J. Anal. Appl. Pyrolysis 56 (2000) 167 178 www.elsevier.

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Analysis of anthraquinoid and indigoid dyes used in ancient artistic works by thermally assisted hydrolysis and methylation in the presence of tetramethylammonium hydroxide
Daniele Fabbri a,*, Giuseppe Chiavari a, He Ling b
a

Laboratorio di Chimica Ambientale, c/o ITIS, Uni6ersita di Bologna, 6ia Marconi 2, ` I-48100 Ra6enna, Italy b Xian Center for the Conser6ation and Restoration of Cultural Property, Xian, Peoples Republic of China

Received 22 November 1999; received in revised form 17 March 2000; accepted 25 April 2000

Abstract Thermally assisted hydrolysis and methylation (THM) in the presence of tetramethylammonium hydroxide (TMAH) was applied to the analysis of red dyes based on the 9,10-anthracenedione skeleton (anthraquinone), namely alizarin, quinizarin, purpurin, carminic acid, and the blue pigment indigo. Alizarin and purpurin are the principal coloured compounds of madder, and carminic acid of cochineal red. Synthetic dyes were dissolved in aqueous TMAH, pyrolysed at 700C for 10 s and the evolved thermochemolytic products were analysed by gas chromatography/mass spectrometry (GC/MS). The resulting chromatograms of all the madder dyes were characterised by the presence of the methyl esters of 1,2-benzenedicarboxylic acid (dimethyl-o-phthalate) and benzoic acid. In addition, thermochemolysis of alizarin and quinizarin afforded 1,2-dimethoxyanthraquinone and 1,4-dimethoxyanthraquinone, respectively. Carminic acid produced a series of methylated fragments deriving from both the substituted anthraquinoid moiety and the glycosidically-Clinked glucose. Finally, the principal thermochemolysis products of indigo were the Nmethylated derivatives of 2-amino benzoic acid methyl ester followed by methylated aniline and indoles. The potential of THM as a rapid technique for the identication of dyes in real samples was illustrated by the analysis of madder lake, cochineal lake, and natural indigo dispersed in a binder. 2000 Elsevier Science B.V. All rights reserved.

* Corresponding author. Tel.: + 39-0544-400870; fax: +39-0544-484001. E-mail address: dfabbri@ambra.unibo.it (D. Fabbri). 0165-2370/00/$ - see front matter 2000 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 5 - 2 3 7 0 ( 0 0 ) 0 0 0 9 2 - 9

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Keywords: Tetramethylammonium hydroxide; Thermally assisted hydrolysis and methylation; Thermochemolysis; Pyrolysis; Gas chromatography/mass spectrometry; Madder; Cochineal; Indigo; Hydroxyanthraquinones

1. Introduction A great variety of organic substances have been used by artists to dye, protect and bind their works [1]. Pyrolysis (Py) coupled with gas chromatography (GC) and/or mass spectrometry (MS) is among the numerous analytical techniques employed for the characterisation and identication of organic materials used in artistic objects [2]. The potential of analytical pyrolysis in the eld of cultural heritage, has increased in recent years with the use of the derivatising agent tetramethylammonium hydroxide (TMAH) in combination with pyrolysis [3], in the so-called TMAH thermochemolysis or thermally assisted hydrolysis and methylation (THM), or more simply pyrolysis-methylation. In the analysis of painting samples, pyrolysis-methylation has mostly been applied to the identication of complex high molecular weight organic materials used as binders and varnishes by artists, such as drying oils [4], proteinaceous materials [5], natural [6] and articial resins [7]. Considering the benets of analytical pyrolysis as a screening methodology, it is worth assessing its applicability to the analyses of pigments and colorants. A few examples have appeared in the literature showing that conventional Py-GC/MS is capable of revealing the presence of inorganic pigments which produce volatile thermal degradation products, such as orpiment (formation of As4, [8]) and cinnabar (formation of elemental Hg, [9]). The study here presented reports on the potential of analytical pyrolysis for the detection of organic dyes used in the past. We focused our attention on madder, cochineal and indigo, which are amongst the most important and oldest organic dyestuffs used in paintings and textiles [1]. The substances responsible for the colour of madder and cochineal are hydroxyanthraquinones and thin layer chromatography [10], high-pressure liquid chromatography [11] and visible or uorescence spectroscopy [12] have been used for their identication. Hydroxyanthraquinones and indigo contain polar groups, which could be methylated improving gas chromatographic separation, therefore we applied Py-GC/MS in combination with TMAH for their analysis.

