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Analytica Chimica Acta

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owards identification of traditional European and indigenousustralian paint binders using pyrolysis gas chromatographyass spectrometry

iffany Reeves, Rachel S. Popelka-Filcoff, Claire E. Lenehan ∗

chool of Chemical and Physical Sciences, Flinders University, Adelaide, South Australia, Australia

i g h l i g h t s

The first paper to examine both Aus-tralian and European paint binders inwithin the same py-GC-MS study.Unique pyrolysis products identifiedfor each binder.Hierarchical cluster analysis showsthat py-GC-MS can be used to classifybinders.

g r a p h i c a l a b s t r a c t

r t i c l e i n f o

rticle history:eceived 13 May 2013eceived in revised form 8 August 2013ccepted 8 September 2013vailable online xxx

a b s t r a c t

We report a pyrolysis GC–MS method capable of analysing Indigenous Australian and European binderstypically used in the manufacture of culturally important painted works. Eleven different traditionalEuropean binders and ten different Indigenous Australian binders were examined. The method allowsdiscrimination between highly complex and impure lipid, resin, polysaccharide, wax, and protein-basedbinders. Each was found to have characteristic pyrolysis products that were unique to the binder mate-

eywords:yrolysisC–MSaint binders

rial, demonstrating the potential for differentiation of these binders on Australian Aboriginal artworkstowards identification and conservation of cultural heritage.

© 2013 Elsevier B.V. All rights reserved.

ndigenous Australianrt conservation

. Introduction

Please cite this article in press as: T. Reeves, et al., Towards identification opyrolysis gas chromatography mass spectrometry, Anal. Chim. Acta (2013)

Art conservators are often required to identify the complexatural materials used in the manufacture of works of art. Sim-

larly, archaeologists and ethnographers are interested in the

Abbreviations: Py-GC–MS, pyrolysis gas chromatography mass spectrometry;C–MS, gas chromatography mass spectrometry; TMAH, tetramethylammoniuimydroxide; HCA, hierarchical cluster analysis; MS, mass spectrometry; IR, infrared;PLC, high performance liquid chromatography.∗ Corresponding author. Tel.: +61 82012191.

E-mail addresses: [email protected] (T. Reeves),[email protected] (R.S. Popelka-Filcoff),[email protected] (C.E. Lenehan).

003-2670/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.aca.2013.09.012

composition of artworks and artefacts. This information assists inboth conserving the items and understanding cultural phenomenaand technologies used throughout history. For example, knowl-edge of the binders present in a painted work can provide valuableinformation on how best to conserve and restore the work withoutcausing damage, as well as provide a basis for authentication anddating of such works based on when and where particular binderswere known to have been used [1–4].

Binders are used to adhere pigment particles together and tothe painted support [5]. They are typically organic macromolecular

f traditional European and indigenous Australian paint binders using, http://dx.doi.org/10.1016/j.aca.2013.09.012

compounds of plant or animal origin, and are composed primar-ily of proteins, lipids, waxes, polysaccharides, or resins [6,7]. Thematerials used as binders vary widely depending on material avail-ability, as well as on the culture and desired artistic style of the artist

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4,8]. Ethnographic studies suggest that the sources of traditionalustralian Aboriginal binders could differ quite substantially from

hose used traditionally in European objects [9], as well as betweenndigenous groups from different regions of Australia. Methods forhe examination and identification of plant and animal residuessed as binders in European artefacts are well documented [10].onversely, the study of Australian objects is difficult as there is

imited research into the identification of Aboriginal Australianinder components, or the original raw materials. Examination ofustralian objects are further complicated as it is known that, afteruropean settlement, many Indigenous Australians began incorpo-ating European-style binders into their artwork [9,10]. Very littles known, however, about exactly when, where, and how theseransitions took place.

Painted works are often very valuable, unique, and of highultural significance, hence sampling from these objects must beimited in order to maintain their integrity [1]. Non-destructivenalytical techniques, such as infra-red spectroscopy (IR) andaman spectroscopy meet this demand, but do not providehe same depth of chemical information as the more destruc-ive chromatographic techniques [4,5]. They are most effectivehen differentiating between the major binder types, rather than

etween binders of the same type [11,12]. Chromatographic tech-iques such as high performance liquid chromatography (HPLC)nd gas chromatography mass spectrometry (GC–MS) allow com-ounds of the same class to be differentiated, even if present inomplex mixtures and matrices. Furthermore, these techniques areapable of analysing all of the different binder types expected in cul-ural paints [4]. HPLC, however, requires the sample to be in liquidorm, which introduces long sample preparation steps that are notmenable to analysis of small binder samples [12–15]. Finding aolvent capable of dissolving the wide range of binder compoundss also quite difficult.

In order to perform analysis by GC–MS, the sample must be ableo be volatilised to a gaseous form; however binders are generallyon-volatile. Consequently GC–MS analysis of binders is mostommonly achieved using a pyrolysis interface; known as Pyroly-is GC–MS (Py-GC–MS) [16,17]. Although Py-GC–MS is destructive,he sample size required for this technique is so small (in therder of 20 �g) that extensive analyses can be performed withoutamaging the integrity of the painted object. Often a small flake ofaint from an ageing painting can be directly analysed without anybservable interference with the object. In general, Py-GC–MS ofaint is performed using in situ sample derivatisation with tetram-thylammonium hydroxide (TMAH) [20]. Upon heating, polar acidsre converted to less polar methyl esters that are easily separatedn the GC. It is these advantages that have led to Py-GC–MS beinghe most commonly used technique for binder analysis [18].

