Fast qualitative method based on electron paramagnetic resonance (EPR) to date Gmelina arborea and Araucaria angustifolia wood

Método cualitativo rápido basado en resonancia paramagnética electrónica para datar madera de Gmelina arborea y Araucaria angustifolia

G. Inés Bolzón-de Muñiz¹, M. Guadalupe Lomelí-Ramírez², Antonio S. Mangrich³, G. Guadalupe Carbajal- Arizaga^4*

¹ Departamento de Engenheria e Tecnología Florestal. Universidade Federal do Paraná. Avenida Lothario Meissner, 632, CEP. 80210-170, Curitiba, Paraná, Brasil.

³ Departamento de Química, Universidade Federal do Paraná, UFPR, Centro Politécnico - Jardim das Américas, 81531-990, Curitiba, PR, Brasil. & Instituto Nacional de Ciência e Tecnologia, Energia & Ambiente, UFBA, 40170-290, Salvador, BA, Brasil.

⁴ Departamento de Química. Universidad de Guadalajara. Marcelino Garcia Barragan 1421, C.P. 44430, Guadalajara, Jalisco, México. ^* Author for correspondence (gregoriocarbajal@yahoo.com.mx)

Received: January, 2013.
Approved: July, 2014.

Abstract

In Brazil, Gmelina arborea is a common tree to produce char and the content of carbon is an important factor to correlate with the energy content or carbon sequestration capability, whereas Araucaria angustifolia occupies 6 % of its original territory and Brazilian laws protect old native specimens, since the commercial use of wood is restricted for young cultivated specimens. Electron paramagnetic resonance (EPR) spectroscopy is used for detection and quantification of organic free radicals (OFR) and, in this study, it was utilized for exploring OFR content along the rings of one log from G. arborea and A. angustifolia trees, both randomly chosen. The amount of OFR correlated with the carbon content in G. arborea and the age of rings in G. arborea and A. angustifolia. The content of OFR in rings of Gmelina arborea was compared with previous analyses of carbon sequestration in the same tree. Without statistical treatment, an inverse relationship between the carbon and OFR content was found. The concentration of OFR in inner rings (older ages) of A. angustifolia was higher than in outer rings (younger ages). This trend was also observed in G. arborea, suggesting that EPR provides qualitative information about the age. Additionally, a Pleistocene sample of A. angustifolia was analyzed and the content of OFR was one order of magnitude higher than that in the young sample. In conclusion, EPR spectroscopy is proposed as a fast qualitative method to identify the age of wood, especially for confiscated wood loads in protected areas of A. angustifolia and to determine the carbon content in wood.

Keywords:  Araucaria angustifolia, electron paramagnetic resonance, organic free radicals, Gmelina arborea.

Resumen

En Brasil, Gmelina arborea es un árbol para producir carbón y el contenido de carbono es un factor importante correlacionado con el contenido de energía o la capacidad de secuestro de carbono, mientras que la Araucaria angustifolia ocupa el 6 % de su territorio original y las leyes brasileñas protegen los viejos especímenes nativos, ya que el uso comercial de la madera está restringido para ejemplares jóvenes cultivados. La espectroscopia de resonancia paramagnética electrónica (EPR) se usa para la detección y cuantificación de los radicales orgánicos libres (OFR, siglas en inglés) y en este estudio se usó para explorar el contenido de OFR en los anillos de un tronco de árbol de G. arborea y otro de A. angustifolia elegidos al azar. La cantidad de OFR se correlacionó con el contenido de carbono en G. arborea y con la edad de los anillos de G. arborea y A. angustifolia. El contenido de OFR en los anillos de G. arborea se comparó con análisis previos de secuestro de carbono en el mismo árbol. Sin tratamiento estadístico, se encontró una relación inversa entre el contenido de carbono y OFR. La concentración de OFR en los anillos internos (de más edad) de A. angustifolia fue mayor que en los anillos externos (de menor edad). Esta tendencia también se observó en G. arborea, lo que sugiere que EPR proporciona información cualitativa acerca de la edad. Además, se analizó una muestra del Pleistoceno de A. angustifolia y el contenido de OFR fue de una magnitud mayor que en la muestra más joven. En conclusión, la espectroscopia EPR se propone como un método cualitativo rápido para identificar la edad de la madera, especialmente para cargas de madera confiscadas en áreas protegidas de A. angustifolia y para determinar el contenido de carbono en la madera.

Palabras clave: Araucaria angustifolia, resonancia paramagnética electrónica, radicales libres orgánicos, Gmelina arborea.