2. Experimental

2.1. Materials
The synthetic dyes alizarin (1,2-dihydroxyanthraquinone, C.I 58000), quinizarin (1,4-dihydroxyanthraquinone, C.I. 58050), purpurin (1,2,4-trihydroxyan-

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thraquinone, C.I. 58205), carminic acid (7-C-a-D-glucopyranosyl-3,5,6,8-tetrahydroxy-1-methyl-2-anthraquinone carboxylic acid, C.I. 75470), indigo (2-(1,3-dihydro-3-oxo-2H-indol-2-ylidene)-1,2-dihydro-3H-indol-3-one, C.I. 73000) were purchased from Aldrich. Standard samples were kindly prepared by Merlini Gianluca of the Cultural Association Quelli del Ponte (Ravenna, Italy) using natural sources (plants and insects purchased from Zecchi, Florence, Italy). The vegetal indigo was extracted from Indigofera tinctoria with basic water and mixed with arabic gum as binder. The madder lake was prepared from the dried root of Rubia tinctorum by extraction of the dyes with hot water and precipitation of the lake with alum cake (as mordant). The lake obtained by decanting supernatant, was air dried and mixed with arabic gum as binder. The cochineal lake was obtained in a similar way from the insect Coccus cacti. Materials and procedures adopted for the preparation of the lakes were similar to those employed in middle age for miniatures [13].

2.2. Methods
Each synthetic dye (1 mg) was dissolved in 100 ml aqueous solution of TMAH (25% w/w, Aldrich). An aliquot of the solution (corresponding to 1050 mg of dye) was placed onto the central part of a quartz tube and pyrolysed at 700C for 10 s at the maximum heating rate. Standard samples (less than 1 mg) were mixed with aqueous TMAH (3 ml) and pyrolysed as described above. Analysis of madder lake was also performed on the dyes isolated by means of the stripping procedure developed by Schweppe [10]. Briey, the original sample was heated at 100C for 1 min with 10% aqueous H2SO4, extracted with ethyl acetate, the organic layer was washed with water and the solvent evaporated. The residue was dissolved in methanol and analysed by absorption spectroscopy or in aqueous TMAH for pyrolysis-methylation experiments.

2.3. Instrumentation
The pyrolyser was a CDS 1000 pyroprobe interfaced to a Varian 3400 GC coupled to a Saturn II ion trap mass spectrometer. The pyrolysis interface and GC injector were kept at 250C. The column ow was 1.5 ml min 1 (at initial temperature) and split ow was 60 ml min 1. The products were separated on a Supelco SPB5 capillary column (30 m 0.25 mm i.d., 0.25 mm lm thickness) programmed from 50 (2 min hold) to 300C at a heating rate of 5C min 1. Mass spectra were recorded at 1 scan s 1 under electron impact at 70 eV, scan range 45 450 m/z. The ion source was kept at 170C. These are standardised conditions routinely employed in our laboratory. The assignment of most of the products was based on match with both the NIST 1992 and Wiley 1996 mass spectrum libraries. Absorption measurements were performed with a PerkinElmer Lambda 16 spectrophotometer.

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3. Results and discussion

3.1. Madder
Madder is a red colouring material extracted from the roots of the madder plant R. tinctorum which contains a mixture of hydroxyanthraquinones, free or in the form of glycosides. The principal dyes occurring in the dried root are alizarin and purpurin, while quinizarin is present in small amount [10]. Numerous colouring substances belonging to the class of hydroxyanthraquinones are present in the roots of other plants of the type of madder (family Rubiaceae) and used as sources of red dyes in the past [10].

3.1.1. Alizarin, quinizarin and purpurin Chromatograms obtained from the THM reaction of synthetic hydroxyanthraquinones are shown in Fig. 1. The proposed structural assignments for the principal peaks are reported in Table 1. Even though their relative abundances were not reproducible, the characteristic products of alizarin and quinizarin were the methylated dye itself, the dimethylester of 1,2-benzenedicarboxylic acid (dimethylphthalate) and the methyl ester of benzoic acid. Several compounds having the same molecular weight of dimethoxyanthraquinone (molecular ion at m/z 268 in their mass spectra) were detected in the chromatograms of both alizarin and quinizarin. These compounds were interpreted

Fig. 1. Reconstructed ion chromatograms obtained from THM GC/MS of madder dyes. (a) Alizarin; (b) quinizarin; (c) purpurin. Peak numbers are those of Table 1.

