You are on page 1of 8

Available online at www.sciencedirect.

com

Microchemical Journal 88 (2008) 186 – 193
www.elsevier.com/locate/microc

Analysis of lignin from archaeological waterlogged wood by direct exposure
mass spectrometry (DE-MS) and PCA evaluation of mass spectral data
F. Modugno a,⁎, E. Ribechini a , M. Calderisi a , G. Giachi b , M.P. Colombini a
a

b

Dipartimento di Chimica e Chimica Industriale, Università di Pisa, Via Risorgimento 35, I-56126 Pisa, Italy
Soprintendenza ai Beni Archeologici per la Toscana, Laboratorio di Analisi-Centro di Restauro, Largo del Boschetto 3, I-50135 Firenze, Italy
Received 3 August 2007; accepted 21 November 2007
Available online 8 January 2008

Abstract
The chemical characterisation of waterlogged archaeological wood is of fundamental importance to understand the degradation processes
undergone by wooden objects and consequently to develop suitable consolidation and conservation procedures. Lignin extracted from archaeological
waterlogged wood samples was characterized using direct exposure electron ionisation mass spectrometry (DE-MS). DE-MS achieves a mass
spectral fingerprint of the sample in a few minutes, avoiding any chemical pre-treatment and requiring only few micrograms of material.
Mass spectral data were put in relation to the chemical composition of lignin and evaluated by means of principal component analysis (PCA). The
preliminary results, presented in this study, demonstrate the feasibility and the potential of DE-MS as a reproducible and rapid screening method for
archaeological waterlogged wood samples.
© 2007 Elsevier B.V. All rights reserved.
Keywords: Archaeological waterlogged wood; Lignin; Direct exposure electron ionisation mass spectrometry (DE-MS); Principal component analysis (PCA)

1. Introduction
The recovery, consolidation and preservation of waterlogged
wooden artefacts, such as shipwrecks and archaeological objects
recovered from underwater environments, is a particularly arduous conservation problem. Under favourable conditions of low
temperature and low oxygen availability, wood artefacts can
survive underwater in a surprisingly good state. Nevertheless, it
has been shown that some species of anaerobic bacteria can
slowly degrade waterlogged wood even under near anoxic
conditions, mainly by eroding the polysaccharides as a source of
nutrients. This leads to the formation of pores and cavities filled
with water, and transforms the wood into a soft and fragile
structure which is likely to collapse when drying [1–4]. Due to the
almost complete loss of cellulosic components, the chemical
characterisation of lignin is of primary importance in the
diagnosis and conservation of waterlogged wood artefacts.
⁎ Corresponding author. Tel.: +39 050 2219303.
E-mail address: frances@dcci.unipi.it (F. Modugno).
0026-265X/$ - see front matter © 2007 Elsevier B.V. All rights reserved.
doi:10.1016/j.microc.2007.11.010

Lignin, a cross-linked poly-phenolic macromolecule (molecular
mass over 10,000), is insoluble in solvents and does not contain
either chains of repeating units or easily hydrolysable bonds [5].
These chemical–physical properties make the chemical analysis
of lignin extremely complex. Infrared spectroscopy [6,7], thermogravimetric methods [8], solid state 13C-NMR [9,10] and
pyrolysis-gas chromatography based procedures [10–14] have
been adopted to deal with this problem in degraded or aged wood.
The use of pyrolysis gas chromatographic techniques in combination with mass spectrometry has proven to be of particular
interest in the study of macromolecules such as lignin, cellulose
and hemicellulose. In particular, Py-GC/MS provides useful data
on the chemical structure of wood components, and has been used
for studying cellulose and lignin and also for investigating decay
processes in archaeological wood [10,12].
During pyrolysis, the macromolecules are depolymerised by
heat and the pyrolysis products can be identified by mass spectrometry, after gas chromatographic separation. Especially in the
case of lignin, pyrolysis produces a variety of phenolic products,
which consist of guaiacyl and syringyl structural units with an

