Characterization of waterlogged wood by NMR and GPC techniques

Anaerobic erosion bacteria can slowly degrade waterlogged wood, causing a loss of cellulose and hemicellulose. During this process, lignin can also be altered. For this reason, the chemical characterization of waterlogged archaeological wood is crucial for both the elucidation of the degradation processes and also the development of consolidation and conservation procedures.The complex structure of wood makes it practically impossible to dissolve wood in its native form in conventional molecular solvents. Ionic liquids can provide a homogeneous reaction medium for wood-based lignocellulosic materials. Highly substituted lignocellulosic esters and phosphite esters can be obtained under mild conditions by reacting pulverized wood dissolved in ionic liquid with either acyl chlorides or dioxaphospholanes in the presence of pyridine. As a result, the functionalized wood develops an enhanced solubility in molecular solvents, allowing for a complete characterization by means of spectroscopic and chromatographic techniques.In this study, archaeological woods and reference sound woods of the same taxa (Quercus and Arbutus unedo), along with the corresponding extracted lignin, were fully characterized by means of phosphorus NMR spectroscopy, two dimensional NMR spectroscopy and GPC analysis. The samples were collected from the Site of the Ancient Ships of San Rossore (Pisa, Italy), where many shipwrecks dating from 2nd century BC to 5th century AD have been discovered.The results highlighted a deeper and faster depolymerization of the polysaccharide matrix against a limited degradation of the lignin fraction. The use of innovative solvent system as the ionic liquid [amim]Cl enables to highlight chemical and morphologic changes in wood composition avoiding further degradation.     

Analysis of lignin from archaeological waterlogged wood by direct exposure mass spectrometry (DE-MS) and PCA evaluation of mass spectral data

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.     

Archaeological wood characterisation by PY/GC/MS, GC/MS, NMR and GPC techniques

The chemical characterisation of waterlogged archaeological wood is of paramount importance in terms of understanding not only the degradation processes of wood in artistic and archaeological objects but also the development of consolidation and conservation procedures. The two different types of archaeological waterlogged wood were characterized using several analytical techniques. The analytical protocol consists in using PY/GC/MS with in situ derivatisation with hexamethyldisilazane, GC/MS of the extractives, ¹H, ¹³C and ³¹P NMR analysis, and GPC. The main results showed that one of the ancient samples was a gymnosperm wood from the Pinaceae family, whereas the other type of wood was an angiosperm. Lignin from the angiosperm was oxidised in a benzylic position of β-O-4 to give oxo functional groups, in primary alcohol moiety to give carboxylic groups. In addition, the formation of a new intermonomeric bond 5-O-4 was observed.     

Fourier-transform Raman spectroscopic study of a Neolithic waterlogged wood assemblage

The use of Fourier-transform Raman spectroscopy for characterising lignocellulosics has increased significantly over the last twenty years. Here, an FT-Raman spectroscopic study of changes in the chemistry of waterlogged archaeological wood of Pinus sp. and Quercus sp. from a prehistoric assemblage recovered from northern Greece is presented. FT-Raman spectral features of biodeteriorated wood were associated with the depletion of lignin and/or carbohydrate polymers at various stages of deterioration. Spectra from the archaeological wood are presented alongside spectra of sound wood of the same taxa. A comparison of the relative changes in intensities of spectral bands associated with lignin and carbohydrates resulting from decay clearly indicated extensive deterioration of both the softwood and hardwood samples and the carbohydrates appear to be more deteriorated than the lignin. The biodeterioration of the archaeological timbers followed a pattern of initial preferential loss of carbohydrates causing significant loss of cellulose and hemicellulose, followed by the degradation of lignin.     

Characterisation of archaeological waterlogged wood by pyrolytic and mass spectrometric techniques

Two techniques based on analytical pyrolysis and mass spectrometry, direct exposure-MS (DE-MS) and pyrolysis-gas chromatography/mass spectrometry (Py-GC/MS), were used to characterise waterlogged archaeological wood and to study degradation patterns of wood in aqueous environments. The two techniques were applied to samples from the excavation of the Site of the Ancient Ships of Pisa San Rossore in Pisa (Italy), and data were compared to those relative to native sound wood of the same species (pine, elm, beech). Both the methods result valuable in the analysis of ancient wood artefacts, avoiding the long wet-chemical procedures that are commonly used in wood analysis, and allowing us to use a minimal sample size. DE-MS achieves a global mass spectral fingerprint of lignin and polysaccharides pyrolysis compounds in few minutes, and the results have been interpreted with the support of principal component analysis (PCA) of mass spectra. Py-GC/MS permits detailed molecular analysis of pyrolysis compounds and highlights some chemical modifications of lignin in archaeological samples, as demethylation of both guaiacyl and syringyl lignin units. Both the techniques demonstrate consistent loss of polysaccharides in archaeological wood.     