INTRODUCTION

Electron paramagnetic resonance (EPR) spectroscopy measures the absorption of electromagnetic waves by paramagnetic species, such as organic free radicals (OFR), allowing their quantification in solid or liquid samples (Budziak et al., 2004; Mangrich et al., 2009; Guedes et al., 2003). EPR spectroscopy is used along with other techniques as a method to date archaeological samples of mega-fauna (Kinoshita et al., 2008; Joannes-Boyau and Grün, 2011) and charred wood (Triantafyllou et al., 2010). The formation of OFR in wood occurs by weathering, mainly when it is exposed to visible or ultraviolet light (Kalnins et al., 1966) or as a response to chemical agents in the environment like fungi or enzymes, including mechanical and physical stress (Hon and Feist, 1981).

The classical method to quantify OFR in plants is based on a colorimetric assay with 1,1-diphenyl-2-picryl-hydroazyl (DPPH) and an ethanol/water extract of the plant, where the OFR reacts and develop a color measured at 515-520 nm (Sithisarn et al., 2005; McCune and Johns, 2007; Rocha-Guzman et al., 2007). Another method is through deoxyribose degradation by OFR, which is also followed by colorimetry (Rocha-Guzman et al., 2007; Rocha-Guzman et al., 2009; Ribeiro et al., 2014).

The usefulness and practicality of the EPR spectroscopy as an alternative route for characterization in forest sciences should be evaluated, since this technique allows fast and chemical reagents-free quantifications of OFR. In this regard, Illman and Bajts (1997) proposed that the single signal detected by EPR with the specific value of the g factor equal to 2.0 could be the fingerprint for wood. Although this task is not feasible since the OFR signal changes slightly from sample to sample, another information can be monitored, such as the amount of humic substances along the composting process of pine sawdust (Budziak et al., 2004).

In this study, samples were collected from Araucaria angustifolia, which currently occupies 6 % of its original territory in Brazil (Oliveira et al., 2007), plus samples from Gmelina arborea Roxb., harvested in the coastal side of Parana State (South Brazil) and used in a previous report (Marcene et al., 2006) to quantify the carbon content. Therefore, the objective of this study was to acquire the EPR spectra of wood samples and evaluate the content of OFR along different rings of both trees, that is to say, along different ages, since each ring correspond to one year of life (Starr and Taggart, 2008). Two additional samples of A. angustifolia were analyzed, and these were used to find a relationship between the OFR concentration and age of trees.

MATERIALS AND METHODS

The G. arborea Roxb. and A. angustifolia (recently harvested and one corresponding to the Pleistocene age) samples were randomly collected from the Department of Forest Engineering and Technology of the Federal University of Parana State, Brazil. The fresh A. angustifolia sample corresponds to a cultivated tree, not a native one.

EPR spectra were obtained in a Bruker EMX spectrometer (Bruker Bio Spin, Germany) operated in the X-band (~9.5 GHz) at room temperature (300 K) in a range of magnetic field between 3485 and 3535 Gauss.

One log from G. arborea Roxb. and one from A. angustifolia was separated in rings and mechanically grinded up to powder sizes below 0.25 cm. The size is an important factor because it allows the particles to fill the EPR quartz tube without leaving voids. The amount of OFR was calculated from the area of the absorption curve in the normal mode taking as starting and end point of the absorption the value above the zero (dashed line). Due to the high concentration of OFR, the signal to noise ratio was not corrected.

The areas of the absorption spectra were compared with the weak pitch reference sample supplied by Bruker with 1.04x10¹³ spin cm^-1 (dark line in Figure 1). Absorption areas for curves with the same shape are related with the amount of free radicals (Lee et al., 2000), which were related with the mass of wood placed in the tube to calculate the number of spin per gram of sample, i.e. number of radicals per gram. This method was applied in samples of humus (Budziak et al., 2004), mangrove sediments (Mangrich et al., 2009), biochar (Angelo et al., 2013), coffe powder (Krakowian et al., 2014), human tissue and polyolefines (Camara et al., 2006).

RESULTS AND DISCUSSION

Free radicals in Gmelina arbórea

The EPR spectra of G. arborea samples presented a clear signal of OFR (Figure 2). The g factor is the characteristic value that helps to identify paramagnetic species. The OFR signal with g ~2.0040 has the spin density probably more localized on oxygen atoms, and with g ~2.0025 has the spin density localized near carbon atoms. OFR at intermediary distance between carbon and oxygen atoms was found in Pinus taeda samples with g=2.0030 (Budziak et al., 2004), associated to lignin (Pryor, 1992; Budziak et al., 2004; Czechowski et al., 2004). The G. arborea samples in our study presented values between 2.0046 and 2.0047, which match with the g factors found for semiquinones (around oxygen atoms) in wood (Yen et al., 1962; Guedes et al., 2003), which can be even higher, g=2.0050 (Zhou et al., 2009). Then, it is reasonable to accept that the EPR signals detected in G. arborea correspond to free radicals in semiquinone structures.

The derived absorption signals were integrated with the WinEPR program (Bruker) and the areas were compared with the area of the weak pitch sample in order to obtain the number of spins that contributed to that signal. By knowing the mass of wood placed in the tube, the number of spins (free radicals) per gram of sample was determined (Table 1).