1. . In DE-MS the sample undergoes a resistively controlled heating on a rhenium filament within the ion source of the mass- 187 spectrometer. in the case of macromolecules. Modugno et al. DE mass spectra of a) lignin extracted from archaeological wood IS. with specific applications in the analysis of wood-derived materials such as terpenic resins [16. / Microchemical Journal 88 (2008) 186–193 aliphatic side chain in position 4 of the aromatic ring [15]. The sample components are desorbed or.F. it can be performed rapidly (a few minutes for analysis and data acquisition). to a greater or lesser degree. and b) lignin extracted from spruce sound wood. and are ionised and analysed as a function of time. Fig. the structural units that make up the macromolecule. and its sensitivity has been shown to be suitable for identifying materials in the archaeometric field. The aim of this study was to use a direct in-source Py-mass spectrometric technique. identified as Pinaceae. It is assumed that pyrolysis products represent. to characterise archaeological waterlogged wood and lignin extracted from archaeological artefacts. pyrolysed over the heating range. DE-MS requires very small amounts of untreated sample (in the order of micrograms).17]. where the availability of sample is often limited and contamination must be avoided. Direct and rapid analytical methods that avoid sample pre-treatment are particularly welcome in the cultural heritage field. namely direct exposure electron ionisation mass spectrometry (DE-MS). in order to explore the potential of this technique to retrieve chemical information on archaeological wood materials and to evaluate decay processes.

The source temperature was at 230 °C. The lignin samples were derived from archaeological wood samples of different ages and origins: . Italy): B44.a wooden piece from the shipwreck Epave du Grand Conglouè recovered near Marseille (France) and dated 2nd century BC.188 F. prop-2-enalguaiacol (M+) Vinylsyringol (M+) Propenylsyringol (M+) Acetylsyringol (M+-CH3) Propenalsiryngol (M+) Sinapylalcohol (M+) Stilbene-type dimer (M+) Stilbene-type dimer (M+) Stilbene-type dimer (M+) β-resinol type dimer (M+) β-resinol type dimer (M+) β-resinol type dimer (M+) G G G G 164 151 152 154 168 167 178 180 194 181 208 210 272 303 332 358 388 418 G G S S S G S S S S S G–G dimer G–S dimer S–S dimer G–G dimer G–S dimer S–S dimer . Orlandi of the Department of Environmental and Earth Sciences of the University of Milano Bicocca (Milan. Israel) and dated 8– 9th century AD. provided by the Archaeological Superintendence of Tuscany: D (Ulmus sp). Recent lignin from spruce (soft wood) and birch (hard wood) extracted with the same procedure was used as references. 2. Modugno et al. Materials and methods 3.5 m deep) in Tantura Lagoon (Haifa. Vanillin (M+-H). 2. The wood was identified as oak (Quercus sp. Moreover. Propylsyringol (M+-CH2CH3). coupled with a Polaris Q ion trap external ionisation mass spectrometer (electron impact ionisation 70 eV). The application of pattern analysis based on principal components (PCA) enables the mass profiles obtained for the various samples to be compared quantitatively in an easily readable manner. Donato (Department of Chemistry of the University of Palermo. SR (hardwood. Paris. 2. The mass spectrometer was scanned over an m/z range of 50–1000. Italy). unknown species).a wooden piece from a shipwreck found under shallow waters (0. the examination of loading plots permits the differences to be correlated to specific chemical features. where it can be used to monitor the state of conservation of the wood in various regions of the timbers. probably from the Pinaceae family [10]. have highlighted that it is possible to relate mass spectral data to the chemical composition of lignin and of wood in sediments [18]. Samples All the samples examined showed the presence of a single peak in the total ion current (TIC) profile. This analytical approach is suitable for fast screening even a large number of samples. DE-MS Samples were analysed by depositing a few particles of powder directly on the direct exposure probe filament by means of a capillary.). [20]. Nipals algorithm) on the covariance matrix of centered data. Italy).D.0 (Addinsoft.2. . The region 50–500 m/z was selected because it contains all the pyrolysis fragments corresponding to lignin monomers and dimers. it nevertheless achieves a mass spectral fingerprint of the samples within a few minutes. It also highlights similarities and differences between samples. and is sensitive to differences in the chemical structure of lignin. The software used was XLSTAT 6. The instrumentation (Thermo Electron Corporation. based on a modification of the milled wood method developed by Holmbom et al. France).19]. provided by the Institute of Maritime Studies of the University of Haifa: sample IS. Each sample was analysed in duplicate. Unsupervised pattern recognition analysis of DE-MS mass spectral data corresponding to the mass range 50–500 m/z was performed by principal component analysis (PCA.3. when applied to wood artefacts of large dimensions. provided by Prof. . Previous analysis had identified the wood as a conifer. Sinapylalcohol (M+-C2H2OH) Propenyl-3-one-guaiacol (M+). Propylguaiacol (M+ -CH2CH3). Results and discussion 2. after row normalisation of the full 50–1000 spectra. It thus has great potential. / Microchemical Journal 88 (2008) 186–193 The few applications of in-source pyrolysis-mass spectrometry (direct temperature resolved mass spectrometry. Lignin extraction and isolation was carried out using a recently published procedure [9]. The archaeological artefacts from the site are dated to a period between the 4th century BC to the 2nd century AD. DTMS) to wood and lignin described in the literature [18. Data analysis Table 1 Attribution of m/z peaks observed in archaeological lignin DE mass spectra m/z Derivation Type of monomer G: guaiacyl S: siringyl 124 138 150 137 Guaiacol (M+) coniferylalcohol (M+-C3H4OH) Methylguaiacol (M+) Vinylguaiacol (M+) Ethyguaiacol (M+ -CH3). The peaks show a variable degree of broadness and in some cases the presence of The lignin was prepared and donated by Professor M. Although directly combining pyrolysis with mass spectrometry does not offer the detailed chemical information accomplished by Py-GC/ MS. Propylguaiacol (M-CH3) Vanillin (M+) Syringol (M+) Methylsyringol (M+) Ethylsyringol (M+-CH3). such as shipwrecks. USA) was made up of a Direct Probe Controller and a Direct Exposure Probe (rhenium filament. I.two samples from bollards excavated in the Site of Ancient Pisa Ships (Pisa. current programmed mode 0 mA to 1000 mA in 2 s then 1000 mA for 60 s). Coniferylalcohol (M+-C2H2OH) Propenylguaiacol (M+) Acetylguaiacol (M+-CH3).1. This first application of DE-MS to archaeological wood shows that it is a fast fingerprint tool that is able to discriminate between hardwood and softwood archaeological wood.