D. Fabbri et al. / J. Anal. Appl. Pyrolysis 56 (2000) 168178 Table 1 Principal THM products of madder dyesa Dye Alizarin m/z 77, 77, 238, 238, 210, 77, 225, 77, 95, 77, 163, 127, 225, 221, 77, 77, 175, 217, 105, 133, 253, 253, 253, 165, 239, 105, 123, 133, 211, 225, 253, 239, 105, 133, 203, 245, 136 163 268 268 268 300 268 136 138 163 300 254 268 268 136 163 218 260 Tentative structural assignment Benzoic acid, methyl ester Dimethyl-o-phthalate Dimethoxyanthracenedione Dimethoxyanthracenedione Dimethoxyanthracenedione Unknown 1,2-Dimethoxyanthraquinone Benzoic acid, methyl ester 1,4-Dimethoxybenzene Dimethyl-o-phthalate Unknown 1-Hydroxy-4-methoxyanthraquinone 9,10-Dimethoxyanthracene-1,4-dione (?) 1,4-Dimethoxyanthraquinone Benzoic acid, methyl ester Dimethyl-o-phthalate Unknown Unknown

171

c 1 3 6 8 10 11 13 1 2 3 7 9 12 14 1 3 4 5

Quinizarin

Purpurin

Peak numbers, numbered in order of increasing retention time, are those reported in Figs. 1 and 3.

as isomers occurring as contaminants in the commercial product and/or methylated tautomers of the dye, which formed in the basic TMAH solution. Dimethyl-ortho-phthalate is by far the most abundant product released from the TMAH thermochemolysis of purpurin (Fig. 1c). Low levels of other methylated fragments were observed in the chromatogram of purpurin, among which the methyl ester of benzoic acid. The fully or partially methylated purpurin was not detected. A possible mechanism of formation of dimethyl-o-phthalate from hydroxyanthraquinones is proposed in Fig. 2, exemplied for quinizarin. Even though in organic solvents quinizarin appears to exist solely in the meso form (1,4-dihydroxy9,10-anthraquinone) [14], in the basic medium it could tautomerise into more or less stable keto enol forms. Among these, b-diketones, even though less stable, undergo rapid alkaline cleavage forming carboxylic acid derivatives. As shown in scheme of Fig. 2, when hydroxide ions attack on carbonyl carbons 9 and 10, the cleavage of the bonds between the carbonyl carbon and the carbon of the dihydroxybenzene ring is expected, with formation of ortho-phthalate and 1,4-dihydroxybenzene, which are subsequently methylated. If the attack of hydroxide ions involve carbonyl carbons 1 or 4, aliphatic structures (ketoacids) are produced which are rapidly degraded under basic/pyrolytic conditions. Quinizarin released 1,4-dimethoxybenzene in high yield, but only trace levels of 1,2-dimethoxybenzene and 1,2,4-trimethoxybenzene were observed in the chromatograms of alizarin and purpurin, respectively. The occurrence of hydroxy groups in ortho position may be responsible for the faster degradation of the

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hydroxybenzene ring observed for alizarin and purpurin with respect to quinizarin, whose hydroxyls are in para conguration. No methylated hydroxy o-phthalic acids were detected in the chromatograms of madder dyes, in agreement with the proposed scheme.

3.1.2. Madder lake The chromatogram obtained from the THM reaction of madder lake, shown in Fig. 3a, was dominated by peaks derived from the binder which was present in

Fig. 2. Proposed mechanism for the formation of the THM products of quinizarin.

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Fig. 3. Chromatograms obtained from THMGC/MS of madder lake. (a) Reconstructed ion chromatogram; and (b) mass chromatogram at m/z 105 and 163 of the original madder lake; (c) reconstructed ion chromatogram of the extracted dyes. Peak numbers are those of Table 1.

much greater proportions than the colorants. The occurrence of dimethylphthalate and the methyl ester of benzoic acid, markers for both alizarin and purpurin, were detected by displaying the chromatograms using the intensity of characteristic ions (m/z 105, and 163, see Fig. 3b), whereas the methylated alizarin was not revealed. In order to evaluate whether the absence of methylated alizarin was caused by matrix effects (mordant and binder), madder dyes were isolated from the lake and pyrolysed with TMAH (see Section 2). The resulting chromatogram, given in Fig. 3c, showed two major peaks resulting from methylated phthalic and benzoic acids, but did not exhibit signals associated with methylated alizarin. Therefore, it has to be assumed that dimethylphthalate was derived principally from purpurin. This nding was conrmed by the absorption spectrum of the extract, which was almost coincident with that of standard purpurin, in agreement with the results of Miliani et al. [1] who reported that purpurin was present at higher levels (85%) than alizarin in madder extracts analysed by spectrophotometry. Even though alizarin is known to be the main anthraquinone of madder root, purpurin may prevail in the nal sample as a consequence of extraction procedures, drying, ageing and chemical treatments in the analytical procedure [10,11].

