borders of the 14th to 16th centuries, Dyes in History and Archaeology 14 (1995) 48–58. [5] I. Karapanagiotis, L. Valianou, Y. Sister Daniila, Chryssoulakis, Organic dyes in Byzantine and post-Byzantine icons from Chalkidiki (Greece), Journal of Cultural Heritage 8 (2007) 294–298. [6] M. Van Bommel, J.H. Hofenk de Graaff, Master dyers to the court of Sicily, Dyes in History and Archaeology, 22/23, publication due 2012. [7] I. Petroviciu, J. Wouters, Analysis of natural dyes from Romanian 19th and 20th century ethnographical textiles by DAD-HPLC, Dyes in History and Archaeology 18 (2002) 57–62. [8] I. Petroviciu, J. Wouters, I. Vanden Berghe, I. Cretu, Dyes in some textiles from the Romanian Medieval Art Gallery, Dyes in History and Archaeology 22/23, publication due 2012.
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[9] I. Petroviciu, J. Wouters, I. Vanden Berghe, I. Cretu, Dye analysis on some 15th Century byzantine embroideries, Dyes in History and Archaeology. 22/23, publication due 2012. [10] I. Petroviciu, I. Vanden Berghe, I. Cretu, J. Wouters, Analysis of Dyestuffs in 15th–17th Century byzantine embroideries from Putna Monastery, Romania, Dyes in History and Archaeology, 24/25, publication due 2012. [11] M. Trojanowicz, J. Orska-Gawrys, I. Surowiec, B. Szostek, Chromatographic investigation of dyes extracted from Coptic texiles from the National Museum in Warsaw, Studies in Conservation 49 (2004) 115–130. [12] J. Wouters, N. Rosario-Chirinos, Dye analysis of pre-combian peruvian textiles with HPLC and DAD, Journal of the American Institute for Conservation 31 (1992) 237–255. [13] I. Petroviciu, F. Albu, A. Medvedovici, LC/MS and LC/MS/MS based protocol for identification of dyes in historic textiles, Microchemical Journal 95 (2010) 247–254. [14] D. Peggie, The development and application of analytical methods for the identification of dyes on historical textiles, PhD thesis, University of Edinburgh (2006). [15] D. Peggie, A. Hulme, Mc. Nab, H.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), Microchemica Acta 162 (2008) 371–380. [16] J. Wouters, A. Verhecken, The Coccid insect dyes: HPLC and computerized analysis of dyed yarns, Studies in Conservation 34 (1989) 189–200. [17] J. Wouters, A. Verhecken, The scale insect dyes (Homoptera:Coccoidea) species recognition by HPLC and Diode-Array analysis of the dyestuffs, Annales de la Société entomologique de France 25 (1989) 393–410. [18] I. Vanden Berghe, Investigation of cochineal dyeing parameters and refinement of the cochineal identification system’, Dyes in History and Archaeology22/23, publication due 2012. [19] 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) 129–144. [20] I. Karapanagiotis, E. Minopoulou, L. Valianou, Y. Sister Daniilia, Chryssoulakis, Investigation of the colourants used in icons of the Cretan school of iconography, Analytica Chimica Acta 647 (2009) 231–242. [21] E. Ferreira, New approaches towards the identification of yellow dyes in 463 ancient textiles, PhD thesis, University of Edinburgh (2001). [22] http://www.organic-colorants.org. [23] H. Schweppe, Handbuch der Naturfarbstoffe, Vorkommen, Verwendung, Nachweis, Nikol Verlagsgesellschaft mbH&Co, KG, Hamburg, 1993, p. 224–228. [24] J.H. Hofenk de Graaff, The colourful past. Origins chemistry and identification of natural dyestuffs, Abegg Stiftung & Archetype Publications Ltd, Riggisberg and London, 2004. [25] D. Cardon, Natural dyes–sources, tradition, technology science, Archetype Publications, London, 2007. [26] J. Wouters, Possible future developments in the analysis of organic dyes, Dyes in History and Archaeology 20 (2005) 23–29. [27] I. Vanden Berghe, M.C. Maquoi, J. Wouters, Dye analysis of Ottoman silk, in: M. van Raemdonck (Ed.), The Ottoman silk textiles from the Royal Museums of Art and History in Brussels, Turhout Brepols, Brussels, 2004, pp. 49–60. [28] N. Enez, H. Böhmer, Ottoman textiles: dye analysis, results and interpretation, Dyes in History and Archaeology 14 (1995) 39–44. [29] F. Brunello, The art of dyeing in the History of Mankind, Vicenza, 1973, p. 175–221.