/ Microchemical Journal 88 (2008) 186–193 shoulders. m/z 151 (guaiacol + CH2CH2+). an ethyl radical and a C2H2OH. The mass spectrum is obtained by time-integration of the main peak in the total ion current (TIC) profile. corresponding to the molecular peak 189 of guaiacol (2-methoxy-phenol). which could derive from several compounds formed in the pyrolysis of guaiacyl lignin including ethylguaiacol. The mass spectra show high complexity. radical respectively. DE mass spectra of a) lignin extracted from archaeological wood SR . m/z 152. 1a and compared with the mass spectra of the reference spruce lignin (Fig. Fig. unknown specie. m/z 137 ([guaiacol + CH2]+). Modugno et al. and b) lignin extracted from sound birch wood. The mass spectra of lignin sample IS from the shipwreck from Israel (softwood) is shown in Fig. This is probably due to the polymeric nature of lignin and the degree of the polymerization of lignin samples collected from the various species of wood. based on the mass spectra of guaiacyl and siringyl compounds determined by Py-GC/MS [10] and available from the literature [18].3'-dihydroxy-4. . The peak at m/z 272 corresponds to the molecular peak of a guaiacyl-guaiacyl dimer compound with a stilbene structure (3.F. as expected in the direct mass spectrometric analysis of the complex mixture of products formed in the pyrolysis of lignin.4'-dimethoxy-stilbene). Both the spectra are characterised by the occurrence of peaks indicative of a guaiacyl lignin: m/z 124. corresponding to the molecular ion of vanillin. by the loss of a methyl radical. 1b). 2. Table 1 lists the fragments identified and the most probable attribution. propylguaiacol and coniferyl alcohol.