Hierarchical cluster analysis (HCA) is a method in which databtained from Py-GC–MS analyses can then be explored [19–24].CA is an example of unsupervised pattern recognition in which allariation in the dataset is considered and clusters are produced byalculating the distances between samples, with similar samplesonsidered to have close distance [22]. Several distance measuresave been utilised for HCA, with the most common being Euclidian24]. Euclidian distance compares absolute values, which in thease of Py-GC–MS analyses can vary considerably with differentample sizes and shapes [25]. Spearman’s Rank Correlation, whichs a distance measure that does not rely on absolute results, butather their relative rank, is therefore more suitable. In Spearman’sank Correlation the signals (for instance peak height at a givenetention time) are given integer values corresponding to the order


f their magnitude (intensity) within a sample or replicate [25].he distance is then calculated using these ranked variables, and

dendrogram produced, in which the distances between samplesre indicated by the lengths of the lines that join them [13]. Care

PRESSa Acta xxx (2013) xxx– xxx

must still be taken, however, when interpreting the dendrogram,to ensure the groups formed are practically significant in thecontext of the investigation. For this reason Complete Linkage,which defines the distance as that between the most dissimilarvariables in the samples, is often used, hence providing the mostconservative distance and minimising the possibility of falsegroupings.

This paper reports a Pyrolysis-GC–MS (Py-GC–MS) method within situ derivatisation with TMAH for the analysis of a suite of tradi-tional Australian and European binders typically used in Indigenousartworks. Hierarchical Cluster Analysis (HCA) [22] using Spear-man’s rank correlation and Complete Linkage showed that themethod was capable of differentiating twenty-one different Aborig-inal and European binders.

2. Materials and methods

2.1. Samples and reagents

Twenty-one different binders representative of those knownto have been used in European or Indigenous Australian paintedworks were analysed during this research. These are describedin Table 1. These materials were obtained in raw form from theSouth Australian Museum (SAM), various art suppliers, and from A.Blee [26]. Those obtained from SAM were approximately 100 yearsold, while the others were all freshly prepared. Aqueous TMAH(25% m/v, 2 �L, Sigma–Aldrich, Australia) was used as the in situderivatising agent for all Py-GC–MS analyses.

2.2. Sample preparation

Solid binding materials were each ground with a mortar and pes-tle to form homogeneous powders. Thin films of the liquid binderswere prepared on glass slides and heated on a hot plate at 50 ◦Cuntil dry (approximately 2 weeks). The milk and egg samples wereair dried at room temperature for 24 h prior to analysis.

Approximately 1–5 �g of the prepared binder sample wasplaced in a quartz capillary vial (CDS Analytical, Inc., USA) contain-ing a small amount of quartz wool. TMAH (2 �L, 25% m/v) was thenadded to the sample using a GC syringe (SGE Analytical Science,Australia). The pyrolysis tube was then placed immediately intothe pyroprobe for analysis. A minimum of three replicate analyseswere performed for each binder.

2.3. Py-GC–MS instrumentation

Py-GC–MS analyses were performed using a CDS Model 5000Series Pyroprobe coupled to an Agilent Technologies 7890A GCSystem (Agilent Technologies, Santa Clara, USA) with an AgilentTechnologies 5975C inert XL EI/CI MSD with triple axis detector(Agilent Technologies, Santa Clara, USA). The GC column was a HP-5MS Ultra Inert capillary column (30 m × 250 �m × 0.25 �m) witha 5% phenyl 95% methyl polysiloxane stationary phase. Helium wasused as the carrier gas with a flow rate of 1 mL min−1, a split ratioof 50:1, and a solvent delay of 4 min. Unless stated otherwise, apyrolysis temperature of 600 ◦C was used throughout. The pyrol-yser accessory temperature was held at 300 ◦C for 3 min duringpyrolysis, while the valve oven and transfer line were held at 300 ◦Cfor all analyses.

The final GC oven temperature conditions were as follows:50 ◦C for 5 min, then ramped to 240 ◦C at 5 ◦C min−1 before being


held for 5 min, then ramped to 300 ◦C at 5 ◦C min−1 and held for afurther 8 min. Electron Ionisation (EI) at 70 eV was used for massspectra collection over the m/z range of 30–350. The MS ion sourceand quadrupole were held at 230 ◦C and 150 ◦C respectively.

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Table 1Raw binder samples used for py-GCMS method development and library compilation.

Sample Genus and species Source

Aborginal bindersBracket orchid juice Dendrobium speciosium Alisa J. Blee [26]Native Australian beeswax Trigona australis Sugar Bag, BrisbaneMacadamia nut oil Macadamia integrifolia Local supermarketEmu oil Not recorded Talyala Emu Farm, Murray Bridge, South AustraliaGoanna fat Varanus gouldii A. Blee [26]Porcupine grass gum Spinifex genus West of Mt. Liebig, South Australia, SAM No.: A17752Acacia gum Not recorded Coopers Creek (Nqurliwajaka), SAM No.: A2182Yacca gum sample 1 Xanthorrhoea tateana Munston, Kangaroo Island, South Australia, SAM No.: A66781Yacca gum sample 2 Xanthorrhoea semiplana West of Clarendon, South Australia, SAM No.: A66782Iron wood Not recorded Mt Zeil, Central Australia, SAM No.: A17755European bindersManila copal Agathis dammara Talas, New York, USAManila elemi – Talas, New York, USASandarac Tetraclinis articulata Talas, New York, USAShellac Kerria lacca Art Materials Pty. Ltd. VictoriaHeat thickened linseed oil – Art Materials Pty. Ltd. VictoriaEuropean beeswax – Talas, New York, USAPoppy seed oil – Art Stretchers Co Pty Ltd, AdelaideWalnut oil – Local supermarket

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.4. Statistical analysis

The assignment of pyrolysis products was based on compar-son of calculated retention indices (RI) with literature values

here they could be obtained [27], as well as the match qual-ty obtained from the Mass spectral software MSD ChemStation.02.02.1431 Software (Agilent Technologies, Inc.), which utilisedhe National Institute of Standards and Technology (NIST) Standardeference Database Number 69 – NIST Chemistry WebBook [27].xperimentally determined RIs could not be obtained for allompounds, and hence RI estimates obtained from the NISTatabase were also used. These are calculated using quantitative

tructure–(chromatographic) retention relationships (QSRR) basedn the functional groups present in the molecules.