Study on the impregnation of archaeological waterlogged wood with consolidation treatments using synchrotron radiation microtomography

In favourable conditions of low temperature and low oxygen concentration, archaeological waterlogged wooden artefacts, such as shipwrecks, can survive with a good state of preservation. Nevertheless, anaerobic bacteria can considerably degrade waterlogged wooden objects with a significant loss in polysaccharidic components. Due to these decay processes, wood porosity and water content increase under ageing. In such conditions, the conservation treatments of archaeological wooden artefacts often involve the replacement of water with substances which fill the cavities and help to prevent collapse and stress during drying. The treatments are very often expensive and technically difficult, and their effectiveness very much depends on the chemical and physical characteristics of the substances used for impregnation. Also important are the degree of cavity-filling, penetration depth and distribution in the structure of the wood. In this study, the distribution in wood cavities of some mixtures based on polyethylene glycols and colophony, used for the conservation of waterlogged archaeological wood, was investigated using synchrotron radiation X-ray computed microtomography (SR-μCT). This non-destructive imaging technique was useful for the study of the degraded waterlogged wood and enabled us to visualise the morphology of the wood and the distribution of the materials used in the wood treatments. The study has shown how deposition is strictly related to the dimension of the wooden cavities. The work is currently proceeding with the comparison of synchrotron observations with the data of the solutions viscosity and with those of the properties imparted to the wood by the treatments.     

Snapshots of lignin oxidation and depolymerization in archaeological wood: an EGA‐MS study

Evolved gas analysis‐mass spectrometry (EGA‐MS) was used for the first time to study archaeological wood, in order to investigate its chemical degradation. The archaeological wood was from an oak pile from a stilt house found in the Neolithic ‘La Marmotta’ village (Lake Bracciano, Rome, Italy). The sampling was performed from the external to the internal part of the pile, following the annual growth rings in groups of five. In addition, sound oak wood and isolated wood components (holocellulose and cellulose) were also analyzed, and the results were used to highlight differences because of degradation. Our study demonstrated that EGA‐MS provides information on the thermo‐chemistry of archaeological wood along with in‐depth compositional data thanks to the use of MS. Our investigations not only highlighted wood degradation in terms of differences between carbohydrates and lignin content, but also showed that lignin oxidation and depolymerization took place in the archaeological wood. Mass spectral data revealed differences among the archaeological samples from the internal to the external part of the pile. An increase in the formation of wood pyrolysis products bearing a carbonyl group at the benzylic position and a decrease in the amount of lignin dimers were observed. These were related to oxidation and depolymerization reactions, respectively. Copyright © 2015 John Wiley & Sons, Ltd.     

Near-infrared spectroscopic observation of the ageing process in archaeological wood using a deuterium exchange method

The ageing degradation of the fine wood structure of dry-exposed archaeological wood was investigated by Fourier transform near-infrared spectroscopy with the aid of a deuterium exchange method. The archaeological wood sample was taken from an old wooden temple in Japan (late 7th century), which has been designated as a UNESCO world heritage site. Comparing the analytical results with those of a modern wood sample of the same species, the ageing process of archaeological wood was clarified as a change in the state of order on a macromolecular structural level. It can be concluded from NIR spectra that the amorphous region, and partially semi-crystalline region, in cellulose, hemicellulose, and lignin decreased by the ageing degradation, whereas the crystalline region in cellulose was not affected by the ageing. The accessibility of the diffusant to effect H/D-exchange was monitored by an OH-related absorption band obtained from FT-NIR transmission spectroscopy and characteristically varied with the ageing process of the wood samples, the absorption bands characteristic of a specific state of order and the diffusion agent. Finally, we proposed a morphological model to describe the variation of the fine structure of the microfibrils in the cell wall with ageing degradation. The state of microfibrils changed loosely by ageing, so that elementary fibrils were arranged loosely under 5 A, whereas several elementary fibrils in the modern wood were arranged in very close proximity under 3 A to each other.     

Reactivity of Waterlogged Archeological Elm Wood with Organosilicon Compounds Applied as Wood Consolidants: 2D 1H–13C Solution-State NMR Studies