137.190 F. and at m/z 167. respectively. DE mass spectra of lignin extracted from archaeological wood B44. identified as Ulmus sp. These peaks could derive from the demethylation reaction of alkylsyringyl monomeric units. 210 deriving from syringyl monomers. the occurrence of peaks at m/z 196 and at m/z 153 is significant. . As can be seen in Figs. 2. This reaction leads to the formation of corresponding catechol derivatives. the peaks at m/z 167 (syringol + CH2+) and at m/z 181 (syringol + CH2CH2+) correspond to the main ion fragments deriving from sinapyl alcohol and alkylsyringol compounds. Due to the fact that demethylation of alkylsyringyl units is a recognized Fig. 4. 208. B44 and D show the presence of peaks characteristic of guaiacyl-syringyl lignin at m/z 124. Modugno et al. 151 deriving from guaiacyl monomers. the peaks at m/z 196 and at m/z 153 correspond to a loss of 14 uma from peaks at m/z 210 and m/z 167. 3. In the mass spectra of sample D (Fig. identified as Quercus sp. 3 and 4. In particular. 181. 4). In fact. the mass spectra of birch lignin (hardwood) and of samples SR. / Microchemical Journal 88 (2008) 186–193 Fig. while the peak at m/z 210 corresponds to the molecular ion of sinapyl alcohol. DE mass spectra of lignin extracted from archaeological wood D. whose fragmentation should form ion fragments at m/z 196 and at m/z 153.

Moreover. and that of peak at m/z 137. decay index for lignin [12]. Conclusions This preliminary study has highlighted that DE-MS is a fast fingerprint tool which is able to discriminate between hardwood . at an intermediate guaiacyl/ syringyl ratio. in addition to the peaks deriving from the guaiacyl and siringyl monomer structures. Neverthless. which is also considered as a lignin degradation marker [12]. where the intensities of each m/z fragment was expressed as percentage of the sum of the intensities of all the m/z fragments in the spectra (row normalisation). can be considered as a rough indication of the S/G ratio for the lignin sample. The analysis of a higher number of samples and the comparison of DE-MS data with PyGC/MS data will permit to better evaluate the role of the formation of catechols in the degradation processes affecting lignin in waterlogged environment [21]. The PC1+ is indicative of syringyl units and the PC1. can be attributed to GG. spruce: reference spruce lignin. corresponding to the molecular mass of catechol. demonstrating the efficiency of the extraction and purification procedure adopted to obtain lignin. Modugno et al. GS and SS dimers with β-β resinol type structures [18]. The DE mass spectra obtained from the archaeological lignin examined and those from the two reference lignin extracted from wood were compared by means of PCA. Amongst these. on the basis of the literature [19] and of the analysis of reference cellulose. deriving from guaiacyl monomers. The archaeological sample D1 was similar to the modern birch lignin. / Microchemical Journal 88 (2008) 186–193 Fig. the most abundant from syringyl monomers. accounting for 78. The ratio between the relative abundance of peak at m/z 167. shown in Fig. the two softwood samples.F. which differ by 30 Da (OCH3). PC3 showed a positive loading for m/z 110. Actually. the following m/z fragments would have been observed: 65. Moreover. 127. if polysaccharides had been present in not negligible amounts. In all the mass spectra obtained for hardwood and softwood lignin. 97. Due to the complexity of the mass spectra obtained in the direct Py-MS analysis of lignin. PCA score plot of PC1 and PC2 of DE mass spectral data. 7. Fig. principal component analysis (PCA) was 191 used as a pattern recognition technique to quantitatively compare the mass spectra obtained and to highlight differences and similarities between samples and correlations between variables.5% of the total variance. SR: D: hardwood lignin from a bollard pertinent to the Roman Harbour of san Rossore (Pisa. 5. 79. softwood samples do not contain a peak at m/z 167. the occurrence of peaks relative to lignin demethylation products can be considered as a way to establish the degree of degradation. birch lignin and DD1. 4. 73. The data matrix was column-centered and the covariance matrix was used for PCA. The loading plot of PC1 (Fig. IS: conifer lignin from a 8–9th century AD shipwreck recovered near Haifa (Israel). were also observed. while hardwood samples show variability in this ratio depending on the species of wood. The results show that the differences in the mass spectra are related not only to the difference between guaiacyl lignin (spruce and IS) and guaiacyl/siringyl lignin (other samples). and they were well differentiated from the hardwood samples. and SR and B44.of guaiacyl units. peaks attributable to ion fragments deriving from dimeric structures of the lignin macromolecule were observed.5% of total variance. 5 shows the scatter plot corresponding to the first two PCs. conclusions cannot be drawn from PC3 values because this PC3 has a low significance (only 9% of total variance) and in fact shows a non negligible variability between replicated samples. Peaks at m/z 153 and 196 are well evident in the mass spectra of sample D. birch: reference birch lignin. 388 and 418. which corresponds to the molecular ion of the G–G dimer dihydroxydimethoxy-stilbene. in fact the reconstructed mass spectra of PC2 and PC3 show that both the axes have significant loadings for m/z 196 and m/z 153. The data matrix was constituted by the mass spectra of the samples (in replicates) in the range m/z 50–500. In the lignin mass spectra. In particular. In samples from hardwood species. but also to the ratio between guaiacyl and syringyl components and to the degree of demethylation undergone by lignin monomers in the course of degradation. the most abundant was at m/z 272. because they contained only guaiacyl lignin. B44: oak lignin from a 2nd century BC shipwreck recovered near Marseille (France). which can be attributed to the fragmentation catechol derivatives formed by the demethylation of the alkylsyringyl units. As can be seen from the mass spectra. The following couples of similar samples can be highlighted: spruce lignin and IS. 6) on the basis of the loadings shows that this axis of variance concerns the ratio between syringyl and guaiacyl markers. spruce and IS. accounting for 78. the series of peaks at m/z 358. peaks at m/z 302 and m/z 332 attributable to the homologue SG and SS dimers. D: elm lignin from a bollard pertinent to the Site of Ancient Pisa Ships (Pisa. m/z fragments deriving from residual cellulose and hemicellulose are not evident. while the archaeological samples B44 and SR formed a separate cluster with high syringyl content. Italy). scored lowest on PC1. Italy). enhance the differences between archaeological (PC3+) and modern lignin (PC3−). It seems that PC2 and PC3 might be indicative of the degree of conservation. 85. The scores of the samples on the PC3/PC1 map.