Chromatographic peak areas were calculated using the MSDhemStation E.02.02.1431 Software (Agilent Technologies, Inc.)nd expressed as a percentage of the total chromatographicrea for each run. Visually comparable chromatograms were alsobtained by range normalising the peak intensities across eachhromatogram using The Unscrambler® X 10.0.1 from CAMO Soft-are, in which the most intense signal was assigned the number 1

nd the lowest assigned 0, and each other data point given valueselative to these limits. The resulting chromatograms were thenveraged over each trial for each binder, using Eq. (1), where xi washe peak intensity at a particular retention time for each of the neplicates.

verage peak intensity = ˙xi

n(1)

In order to perform HCA, the raw peak areas of peaks found to bendicative of each of the five major binder types (polysaccharides,esins, lipids, waxes, and proteins), were first range normalisedsing The Unscrambler® X 10.0.1 from CAMO Software. HCA usingpearman’s Rank Correlation and Complete Linkage was then per-ormed on this dataset.

. Results and discussion


.1. Method development

Initial Py-GC–MS conditions were based upon those of Wangt al. [23] who had reported a method for the analysis of shellac. In

Art Stretchers Co Pty Ltd, AdelaideLocal supermarketLocal supermarket

order to examine the effect of pyrolysis temperature on the result-ing chromatograms, Manila copal, European beeswax, shellac, andsandarac were each analysed at pyrolysis temperatures of 400,600, and 750 ◦C [10,22,23]. For all binders, increasing the pyrolysistemperature from 400 ◦C to 750 ◦C resulted in a greater numberof components eluting within the first 20 min of the analysis,with fewer components eluting later in the run. A temperature of750 ◦C, however, resulted in a number of overlapping peaks withinthis region. This is consistent with increased thermal degradationat higher temperatures. A pyrolysis temperature of 600 ◦C wasused for the remainder of the study as this represented a goodcompromise with all components eluting within the 60 min runtime and limited overlap of early eluting peaks.

Using the conditions reported by Wang et al. [23], the Europeanbeeswax resulted in the formation a small number of products thatwere not eluted within the run time and were carried over into thenext analysis. Consequently, the GC oven temperature gradient wasmodified to that of Bonaduce and Columbini [32], who had reportedthe analysis of European beeswax. These conditions removed thecarry-over effects, but produced extensive peak overlap. The finalGC temperature gradient was a mixture of the two methods, andallowed adequate separation throughout the pyrogram. It shouldalso be noted that the solvent delay (the time between the startof the GC run and the acquisition of data) was also increased from1.5 min to 4 min when compiling the binder library, so that anysignals from the TMAH itself would be ignored.

3.2. Binder chemistry

Typical range-normalised, averaged, and offset chromatogramsof each binder analysed using the developed Py-GC–MS method aregiven in Fig. 1. As can be seen, each binder resulted in distinctly dif-ferent pyrograms. Furthermore, visual inspection showed that thebinders could be identified as belonging to different correspondingbinder classes (waxes, lipids, proteins, resins and polysaccharides)based on their pyrogram profiles. Despite the obvious differencesbetween pyrograms, peaks corresponding to hexadecanoic acidmethyl ester and octadecanoic acid methyl ester were present


at 34.6 min and 38.5 min respectively in every binder pyrogram.The RIs for these peaks were 1934 and 2226 respectively, whichwere very close to those obtained in previous experiments in lit-erature for these compounds (1928 and 2139 respectively), hence

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ig. 1. Range-normalised, averaged, and offset chromatograms of lipid-based,esinous, polysaccharide, wax-based, and proteinaceous binders obtained by py-C-MS analysis using the developed method and a pyrolysis temperature of 600 ◦C.

upporting their identification [27]. These compounds wereethanolysis products of fatty acids found within the natu-

al products, and were commonly carried over between runs.ttempts to remove this carry-over through column cleaningnd high temperature burn-off, as well as replacement of theC inlet and liner were performed with limited success, hence


uggesting that the carry-over was as a result of memory effectsrom the Pyrolyser-GC interface, which are commonly observedn oil analysis [8,28]. The peak areas of these compounds wereherefore excluded in the calculations of percentage peak areas

able 2entative identification of pyrolysis products unique to each polysaccharide binder, with avetention times (RT), molecular ions (M+), and retention indices (RI).

Binder Proposed Pyrolysis Product

Yacca

1-(2-Hydroxy-4-methoxyphenyl)-ethanone

1,2-Dimethoxy-4-(2-methoxyethenyl)benzene

2′ ,4′-Dimethoxyacetophenone

3-(4-Methoxyphenyl)-2-propenoic acid methyl ester

2,4,6-Trimethoxyacetophenone

3,4,5-Trimethoxy-benzamide

OrchidButanedioic acid dimethyl ester

Orchid unknown 1

IronwoodIronwood unknown 1

2,3-Di-O-methylpentopyranose

Ironwood unknown 1

Acacia

Acacia unknown 1

Acacia unknown 2

1,2,3,4,5,6-Hexa-O-methyl-myo-inositol

Acacia unknown 3

Acacia unknown 4

ll Literature RIs obtained from National Institute of Standards and Technology (NIST) [2a based on experimental data using the Van den Dool and Kratz equation.b calculated estimate based on functional groups


and when conducting HCA (Section 2.4). This meant, however,that the calculation of fatty acid ratios (a common method for lipiddifferentiation [3,21,29,30]) could not be utilised.

In general, waxes produced peaks predominantly after 45 mincorresponding to the series of long-chain hydrocarbons com-monly identified in wax analysis [31–34], while the proteinpyrolysis products were eluted between 30 and 40 min, typicalof well-known pyrolysis products of amino acids [35–37]. Oilsproduced two groups of peaks; one at approximately 9 min due tomethanolysis derivatives of the glycerol backbone of triglycerides,and the other group between 30 and 40 min corresponding tofatty acid methyl esters. The animal products goanna fat and emuoil also produced these compounds, as will be discussed in latersections. Resins produced distinctive groups at around either 40 or60 min, depending on whether they were di- or tri- terpenic resinsrespectively. The polysaccharide binders were less easily grouped,most likely because plants contain a wide variety of carbohydrates[38]. Acacia gum, and orchid juice did, however, share a commonpyrolysis product at 22.1 min, being 1,2,4-trimethoxy benzene; acommon pyrolysis product of carbohydrates [39]. The RI of thispeak was 1376, which correlated well with the RI of 1378 obtainedfor this compound by other researchers [40], hence supporting itsidentification.