3.2. Cochineal red

Cochineal red is a dyestuff obtained from the insect Dactylopius coccus Costa (Coccus cacti ). Several red dyestuffs from scale insects were used in the past,

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among them Kermes and Lac dye, and cochineal red, which became increasingly popular after its discovery in Mexico owing to its superiority to other insect dyes [1]. Insect dyes are composed of hydroxyanthraquinones, such as laccaic acids, kermesic acid and carminic acid. Carminic acid is the most abundant anthraquinoid dye found in cochineal red.

3.2.1. Carminic acid The chromatogram obtained from TMH of carminic acid is shown in Fig. 4 and the tentative structural assignments of the main peaks are reported in Table 2. The most intense peak was derived from the glucose unit and was identied as a methylated inositol. Glucose is known to produce principally methylated deoxyglucuronic acids (saccharinic acids) when subjected to pyrolysis in the presence of TMAH [15], but their presence was not detected in the chromatogram of carminic

Fig. 4. Reconstructed ion chromatogram obtained from THM GC/MS of carminic acid. Peak numbers are those of Table 2. Table 2 Principal THM products of carminic acid (cochineal)a m/z 59, 45, 71, 121, 75, 190, 206, 75, 178,
a

Tentative structural assignment 75, 103 71, 103 115, 131 149, 180 101, 144 203, 218 207, 238 101, 111 265, 296 2-Methoxypropanoic acid, methyl ester 2,4-Dimethoxybutanoic acid, methyl ester Unknown 2-Methyl-6-methoxybenzoic acid, methyl ester (?) Hexa-O-methyl-myo-inositole Unknown 3-Methyl-5-methoxy-1,2-benzenedicarboxylic acid, dimethyl ester (?) Methylated glucose derivative 3-Methyl-5-methoxy-1,2,4-benzenetricarboxylic acid, trimethylester (?)

c 1 2 3 4 5 6 7 8 9

Peak numbers are those reported in Figs. 4 and 5.

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acid. This nding was explained by the fact that glucose is linked to the anthraquinone ring through a C-linkage instead of the more common O-linkage, thus the aldehydic functionality necessary for the saccharinic acid formation is lacking. The thermochemolytic compounds released from the anthraquinoid moiety of carminic acid gave mass spectra not included in the libraries, thus their identication is only tentative. Differing from the madder dyes discussed above, carminic acid did not yield dimethyl-o-phthalate, in accordance with the fact that both benzene rings bear substituents. Adopting the fragmentation pattern proposed for quinizarin (Fig. 2), methylated derivatives of 3-methyl-5-hydroxy-1,2,4-benzenetricarboxylic acid should be expected in place of the methylated 1,2-benzenedicarboxylic acid typical of madder dyes. Consistently with this hypothesis, a compound (peak 9) was detected at high retention times which was tentatively identied as the fully methylated 3-methyl-5-hydroxy-1,2,4-benzenetricarboxylic acid, its mass spectrum being characterised by a molecular ion at m/z 296 and a base peak at 265 (loss of a methoxy group). Compounds 7 and 4 were thought to be virtually derived from compound 9 following the loss of one and two carboxymethyl groups, respectively. Thus, peak 4 and 7 were tentatively identied as the methyl esters of 2-methyl-6-methoxybenzoic acid and 3-methyl-5-methoxy-1,2-benzenedicarboxylic acid, respectively, but other isomeric structures differing for the position of the carboxymethyl groups could be assigned as well. Compound 9 and its derivatives may be considered characteristic markers of scale insect dyes, since the structural unit of the anthraquinoid molecule from which they are thought to be derived is common to other insect dyes, such as kermesic acid (3,5,6,8-tetrahydroxy-1-methyl-2-anthraquinone carboxylic acid) and avokermesic acid (3,6,8-trihydroxy-1-methyl-2-anthraquinone carboxylic acid, laccaic acid D).