and this method would seem to be suitable for studying degradation processes in lignin contained in archaeological wood. Further comparative investigations are in progress. Loading plots of the first three PCs. .192 F. 6. and softwood archaeological wood and which is sensitive to differences in the chemical structure of lignin. / Microchemical Journal 88 (2008) 186–193 Fig. Modugno et al. They involve a larger number of archaeological and reference samples. The mass spectra obtained can be quantitatively evaluated and compared by means of principal component analysis.

G.). in preparation. J. Ribechini. 3. in: P. Tomellini. T.J.F. pp. 193 [4] L. M. 11 (1987) 437–450. Ribechini. Swedish National Maritime Museums. [18] E. [20] B. Hoffmann. [14] V. J. 1990. Evershed.W.K. R. D: elm lignin from a bollard pertinent to the Roman Harbour of San Rossore (Pisa. Vasileb. P. Lignin attack during eucalypt wood decat by selected basidiomycetes: a Py-GC/MS study.C. in: R. [19] C. [11] C. Prof. I. Orlandi. Modugno. [13] J. Rapid Communications in Mass Spectrometry 20 (2006) 1787–1800.M. NMR and GPC techniques. Zoia. Direct exposure electron ionization mass spectrometry and gas chromatography/mass spectrometry techniques to study organic coatings on archaeological amphorae. Nilsson. Boon.W. [8] I. [17] F. C. fresh and waterlogged woods. Lucac. Archaeological Wood.P. Forest Product Chemistry. Helsinki. Saiz-Jimenez. 2001. Modugno. Martinez. Structural characterization of lignin polymers by temperature-resolved in-source pyrolysis-mass spectrometry and Curie-point pyrolysis-gas chromatography/mass spectrometry. Barbour (Eds.). Barbour (Eds. . R. [15] C. Colombini. M. Rawell.P. Stenius (Ed. Rawell. M.P. De Leeuw. Lignin pyrolysis products: their structures and their significance as biomarkers. American Chemical Society. B44: oak lignin from a 2nd century BC shipwreck recovered near Marseille (France). C. E. PCA score plot of PC1 and PC3 of DE mass spectral data. Properties and Preservation. GC/MS. Donato (University of Palermo) for having provided the archaeological samples. E-L. Journal of Analytical and Applied Pyrolysis 64 (2002) 421–431. Hessels. Lignin degradation by white rot fungi on spruce wood shavings during short-time solid-state fermentations monitored by near infrared spectroscopy. T. R.M. Washington. Chemical characterisation of recent and buried wood by analytical pyrolysis. Vinciguerra. i Tra ̂n. M. Journal of Chemical Education 76 (1999) 1076–1079. American Chemical Society.P.A. 1992. Foray. F. Boon.F. G. A. 7. Enzyme and Microbial Technology 39 (2006) 1476–1483. SR: hardwood lignin from a bollard pertinent to the Roman Harbour of San Rossore (Pisa. Biological degradation of wood. Methods in Lignin Chemistry.M. Saiz-Jimenez. Economic support was from the Italian MIUR findings (PRIN Cofin05). J. J. N. Zoia. E. [3] R. 2000. Analytical Chemistry 74 (2002) 4386–4390. Crina. W. Modugno et al. spruce: reference spruce lignin. Gradinger. J. Dence (Eds. Van der Hage. Hedges. A. Hafors. Caple. Colombini. 