Closer examination of the resulting mass spectra revealed thatmost binders examined had at least one pyrolysis product thatwas indicative of that particular substance. These are given inTables 2–6, presented separately according to binder class and arediscussed below. The reliability of the QSRR estimates depends onthe quality of the retention index data used as input for the calcu-lation. As a result these calculated values can be subject to error.Additionally, the NIST database does not provide an error estimatefor the reported values. Thus it is difficult to determine whether ameasured RI is indeed within the required range of the reported RI.

3.2.1. Polysaccharide bindersYacca, orchid, ironwood and acacia are all examples of polysac-

charide containing binders. The yacca gums each produced variousacetophenones, which were most likely formed during the pyrol-ysis of chalcones; components of many small plants [41]. The


retention indices of these products also closely matched those pre-viously obtained for each corresponding compound [27], and thematch qualities were all quite high, indicating that sufficient sep-aration had been obtained using the developed method to allow

erage peak areas of at least 1% of the total pyrographic peak area, with corresponding

M+ RT (min) RI Lit. RI

166 23.9 1447 1438a

194 25.7 1520 1455b

180 27.1 1580 1586a

192 29.3 1678 1546b

210 30.2 1719 1596b

211 31.4 1775 1770b

146 12.1 1041 1036a

294 24.9 1487 –

294 19.9 1294 –178 20.8 1328 1359b

216 22.3 1384 –

175 10.1 982.8 –138 10.4 991.1 –264 25.8 1525 1541a

204 29.8 1700 –250 44.8 2614 –

7].

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Table 3Tentative identification of pyrolysis products unique to each resinous binder, with average peak areas of at least 1% of the total pyrographic peak area, with correspondingretention times (RT), molecular ions (M+), retention indices (RI), and corresponding literature RIs (Lit. RI).

Binder Proposed pyrolysis product M+ RT (min) RI Lit. RI

Shellac

7-Hydroxy-octadecanoic acid methyl ester 314 33.3 1868 2239b

Shellac unknown 1 306 36.5 2034 –3,5-Di-tert-butyl-4-hydroxycinnamic acid 276 36.7 2045 2231b

Laccijalaric acid methyl ester 306 37.1 20672-[[2-[[2-[(2-Pentylcyclopropyl)methyl]cyclopropyl]methyl]cyclopropyl]-methyl]-cyclopropanebutanoic acid methyl ester

374 38.8 2249

9,10,12-Trimethoxy-octadecanoic acid methyl ester 388 38.9 2256 2411b

Jalaric acid methyl ester 336 39.6 2307Shellac unknown 2 342 41.1 2396 –Shellac unknown 3 232 42.7 2494 –

Spinifex

Naphthalene 128 16.7 1184 1191a

Spinifex unknown 1 426 58.3 3307 –1-(5,5,8a-Trimethyl-2-methylenedecahydro-1-naphthalenyl)-3-methyl-3-pentanol 292 58.5 3322 2026b

Spinifex unknown 2 292 58.7 3336 –Spinifex unknown 2 424 58.8 3344 –�-Amyrin 426 59.0 3358 3337a

Spinifex unknown 3 502 60.5 3469 –Spinifex unknown 4 341 61.0 3506 –Spinifex unknown 5 287 61.2 3519 –

Manila elemi

�-Terpineol 154 17.0 1193 1189a

Elemol 222 26.5 1554 1547a

Elemicin 208 26.7 1563 1560a

Isoelemicin 208 28.9 1660 1650a

Manila elemi unknown 1 222 32.4 1824 –�-Amyrin 426 60.6 3476 3376a

Manila elemi unknown 2 442 60.8 3491 –Oleanolic acid 456 61.0 3506 3242b

Manila elemi unknown 3 338 61.3 3525 –Betulin 442 62.2 3584

Manila copal

Agatholic acid 304 38.6 2234 2276b

Methyl pimarate 316 41.6 2427 –Manila copal unknown 1 304 43.2 2524 –Manila copal unknown 2 362 43.8 2559 –19-Hydroxy-labda-8(20)-13-dien-15-oic acid methyl ester 304 44.3 2588 2429b

Methyl1,4a-dimethyl-6-methylene-5-(3-methyl-5-oxohexyl)decahydro-1-naphthalenecarboxylate

348 44.7 2609 2372b

Manila copal unknown 3 342 45.0 2623 –Methyl athecate 362 45.7 2656 2453b

Sandarac

�-Pinene 136 8.29 932.8 922a

Kaur-16-en-18-oic acid methyl ester 316 39.2 2279 2056b

Methyl1,4a-dimethyl-6-methylene-5-(3-methyl-5-oxohexyl)decahydro-1-naphthalenecarboxylate

348 42.6 2488 2372b

Unknown sandarac 1 332 42.7 2494 –Unknown sandarac 2 316 43.3 2530 –Decahydro-5-(methoxycarbonyl)-�,5,8a-trimethyl-2-methylene-1-naphthalenepentanoic acid 364 44.0 2570 2403b

12-Hydroxy-13-isopropyl-podocarpa-8,11,13-trien-3-one 300 44.9 2618 2396b

A ST) [27

fatcotp

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ll Literature RIs obtained from National Institute of Standards and Technology (NIa Based on experimental data using the Van den Dool and Kratz equation.b Calculated estimate based on functional groups.

or accurate identification. The only pyrolysis product that had retention index differing from its expected value was 2,4,6-rimethoxyacetophenone; having an RI of 1719 compared to thealculated RI of 1519 [27], despite having a high MS match qualityf 90.5%. This discrepancy could be due to the errors associated withhe QSRR model used to calculate the literature RI, as mentionedreviously.

Ironwood was found to produce 2,3-di-O-methylpentopy-anose, which is the methyl derivative of a common monosaccha-ide in wood plants [38], as well as a compound at 22.3 min thatould not be identified using the available database. Both ironwoodnd orchid juice also produced unknown compounds at 19.9 and4.9 min respectively that were both identified by the databases 2,4,5,6,7-pentamethoxyheptanoic acid methyl ester. The great


ifference in their retention times, however, suggested that thesewo peaks could not be due to the same compound. The reten-ion indices of these pyrolysis products (1487 and 1294 for those at4.9 and 19.9 min respectively) were also quite different from the

].

