# Analytical Pyrolysis and Mass Spectrometry to Characterise Lignin in Archaeological Wood
## 3 Results and Discussion
### Direct Exposure Mass Spectrometry (DE-MS)
In particular, the fragment with \(m/z\) 151 is due to the 4-(1-oxomethyl)-guaiacol ion, whereas \(m/z\) 181 corresponds to the ion with an additional methoxyl functionality, the 4-(1-oxomethyl)-syringol ion In fact, the former can be generated by the fragmentation of vanillin, acetovanillone, propenylguaiacol, guaiacyl vinyl ketone, vanillin acid, and vanillic acid methyl ester, whereas the latter is formed during the fragmentation of syringaldehyde, acetosyringone, propiosyringone, syringyl vinyl ketone, syringe acid, and syringe acid methyl ester. Figure 1: DE-MS mass spectra of a reference sound oak sample (**a**), samples of archaeological oak wood from Oak 6 from Biskupin site (**b**) and La Marmotta (2212-B) (**c**). By comparing the fragments deriving from the oxidised lignin products with those at \(m/z\) 137 and 167, it is possible to have an estimation of the oxidation level of this polymer. In fact, the increase in the relative abundance of the peaks at \(m/z\) 151 and 181 with respect to the peaks at \(m/z\) 137 and 167 was evident in the archaeological samples compared to the reference, and in particular for La Marmotta sample, indicating an increase in lignin oxidation [33]. The level of lignin depolymerisation is another index of its degradation. The peaks related to the lignin polymerisation state are those at \(m/z\) 272, 302, and 332, which correspond to dimers (Table 2). In fact, these fragments were negligible in the mass spectra obtained from La Marmotta sample (Figure 1c), indicating that lignin degradation reactions involved partial depolymerisation, resulting in the cleavage of these dimeric bonds. On the other hand, in the Biskupin samples, dimer fragments were evident in the mass spectra, highlighting a better preservation of lignin. In fact, the presence and relative intensity of the lignin dimer fragments in the pyrolysis mass spectral profiles can be used as a probe to estimate lignin depolymerisation, but they can also give information on the state of lignin-carbohydrate bonds. The fact that dimer fragments were more evident in the Biskupin sample than in the sound wood sample can be interpreted as an indication of degraded polysaccharides, which make lignin more prone to undergo extensive pyrolytic degradation. Despite the qualitative information that can be directly obtained from observing the MS spectra, DE-MS analysis is generally used to obtain a high number of spectra, which still contain much information hidden underneath complex data. PCA is therefore used as an effective pattern recognition tool in order to compare the results and highlight differences and similarities among the samples and correlation between variables. Figure 2 shows the score plot obtained for the first two principal components (PCs), which accounted for ca. 74% of the total variance. It is evident that PC1 is correlated to the intensities of the peaks generated from the syringl units (positive values of the PC1) and correlated negatively to the guaiacyl ones (\(m/z\) 124, 137). Since the pine wood samples do not contain syringl-lignin, they are placed in the negative range of PC1. Therefore, PC1 discriminating between softwoods and hardwoods gives a rapid indication of the type of wood and of the ratio between the amounts of syringl and guaiacyl fragments. Due to the difference in lignin composition, softwood samples are located at low values of PC1, whereas hardwood samples are located at higher values of PC1. PC2 in the range of positive values is correlated to the fragments produced during the fragmentation of the polysaccharide fraction (\(m/z\) 55, 69, 73, 85, 97, 114, 126) [54]. This main component differentiates samples on the basis of holocellulose content and can provide an indication of wood degradation. All the waterlogged archaeological samples are positioned at the bottom of the PC2/PC1 score plot. This indicates their lower polysaccharide content when compared to reference samples of the same species.