Some organosilicon compounds, including alkoxysilanes and siloxanes, proved effective in stabilizing the dimensions of waterlogged archaeological wood during drying, which is essential in the conservation process of ancient artifacts. However, it was difficult to determine a strong correlation between the wood stabilizing effect and the properties of organosilicon compounds, such as molecular weight and size, weight percent gain, and the presence of other potentially reactive groups. Therefore, to better understand the mechanism behind the stabilization effectiveness, the reactivity of organosilicons with wood polymers was studied using a 2D ¹H–¹³C solution-state NMR technique. The results showed an extensive modification of lignin through its demethoxylation and decarbonylation and also the absence of the native cellulose anomeric peak in siloxane-treated wood. The most substantial reactivity between wood polymers and organosilicon was observed with the (3-mercaptopropyl)trimethoxysilane treatment, showing complete removal of lignin side chains, the lowest syringyl/guaiacyl ratio, depolymerization of cellulose and xylan, and reactivity with the C6 primary hydroxyls in cellulose. This may explain the outstanding stabilizing effectiveness of this silane and supports the conclusion that extensive chemical interactions are essential in this process. It also indicates the vital role of a mercapto group in wood stabilization by organosilicons. This 2D NMR technique sheds new light on the chemical mechanisms involved in organosilicon consolidation of wood and reveals what chemical characteristics are essential in developing future conservation treatments.     

Analytical pyrolysis vs. classical wet chemical analysis to assess the decay of archaeological waterlogged wood

The macromolecular complexity of wood limits the possibility of obtaining complete chemical information on its alteration in archaeological objects. This paper compares the results obtained in the characterisation of the components of archaeological wood by a classical wet chemical method and by an instrumental method based on pyrolysis in presence of hexamethyldisilazane coupled with gas chromatography/mass spectrometry, Py(HMDS)–GC/MS. We compare the results obtained with the two methods quantitatively. This enables us to evaluate the efficiency of Py(HMDS)–GC/MS in assessing the chemical composition and the state of conservation of degraded wood. The material analysed consisted of reference sound wood and waterlogged wood from the ?ó?te historical site, located on a small island on Lake Zarańskie in Poland. The samples are from the remains of settlements dating to a period between the 9th and the 12th centuries AD.     

High-resolution multiparametric MRI of contemporary and waterlogged archaeological wood

High-resolution NMR images on three different present-day wood samples and an archaeological wood specimen were presented and discussed. Although the spatial resolution is still low to perform dendrology for the exact identification of the wooden species, the T₂-spin-density weighted images exhibit contrasts that are in excellent agreement with optical histological images. On the other hand, T₂* and T₁-weighted images provide physiological information that is not obtainable by the usual light microscopic investigations. Moreover, the diffusion-weighted images show the anisotropic behaviour of the water diffusion coefficient quantified along and perpendicularly to vascular bundles (vessels and tracheids), which can be related to the morphology and size of wooden microstructure. This work suggests that high-resolution multiparametric MRI may be a useful tool to increase the information obtainable from the waterlogged archaeological wood remains in a completely non-invasive and non-destructive approach. Therefore, it would be desirable to further develop the hardware and functional characteristics of MRI scanners to improve their potential application in the field of wooden cultural heritage.     

Study of artificially degraded woods simulating natural ageing of archaeological findings

Simulation of waterlogged archaeological woods was carried out by immersion of fir and chestnut wood samples into sea water at different temperatures (room temperature and 40°C). The effects of metals in contact with woods were simulated by inserting in some specimens of the two types of wood copper or iron nails, the most important metals from the archaeological point of view. The effects of this ageing simulation on woods were studied by different characterization methods. At first we have performed gravimetric analyses, controlling the mass increase of immersed wood in function of the time of immersion and the temperature of the bath. Then, thermogravimetry, differential thermal analysis, differential scanning calorimetry in oxygen flux were used. The alteration of wood was observed by means of the peak temperatures of DTA, DTG and DSC variation and by the mass losses observed during heating, evaluated on the basis of the measured thermal data. The samples were woods powder obtained by milling. Complementary characterization of the woods was performed by evaluating the crystallinity of cellulose by means of X-ray powder diffraction. The change in colour of woods during ageing was checked by means of spectrophotometric measurements in the visible region. X-ray fluorescence was used to investigate the penetration of metals into wood samples. An artificial ageing treatment with NaOH and O₃ was also performed. Finally, a comparison between the effects of artificial alteration realised in our specimens and natural degradation observed in archaeological woods, was performed.     

Determining the selective impregnation of waterlogged archaeological woods with poly(ethylene) glycols mixtures by differential scanning calorimetry

The differential scanning calorimetry (DSC) technique was demonstrated to be a reliable and fast tool for the investigation of the selective impregnation of archaeological woods with poly(ethylene) glycols (PEGs) mixtures. To this aim, waterlogged archaeological woods were impregnated by using aqueous mixtures of PEG 4000 and PEG 400 as well as mixtures of these polymers in the melt state. The efficiency of the treatments was also estimated by determining the total consolidant content entrapped into the cavities of degraded wood by means of DSC and thermogravimetry.     