1604 obtained in literature [27], and hence further investigationis required. Despite difficulties with their identification, however,these compounds were produced reproducibly by each binder, andhence could still be used for their differentiation. Orchid juice wasalso found to produce butanedioic acid dimethyl ester, which isa methyl-derivative of succinic acid, which plays a critical role inthe citric acid cycle in plants [41]. The citric acid cycle is a com-mon process, and hence this compound may also be present inother materials, and hence other binders. Further investigation intoother binding materials is therefore required to determine if thiscompound is indeed an effective marker for orchid juice.

Acacia produced 1,2,3,4,5,6-hexa-O-methyl-myo-inositol,which is characteristic of carbohydrate pyrolysis [38], as well asseveral compounds that could not be identified using the available


database. The RI for this product (1525) matched its expected value(1541) quite well, despite having a very low MS match quality of16.6%. This could have been a result of peak overlap, which wouldhave given rise to foreign peaks in the MS, hence decreasing the

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Please cite this article in press as: T. Reeves, et al., Towards identification of traditional European and indigenous Australian paint binders usingpyrolysis gas chromatography mass spectrometry, Anal. Chim. Acta (2013), http://dx.doi.org/10.1016/j.aca.2013.09.012



6 T. Reeves et al. / Analytica Chimica Acta xxx (2013) xxx– xxx

Table 4Tentative identification of pyrolysis products unique to the wax binders, with average peak areas of at least 1% of the total pyrographic peak area, with correspondingretention times (RT), molecular ions (M+), and retention indices (RI).

Binder Proposed Pyrolysis Product M+ RT (min) RI Lit. RI

European beeswax

1-Octadecanol methyl ether 284 36.5 2034 2032a

Methyl 14-methoxyhexadecanoate 300 37.7 2100 1990b

European beeswax unknown 1 296 44.6 2604 –n-Tetracosanol 354 47.5 2735 2711a

Octacosane 394 49.3 2807 2800b

European beeswax unknown 2 354 52.2 2939 –European beeswax unknown 3 396 55.8 3142 –European beeswax unknown 4 380 56.8 3205 –European beeswax unknown 5 410 59.3 3380 –European beeswax unknown 6 480 61.5 3538 –

Native beeswax

Native beeswax unknown 1 222 27.8 1610 –3-(4-Methoxyphenyl)-2-propenoic acid, methyl ester 192 29.3 1678 1546b

Ketodihydrogendunin 440 38.4 2218Native beeswax unknown 2 317 39.0 2264 –Native beeswax unknown 3 316 40.5 2361 –Native beeswax unknown 4 316 40.6 2366 –Native beeswax unknown 5 324 41.3 2408 –Dihydroagathic acid 336 42.5 2482 2583b

2-(9-Octadecenyloxy)-ethanol 312 42.9 2506 2336b

Native beeswax unknown 6 312 43.1 2518 –Native beeswax unknown 7 402 57.4 3246 –�-Amyrin 426 59.0 3358 3337a

All Literature RIs obtained from National Institute of Standards and Technology (NIST) [27].a Based on experimental data using the Van den Dool and Kratz equation.b Calculated estimate based on functional groups.

Table 5Tentative identification of pyrolysis products unique to the oil binders, with average peak areas of at least 1% of the total pyrographic peak area, with corresponding retentiontimes (RT), molecular ions (M+), and retention indices (RI).


Linseed oil

N,N,N′ ,N′-tetramethyl-1,2-ethanediamine 116 14.8 1122Linolenic acid methyl ester 292 37.1 2067 2105a

Linseed unknown 1 310 37.7 2100 –Linseed unknown 2 310 38.1 2187 –Linseed unknown 3 310 38.6 2234 –Linseed unknown 4 290 38.9 2256 –Linseed unknown 5 294 41.3 2408 –

Macadamia Nut Oil 1,2,3-Trimethoxy propane 134 7.18 902.0 781.0b

Poppy seed oil8-Methoxyoctanoic acid methyl ester 188 21.5 1354 1258b

Methyl-9,10-epoxystearate 312 41.2 2402 2129b

Poppy seed unknown 1 322 43.6 2547 –

All Literature RIs obtained from National Institute of Standards and Technology (NIST) [27]:a based on experimental data using the Van den Dool and Kratz equation.b calculated estimate based on functional groups

Table 6Tentative identification of pyrolysis products unique to animal product binders, with average peak areas of at least 1% of the total pyrographic peak area, with correspondingretention times (RT), molecular ions (M+), and retention indices (RI).


Egg YolkEgg yolk unknown 1 294 38.6 2234 –Egg yolk unknown 2 294 38.7 2241 -

Goanna Fat Goanna unknown 1 226 39.8 2319 –

Milk

Hexanoic acid methyl ester 130 8.21 930.5 924.0a

1,2,4-Trimethoxy-butane 148 13.2 1073 881.0b

3-Hydroxy-5-methoxy-benzenemethanol 124 18.6 1249 1446b

Decanoic acid 186 20.8 1328 1368a

Milk unknown 1 219 25.1 1495 –2,4,5,6,7-Pentamethoxyheptanoic acid methyl ester 294 25.3 1503 1604b

Dodecanoic acid methyl ester 214 25.9 1529 1526a

Rabbit skin glueDihydro-1,3,5-trimethyl-2,4(1H,3H)-pyrimidinedione 198 24.3 1463 1422b

Uric acid 210 30.0 1710 1810b

Rabbit skin glue unknown 1 154 31.2 1766 –

All literature RIs obtained from National Institute of Standards and Technology (NIST) [27]:a Based on experimental data using the Van den Dool and Kratz equation.b Calculated estimate based on functional groups

Refs. [26,27].