3.2.2. Cochineal lake As in the case of madder lake, the mass chromatogram obtained from the THM reaction of the cochineal lake was dominated by peaks originated from the matrix (Fig. 5a). The occurrence of markers of carminic acid was revealed in the mass chromatograms using the principal ions of the mass spectra of compounds 4 and 9 of Table 2, as depicted in Fig. 5b and c. Because carminic acid is strongly fragmented upon thermochemolysis, the intensity of markers can be very low and interference may become important. For example, the characteristic ions at m/z 265 and 296 of compound 9, are also present in the mass spectrum of oleic acid methyl ester which has a similar retention time. In this case, the identication of compound 9 was conrmed by the detection of the more specic ion at m/z 178 in the mass chromatogram (Fig. 5c). 3.3. Indigo 3.3.1. Synthetic dye THM of synthetic indigo afforded the methyl esters of 2-(dimethylamino)-benzoic acid and 2-(methylamino)-benzoic acid (Fig. 6a and Table 3) as principal

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Fig. 5. Chromatograms obtained from THMGC/MS of cochineal red. (a) Total ion current, mass chromatograms; (b) at m/z 149 and 180; and (c) at m/z 178.

Fig. 6. Chromatograms obtained from THMGC/MS of (a) synthetic indigo; and (b) (c) natural indigo mixed with arabic gum. Peak numbers are those of Table 3.

products. The methylated products arising from the decarboxylation (i.e. benzeneamine and its methylated derivatives) and deamination (i.e. benzoic acid methyl ester) of 2-aminobenzoic acid were detected at lower levels. The chromatogram was also characterised by the presence of methylated indole derivatives. As in the case of hydroxyanthraquinones, the formation of the carboxylic group can be explained as occurring from the attack of the hydroxide ion on carbonyl carbons of indigo.

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3.3.2. Natural dye The mass chromatogram obtained from the pyrolysis-methylation of natural indigo dispersed in arabic gum is presented in Fig. 6b. Even though the pyrogram was again dominated by peaks deriving from the binder, signals associated with indigo markers (peaks 6 and 8) could be clearly distinguished. The detection of markers arising from the dye can be made more evident by plotting mass chromatograms using characteristic ions as shown in Fig. 6c.

4. Conclusions This study shows that THM in connection with GC/MS can be effectively used for the identication of anthraquinoid and indigoid dyes dispersed in complex mixtures. Through the analysis of synthetic model compounds, a series of methylated benzenecarboxylic acid derivatives were tentatively identied as key thermochemolysis products whose formation was supposedly promoted by the attack of hydroxide ions on carbonyl carbons of the dye. Characteristic markers of indigo (e.g. 2-(methylamino)-benzoic acid, methyl ester), madder (e.g. dimethyl-o-phthalate) and cochineal (e.g. methyl-methoxybenzoic acid, methyl ester) were detected in the chromatograms of the corresponding standard samples, containing the natural dye dispersed in a matrix (mordant and binder). However, the specicity of the markers should be carefully examined for a proper interpretation in real cases. For example, dimethyl-o-phthalate may be considered a good marker for madder-like pigments, readily distinguishable from insect dyes, which do not release dimethylphthalate upon pyrolysis with TMAH. On the other hand, materials obtained from phthalic acid, like alkyd resins, are very common in modern applications, e.g. as varnishes in restorations, and their presence may interfere with the analysis of madder. Interference problems are increased by the paucity of the sample, a typical situation encountered in the
Table 3 Principal THM products of indigoa m/z 65, 50, 77, 77, 103, 92, 148, 104, 77, 118, 118,
a

Tentative structural assignment 66, 93 76, 103 106, 107 120, 121 130, 131 119, 151 164, 179 132, 165 144, 145 146, 161 146, 161 Benzeneamine Benzenenitrile N-methyl-benzenamine N,N-dimethylbenzenamine 1-Methylindole 2-Amino-benzoic acid, methyl ester 2-(Dimethylamino)-benzoic acid, methyl ester 2-(Methylamino)-benzoic acid, methyl ester Dimethylindole 3-Methoxy-1-methylindole (?) 1,2-Dimethylindol-3-one (?)

c 1 2 3 4 5 6 7 8 9 10 11

Peak numbers are those reported in Fig. 6.

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analysis of museum objects with destructive techniques. Data resulting from pyrolysis may be complemented by the use of conventional and time-consuming techniques for a more reliable and accurate identication of the pigments. In conclusion, THM in combination with GC/MS proved its potential as a simple and rapid approach to obtain preliminary information on the nature of the principal classes of organic materials used by artists, that is binders, protectives and pigments.

Acknowledgements Thanks are given to Gianluca Merlini for kindly providing the standard samples. DF and GC thank the University of Bologna and the National Council of Research (Progetto nalizzato Beni Culturali contract number 96.01097.PF36) for nancial support. The work was partly done within the Peoples Republic of ChinaItaly collaboration (project of the Italian Institute for Africa and Orient).

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