225. High-resolution solid-state CPMAS NMR study of archaeological woods. [12] P. Microchemical Journal 85 (2006) 164–173. [9] M. Archaeological wood characterisation by PY/GC/MS. Tolppa. Advances in Chemical Series. 107–172.J. [2] B. Campanella. [7] B. M. Blanchette.P. M. 35–65.J. 141–174. Acknowledgments The authors wish to thank Dr. Archaeological Wood. Polymer Degradation and Stability 80 (2003) 83–91.J. Journal of Thermal Analysis 37 (1991) 1923–1932. Fackler. Journal of Analytical and Applied Pyrolysis 25 (1993) 149–183. birch: reference birch lignin. M. Crestini. L.L. Mulder.W. Gutierrez. Holmbom. J.L. B. Orlandi. Bistoni. Chemistry. Y. 40 (2005) 675–687.A. Italy). I. Israel). Jones. Structure and degradation processes for waterlogged archaeological wood. De Leeuw. [21] M. Saiz-Jimenez. Ogilvie. C. [6] K. Barker. vol. Owen. Martinez. Journal of Mass Spectrometry. in: R. Del Rio. C. IS: conifer lignin from a 8–9th century AD shipwreck recovered near Haifa (Israel). Evidence for demethylation of syringyl moieties in archaeological wood using pyrolysis-gas chromatography/mass spectrometry. de Leeuw. Orlandi (University of Milano Bicocca) for having extracted lignin from archaeological woods. “Study of degradation processes of lignin in archaeological waterlogged wood by pyrolytic techniques and NMR spectroscopy”. Springer-Verlag. M. Kahanov (University of Haifa.F. Brebub. Hinterstoisser. Modugno. M. Tomassetti. Analytical methods. Fapet Oy. Vasa Studies 18.T.F. Sgherri. 225.).R. Speranza. A.J. Modugno. Sweden. Sandua. Chemistry. [16] M. Finland. Properties and Preservation. Colombini. accounting for 72. Napoli. Italy). pp. R.I. Organic Geochemistry 10 (1985) 869–876. pp. L. C. Abad. Lin. the aim being to establish the value of this technique in determining the extent of degradation of lignin. Pyrolysis-gas chromatography-mass spectrometry of isolated. Rapid Communications in Mass Spectrometry 14 (2000) 71–79. Conservation of the Swedish warship Vasa from 1628. Lucejko. F. Advances in Chemical Series. Stenius. Thermogravimetric study on the ageing of lime wood supports of old paintings. A. C. 1990. [10] M. Bardet. Stockholm. Berlin. Thermoanalysis of ancient. Q. E. I. Organic Geochemistry 6 (1984) 417–422. J.K. van Bergen. M. Wood decay characterization of a naturally infected London plane-tree in urban environment using Py-/GC/MS. M. / Microchemical Journal 88 (2008) 186–193 Fig. References [1] P. Identifying softwoods and hardwoods by infrared spectroscopy. Washington.A. Journal of Analytical and Applied Pyrolysis 78 (2007) 228–231.M. Sandud. Journal of Analytical and Applied Pyrolysis.2% of total variance. F. A.E.C. Colombini. Poole. Petrucci. A. M. and Prof. Chemical study of triterpenoid resinous materials in archaeological findings by mean of direct exposure electron ionization mass spectrometry (DE-MS) and gas chromatography mass spectrometry (GC/MS). [5] Y. synthetic and degraded lignins. Tolppa. R. Sardelli. Daniel.). Comparison of pyrolysis data with 13C NMR and wet chemical data. Papermaking Science and Technology.