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atch quality without altering the RI. The compound that wasluted at 29.8 min was identified by the database as 2,3,4-trimethylevoglucosan, which is also characteristic of carbohydrate pyrol-sis [38], however the literature RI for this compound was 1404;pproximately 300 less than that obtained here. This literature RIas obtained from experiments using a similar column to thatsed here (a VF-5MS capillary column), however the GC temper-ture gradients used were quite different [27]. It is known thathe applicability of retention indices decreases as the complexityf the temperature ramp increases, hence leading to inaccuracies42]. Further identity confirmation is therefore required.

.2.2. Resinous bindersShellac, Spinifex, sandarac, manila elemi, and manila copal

ere evaluated. Within these there appeared to be three distinctroups; those containing diterpenes, triterpenes, and sesquiter-enes. Sandarac and manila copal, both produced methyl transommunate, methyl isopimarate, methyl sandaracopimarate,gatholic acid and methyl athecate, which are known pyrolysisroducts of diterpenic resins (data not shown due to the presencef these compounds in more than one binder) [43]. They could beifferentiated, however, by a very distinctive peak at 8.29 min inhe sandarac pyrogram, corresponding to �-pinene, a known mainomponent of terpenes [44]. Sandarac also produced kaur-16-en-8-oic acid methyl ester, which has been previously isolated as

component of plant terpenoids [45]. Two naphthalene-relatedompounds, decahydro-5-(methoxycarbonyl)-�,5,8a-trimethyl--methylene-1-naphthalenepentanoic acid and methyl,4a-dimethyl-6-methylene-5-(3-methyl-5-oxohexyl)decahydro--naphthalenecarboxylate were also produced. Naphthalenes arenown pyrolysis products of several resins, and hence this resultas to be expected [46]. The peak at 43.3 min in the pyrogram of

andarac could not be identified using the available database, how-ver its mass spectrum had ions at 346, 189, and 121 m/z, whichs characteristic of dimethyl agathate; known to be formed fromandarac [43]. The retention index of this compound could not,owever, be obtained for comparison, and hence no conclusionss to its identity can be made at this time. The peak at 44.9 minas identified as 12-hydroxy-13-isopropyl-podocarpa-8,11,13-

rien-3-one by the database, however its RI of 2618 did not closelyatch the calculated value of 2396 [27], and hence further identity

onfirmation is required. Despite the difficulties in identifying thisompound, it was produce reproducibly, and hence could still acts a marker for sandarac.

Manila copal produced an unidentified peak at 43.2 min withass spectral ions at 348, 303, 243, 221, and 189 m/z, which is

haracteristic of methyl agatholate; a known pyrolysis product ofanila copal [43]. Again, the retention index of this compound

ould not be obtained, and hence further identity confirmations required. The product which was eluted at 43.8 min in thehromatogram of manila copal had an almost identical mass spec-rum to that of methyl athecate, which was eluted at 45.7 minn manila copal. It is therefore possible that this compound wasn isomer of methyl athecate, which is well known in litera-ure [43,47]. The RIs for both methyl athecate and its suspectedsomer (2656 and 2559 respectively), however, were both differ-nt from the calculated value of 2453 for methyl athecate [27].gain, this expected RI was an estimate rather than an experi-ental value, and hence could have been inaccurate and hence

urther investigation to confirm the compound identity is required.imilarly to sandarac, manila copal also produced the naphthalene-elated compound of methyl 1,4a-dimethyl-6-methylene-5-(3-


ethyl-5-oxohexyl)decahydro-1-naphthalenecarboxylate, whichs expected for during resin pyrolysis [48]. The retention index forhis product was 2609, however, which was quite different to thealculated literature value of 2372, and hence further identification

PRESSa Acta xxx (2013) xxx– xxx 7

confirmation is required. Again, despite of difficulties in identifica-tion, 8 pyrolysis products were identified as being unique to manilacopal in the context of the chosen binders. These findings were verypromising, because it is known that sandarac and manila copal canbe very difficult to differentiate chemically [47].

Manila elemi produced most of the compounds expected fromelemi pyrolysis; being �-terpineol, elemicin, elemol, and �-amyrin[49,50]. �-amyrin was also expected but not observed, which couldhave been due to co-elution of this compound with its isomer, �-amyrin, which has very similar chromatographic behaviour [49].Manila elemi was also found to produce oleanolic acid, which ischaracteristic of triterpenoid resins with an oleanane skeleton [49].A compound with a mass spectra resembling that of betulin wasalso detected at 62.2 min, however no retention indices obtainedusing the same column as that used here could be obtained in liter-ature, and hence further identity confirmation is required [27,42].

Spinifex was also found to produce �-amyrin, which is a knownpyrolysis product of triterpenoid resins, as mentioned previously.To this authors knowledge this was the first in-depth chemi-cal analysis of spinifex, and hence the first chemical indicationthat spinifex gum is a triterpenoid resin. Two naphthalene-relatedcompounds were also produced at 16.7 and 58.5 min, which,as mentioned previously, is highly characteristic of a varietyof resins [51]. The compound eluted at 58.5 min was identifiedby the database as 1-(5,5,8a-Trimethyl-2-methylenedecahydro-1-naphthalenyl)-3-methyl-3-pentanol, however its retention indexof 3322 did not correlate with the 2026 obtained for this compoundin literature [27]. This discrepancy could be due to this literaturevalue being based on the QSRR model, or this could be then wrongidentification, and hence further investigation is required. Despitethese difficulties in identification, these 10 pyrolysis products thatwere unique to spinifex, in the context of the chosen binders, wereall formed reproducibly, and hence could be used effectively for itsdifferentiation from the other binders.

Shellac produced different products again, such as octadecanoicacid methyl esters from the fatty acid component of shellac [20,49],and two products at 37.1 and 39.6 min, which were unable to beassigned using the available database. These products had molec-ular ions of 306 and 336 m/z respectively, and their mass spectraclosely matched those of laccijalaric and jalaric acid methyl esters,which are well-known pyrolysis products of the sesquiterpenecomponent of shellac [20]. Similarly, the peak at 33.3 min was iden-tified by the MS database as 7-hydroxyoctadecanoic acid methylester, however it also had a mass spectrum very similar to thatof 9,10,16-trimethoxyhexadecanoic acid methyl ester, having ionsat 157 and 127 m/z corresponding to characteristic �-cleavage atC-9 and C-10 [52]. This compound is a known methanolysis prod-uct of aleuritic acid; a main component of shellac, however itsliterature retention index could not be obtained for comparison,and hence further identification is required [8,20]. 2-[[2-[[2-[(2-pentylcyclopropyl)methyl]cyclopropyl] methyl]cyclopropyl]-methyl]-cyclopropanebutanoic acid methyl ester was most likelythe partial cyclisation product of hydroxylated fatty acids, how-ever RIs could not be found in the literature for this compound,and hence its identity would have to be confirmed. Butolic acid(6-hydroxytetradecanoic acid), another main component of shel-lac, was not detected using this method. This could have been theresult of peak overlap early in the run, resulting in this co-elutionof this compound with other products.