Evidence for demethylation of syringyl moieties in archaeological wood using pyrolysis-gas chromatography/mass spectrometry

Archaeological oak (Quercus sp.) wood samples, ranging from 16(th) C. AD to 6000 BP, were studied using flash pyrolysis-gas chromatography/mass spectrometry to obtain insight into angiosperm lignin degradation. The pyrolysates revealed evidence of a number of 3-methoxy-1,2-benzenediol derivatives, methoxycatechols, directly related to 2,6-dimethoxyphenol, syringyl, moieties which are characteristic building blocks of angiosperm lignin. Mass spectra and mass chromatograms of these compounds are reported. The finding of these characteristic pyrolysis products in well-preserved archaeological wood provides unequivocal evidence that demethylation of syringyl units occurs very early in wood degradation. It is highly likely that the absence of abundant 3-methoxy-1, 2-benzenediols in degrading plant materials containing angiosperm lignin relates to the lability of these newly formed moieties.     

Oxygen consumption by conserved archaeological wood

Rates of oxygen consumption have been measured over extended time periods for 29 whole samples of conserved, archaeological wood and four samples of fresh, unconserved wood, at 50 % relative humidity and room temperature. Samples from the Swedish Warship Vasa and the Danish Skuldelev Viking ships are included. Most rates were close to 1 μg O₂ (g wood)^?1 day^?1 and the process persisted for several years at least. Consumption of oxygen is related to change in chemical composition, which is, in turn, related to degradation. It is thus demonstrated that despite conservation, waterlogged archaeological wood continues to degrade in a museum climate.

A comparative thermogravimetric study of waterlogged archaeological and sound woods

Waterlogged archaeological woods Pinus pinaster and Fagus sylvatica L. were analyzed by using TG technique. Degradation processes ascribable to the holocellulose decay were evidenced at nearly the same temperature for sound and archaeological samples. The residual matters at 600 and 900 °C of the sound woods are much lower than those of archaeological waterlogged woods in agreement with the presence of inorganic materials encapsulated during the burial into the marine environment. It was proposed a new protocol to rapidly calculate the maximum water content parameter, which is related to the wood degradation state. TG experiments at variable heating rates were performed to obtain kinetic parameters for the degradation process. The Flynn–Wall–Ozawa and Friedman approaches allowed us to calculate the activation energy, which is significantly different for the sound and the archaeological woods.     

Thermoanalysis of ancient,fresh and waterlogged woods

TG, DTG and DSC curves are presented and analytical results, obtained by thermogravimetry and DSC calorimetry, of ancient, fresh and waterlogged wood samples, are discussed. The criteria by which the percent content of lignin, cellulose, water and ashes (residue after heating at 700) were obtained by thermogravimetry, are outlined. Finally DSC curves are reported and the degradation which happen in some wood samples evidenced, especially in the case of waterlogged wood.

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Zusammenfassung Die Differenzierung von historischen, neuzeitlichen und gewÄsserten Holzproben mit den Methoden der Thermogravimetrie und DSC wurde untersucht und diskutiert. Die Kriterien, nach denen der Gehalt an Lignin, Cellulose, Wasser und Asche (Rückstand nach Erhitzung auf 700C) aus TG-Kurven abzuleiten sind, wurden erlÄutert und die Befunde mit den Resultaten einer nasschemischen Standardmethode verglichen. DSC-Kurven demonstrieren den chemischen Abbau der unterschiedlich gealterten respektive gewÄsserten Holzproben.

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The authors thank Dr. C. Meucci and Dr. S. D'Urbano, of the Central Institute of Restoration (ICR) Roma, which supplied the waterlogged wood samples. This work was supported by the National Research Council (CNR) of Italy.     

The formation of beta-beta structures in lignin biosynthesis--are there two different pathways?

Based on results from 2D NMR studies, both pinoresinol and secoisolariciresinol structures were found to be present in native lignin from spruce wood as well as in spruce kraft lignin and residual kraft pulp lignin. These two structures constitute the major types of beta-beta inter-unit linkages present in spruce lignin, but their formation in the lignin polymer may follow different pathways leading to their different bonding patterns with the rest of the lignin polymer. The mechanisms involved are discussed.     

Lignin derived oils from the fast pyrolysis of poplar wood

A pyrolysis oil obtained from the fast pyrolysis of poplar wood was subjected to mild hydrolysis and an aqueous and a non-aqueous fraction recovered. The non-aqueous fraction (pyrolytic lignin), a brown powder, was 23% of the oil, or 16% of the wood, corresponding to about 80% volatilization of the lignin content. Nuclear magnetic resonance spectra of this pyrolytic lignin were obtained, and its structure appears to be very similar to that of steam exploded poplar lignin. Methoxy content is relatively high and syringyl units appear to predominate over guaiacyl units. The pyrolytic lignin appears to be somewhat more degraded than steam exploded lignin and is probably lower in molecular weight.