3.2.3. WaxesIt was also found that the wax binders could be easily dif-


ferentiated. Both native and European beeswaxes produced acharacteristic series of long-chain hydrocarbons, but the nativewax also produced �-amyrin and dihydroagathic acid, which arecharacteristic of resinous binders, as mentioned previously. Two

dx.doi.org/10.1016/j.aca.2013.09.012

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losely eluting compounds at 40.5 and 40.6 min were also detectednd identified by the database as methyl trans communate, anotherharacteristic product of resins [43]. Their similar mass spectrauggested that they may be isomers of each other, however no liter-ture retention indices for these compounds could be obtained, andence further confirmation is needed. The native wax analysed hadot been purified after collection, and hence would have containedropolis; a plant resin collected by bees and mixed with theirax for use in hive building [53,54]. The European wax analysed,owever, had been purified, and hence would not have containedhese components. Further research into binder history would beequired in order to determine whether the raw or purified formsf the waxes were actually used as binders in painted works.

There were multiple pyrolysis products of both waxes that couldot be identified using the available database, as shown in Table 6.

t is unlikely that this was due to peak overlap in this case becauset was observed that the beeswax peaks were the best resolved ofll of the binders. It is therefore possible that the single MS wasncapable of distinguishing compounds with similar mass spec-ra, or that these compounds were not part of the database used.andem MS would be required in order to properly identify theseompounds. Similarly, a literature RI for the compound at eluting8.4 min in the Australian beeswax could not be obtained, andence its identity would also have to be confirmed.

.2.4. OilsVery few unique peaks were identified for the oil binders, and

hose that were identified were quite small, having an averageercentage peak area of 1.63% of the total pyrographic peak area.alnut oil, for instance, did not produce any unique pyrolysis prod-

cts with percentage peak areas above 1%. This was most likely theesult of oil binders consisting predominantly of triglycerides, withther compounds, such as hydrocarbons, phenolic compounds, andax esters contributing to only 2–5% of the volume [55]. The dif-

erences between oils based on these components alone wouldherefore be quite minimal, as has been found in many previ-us studies [1,29,47,56–58]. This result highlights the usefulnessf comparison of fatty acid methyl ester ratios in lipid differen-iation. Care should, however, be taken in light of the commonccurrence of carry-over from these compounds, as was observedn this research.

The identifying pyrolysis products that were detected in eachinder could have been a result of a range of factors. For instance,ll lipid binders contain triglycerides, and hence would be expectedo produce methyl derivatives of the glycerol backbone of triglyce-ides during pyrolysis with TMAH. Here the dimethoxy derivativef glycerol was produced by each lipid binder, but only macadamiaut oil produced the trimethoxy derivative. This could have beenue to differences in the shape or viscosity of macadamia oil that

ed to higher methylation efficiency occurring reproducibly in thisinder than in others.

Linseed oil was found to produce 3 consecutive peaks at 37.7,8.1, and 38.6 min that were all identified as methyl 15-hydroxy-,12-octadecadienoate. These products were eluted at differentimes, however, and hence could not be the same compound. Theroduct eluting at 38.6 min had an RI closest to the 2255 expectedor methyl 15-hydroxy-9,12-octadecadienoate [27], hence indicat-ng that this could be the correct identification for this compound.t is possible that the other two compounds were geometric iso-

ers of this or a different unknown compound, hence resultingn their similar retention times and mass spectra. A product at4.8 min was identified by the database as N,N,N′,N′-tetramethyl-


,2-ethanediamine. The only available literature RI value wasbtained from isothermal GC analysis using a packed column,hich is very different to the conditions used here, and hence

t could not be used for comparison [27,42]. The occurrence of


this nitrogen-containing compound, however, was unusual for thepyrolysis of linseed oil. It could have been produced as a result ofthe pyrolysis of the non-triglyceride components of the oil, or, morelikely, it could have been a product of the TMAH methylating agent.The high viscosity of linseed oil compared to the others could resultin incomplete methylation occurring, hence allowing the TMAH topyrolyse with itself.

Poppy seed oil was found to produce three unique pyrolysisproducts, with one at 43.6 min being unidentifiable using the MSdatabase. 8-Methoxyoctanoic acid methyl ester was identified at21.5 min with a high match quality of 91.7%, and an RI of 1354compared to the calculated 1258 from literature. These values werequite close considering the literature RI was obtained using QSRRmodels [27]. This compound was most likely a product from thetriglyceride component of this oil. Methyl-9,10-epoxystearate wasalso identified at 41.2 min, and could have been a product of partialcyclisation of a hydroxylated fatty acid [59]. The RI for this prod-uct of 2402 was, however, quite different to the calculated value of2129, and hence further identity confirmation is required.

3.2.5. Animal productsThe animal products, egg yolk, milk, emu oil, goanna fat, and

rabbit skin glue each consist of multiple components comprisinglipids, carbohydrates, and proteins. During this study it was foundthat only the lipid component of the egg yolk could be detected,resulting in the production of a pyrogram quite similar to those ofthe oils described previously, as can be seen in Fig. 1. The pyrol-ysis products produced solely by this binder in this study weretwo closely-eluting compounds at 38.6 and 38.7 min, each withvery similar mass spectra. Both of these products were identifiedas linoleic acid by the MS database, however the match quality wasonly approximately 20% for each, and their retention indices wereroughly 200 units above those expected for linoleic acid [27]. It istherefore likely that this is not a correct identification, and henceconfirmation using tandem MS is required. Despite of this, thesetwo peaks were both produced reproducibly by the egg binder, andhence could be used for its identification.

Milk was found to produce pyrolysis products from both thesugar and the lipid components of milk; exhibiting 7 identifyingpyrolysis products. The compounds produced at 8.21, 20.8, and25.9 min matched their corresponding expected RIs most closely;having a maximum difference of 40, and were most likely pyrolysisproducts of the lipid components of milk. The RI for the prod-uct identified by the database as 2,4,5,6,7-pentamethoxyheptanoicacid methyl ester, another product of lipid pyrolysis, however,was 100 units lower than its expected value [27]. Again, this lit-erature RI was a prediction, rather than based on experimentalvalues, and hence could have been inaccurate. Another product,3-hydroxy-5-methoxy-benzenemethanol, had a similar structureto the pyrolysis products of many of the polysaccharide binders,such as 2,3,4-trimethyl levoglucosan, 1,2,3,4,5,6-hexa-O-methyl-myo-inositol, which was produced by the polysaccharide acacia.This suggested that this product was a result of pyrolysis of thecarbohydrate, or sugar, component of the milk [34,38].

Rabbit skin glue was found to produce compounds from itsprotein component, such as distinctive nitrogen-containing ringsformed by the pyrolysis reaction of two amino acid monomers[35–37]. Emu oil did not produce any unique pyrolysis products,and hence further research into the differentiation of this binderis required. Finally, goanna fat was found to produce one uniquemarker compound that was eluted at 39.8 min, which could notbe identified. This product did, however, occur reproducibly, and


hence could still be an effective marker for goanna fat.It is important to stress that although the products listed

throughout this paper were produced by only one of the bindersanalysed, it is possible that they could also be produced by binders

dx.doi.org/10.1016/j.aca.2013.09.012




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ot included. They are therefore only unique in the context of thistudy. The pyrolysis products identified for spinifex, for instance,re very similar in structure to those obtained from the pyrolysis ofammar and mastic, two other commonly used binders [49]. Sim-

larly, the identified markers for manila elemi are also producedy other elemi resins not included here [44]. The binders analysedherefore need to be expanded to assess the differentiation abilityf the method for all binders. Similarly, it is possible that the iden-ified markers may also be produced by other materials not useds binders. Amber, for instance, which is fossilised terpenoid resin60], is known to produce butanedioic acid during pyrolysis, the

ethyl ester of which has been listed here as a marker for orchiduice [59]. This compound is also known to be a metabolite of respi-ation in animal tissues [61]. The likelihood of non-binder materialseing present in a paint sample that could also form these mark-rs must therefore be assessed when using the method to identifynknown real samples. These limitations mean that the observa-ion of these marker compounds, whilst indicative of binder type,annot be used alone in determining the binder identity. As suchdditional data within the pyrogram is needed. Pattern recognitionechniques such as HCA, can determine if the binders are able to berouped into different subsets on the basis of their pyrogram data.his then provides the foundation for development of models forhe assignment of unknowns to class groups.

.3. Hierarchical cluster analysis

The resulting dendrogram from HCA of all five replicates of eachf the analysed binders, performed using Spearman’s Rank Corre-ation and Complete Linkage is given in Fig. 2. The replicates forach binder were all grouped together with relative distances ofpproximately zero, hence indicating a high level of reproducibilityn peak area magnitude orders had been achieved between trials.


here were a few binders for which trials were slightly more dis-antly linked, such as orchid juice, which was most likely due to theon-homogeneous nature of these natural samples. These dis-ances between trials were still, however, much less than those

ts unique to each binder and binder type, from the analyses of all library binder

calculated between different binders, hence indicating that thedeveloped Py-GC–MS method was effective in differentiatingbetween each of the binders.

The major binder types were also grouped together, for instancethe oils were all placed on the same branch at the bottom whilethe diterpenoid resins were on a separate branch at the top of thedendrogram. The developed Py-GC–MS method, and the selectedchromatographic peaks, had therefore been shown to be effectivein differentiation of polysaccharide binders of the same age, andbetween oil, resin, wax, and protein binders of the same age.

Consideration must be given, however, when interpreting theresults as the polysaccharide binders were aged samples whilstthe remaining were freshly prepared. It is possible that additionaldistinctive markers for polysaccharides could be detected in freshsamples, and similarly the markers identified for the fresh bindersmay not still be present in aged samples, due to factors such asUV light degradation, and exposure to pollutants and moisture[33,62]. Further research into the stability of these markers overtime must therefore be conducted to assess the true distinguish-ing power of the developed method. Similarly, real paint sampleswill generally contain pigments, as well as possibly a mixture ofmultiple binders, and hence these compounds could interfere withthe detection of the identified markers [33,62]. Some pigments, forinstance, have been observed to form complexes with amino acidsduring hydrolysis derivatisation, hence impeding their Py-GC–MSanalysis [1,32,33]. Research is therefore currently being conductedto determine, initially, the effect of ochre on binder differentiationusing this method. The method must then be applied to the analysisof real paint samples to assess its suitability in real scenarios.

4. Conclusions

A Py-GC–MS method capable of differentiating between


polysaccharide, lipid, resin, wax, and protein based binders, aswell as between binders of the same chemical class, was devel-oped. A chromatographic library of 21 raw binders traditional toEuropean and Australian Aboriginal cultures was compiled and

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nique pyrolysis products were identified for each material. Thisethod represents the first step in providing valuable information

or conservation, restoration, authentication, and dating of previ-usly un-characterised painted works from Australian Aboriginalultures.

cknowledgements

The authors would like to thank Alisa Blee, David VincentFlinders University) and Keryn Walshe, Tara Dodd, Philip Clarke,nd Philip Jones (South Australian Museum) for assistance withollecting, preparing and analysing many of the binder samples.r. Rachel S. Popelka-Filcoff would also like to thank the Australian

nstitute of Nuclear Science and Engineering (AINSE) Research Fel-owship. Tiffany Reeves would like to thank South Australian (SA)

ater, the Playford Memorial Trust, and the Ferry Trust in Adelaideor their generous financial support throughout her studies.

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