The magnitude of free radicals concentration in all the samples was around 10¹² spin g^-1 and is lower than the concentration of free radicals in fresh or composted samples of P. taeda (10¹⁵ spin g^-1) (Budziak et al., 2004). Figure 3A shows the relationship between the free radicals concentration and the rings formed in different ages of the tree. Although there is not a linear behavior in this experimental data (R²=0.54), it is clear that the higher amount of free radicals appear in inner rings (older ages). If the dispersed data were eliminated (Figure 3B), such as values of rings 6 and 8, the recalculated central tendency is close to linearity (R²=0.98).

It is not unusual to find lower concentration of free radicals in younger rings (outer) since the metabolism takes place there (Starr and Taggart 2008) and it avoids formation of free radicals or eliminates them. This fact was probed when wood samples were analyzed by EPR just after collecting them from the tree. In the first minutes, negligible EPR signals were recorded, whereas the intensity of free radicals increased with time up to 2 h and then became constant (Hon et al., 1980). In Gmelina samples the time was not considered since all the samples had much more than 2 h, and then the amount of free radicals is not supposed to change (Hon et al., 1980). Similarly, a large difference in the water content could lead to differences in the intensities (Humar et al., 2006), but the same drying conditions were applied to all the samples in this work.

Although the physiological processes regulate the formation of free radicals in the external rings, there are opposed phenomena that induce the formation of radicals in outer rings such as the incidence of UV-VIS light (Kalnin et al., 1966), and even the indirect exposition to visible light increases the amount of free radicals in wood (Kalnin et al., 1966). In our study, free radicals were found in the outer rings (rings 3-5, Figure 3A), which are not hermetic systems; thus, the diffusion of compounds with the ability to quench radicals from rings in the middle of the stem, (rings 6 and 8, Figure 3A), can be an explanation for the lower OFR in those samples. In the case of the inner rings (rings 9 and 10 in Figure 3A), the reason that could hold the presence of the highest amount of radicals is that those rings retain oldest and dead mater formed by metabolism products such as semiquinone structures (Starr and Taggart, 2008).

Additional information appeared when the amount of radicals in the rings was compared with the carbon content of the same rings reported previously (Marcene et al., 2006). When both values were plotted, an inverse relationship between them was detected (Figure 4). It is remarkable that the carbon content is the highest between rings 3 and 5, where the amount of OFR is the lowest, whereas the carbon content is lower in rings 9 and 10 with a larger amount of OFR. An explanation for these responses could be that in samples with lower carbon content there is higher content of oxygen, which has more ability to stabilize OFR. Although between rings 6 and 8 the tendency is disrupted, the extremes suggest that the inverse relationship could be used to perform a fast determination of carbon content with a simple and easy EPR analysis, but additional experiments with a larger number of samples must be carried out in order to prove this response.

Organic free radicals in Araucaria angustifolia

The values of the g factor are practically the same as those found in G. arborea and the EPR signal can be associated to semiquinone structures, as it was described for G. arborea. At first sight, the dispersion of the values shows no correlation between the free radical concentration profiles in the rings of A. angustifolia (Figure 6A) with the age of the rings (R²=0.17), as if there were irregularity in the composition of the rings or diffusion of compounds between them. However, if the extreme results for rings 3, 4, 7, 8 and 12 are discarded, the new graph (Figure 6B) will show a linear trend similar to that found in G. arborea (R²=0.94), where the concentration in inner (i.e. older) rings is higher with a radical concentration around 2x10¹² spin g^-1, whereas the outer rings (1 and 2) contain 2x10¹¹ spin g^-1, which a difference of one order of magnitude. This could be explained with the same argument used previously for G. arborea, i.e., the low amount of radicals in the outer rings (1 and 2) can be associated to regulatory mechanisms favored by the metabolic activity in the external region.

Araucaria angustifolia samples from pleistocene age

With the aim to relate the concentration of free radicals in the samples of A. angustifolia listed in Table 2 with different specimens, two additional samples were analyzed by EPR corresponding to a recent cultivated Araucaria and one found from the Pleistocene, both without separating the rings but mixing all of them.

The results in Table 3 indicate that the recently cultivated A. angustifolia has 1.64x10¹¹ spin g^-1, which is a low value but it matches with the order of magnitude found in the different rings (Table 2) that could be a reference value for recent samples. Besides, the concentration of radicals in the sample of the Pleistocene rises to 2.30x10¹³ spin g^-1, and it is not within the mean values found for recent samples. This information leads to infer that the EPR spectrometry would be a qualitative technique for showing, in a short time, differences between old and new samples of A. angustifolia trees.

CONCLUSIONS

The free radicals found in Gmelina arborea correspond to semiquinone structures, according to the EPR spectroscopy g factor values. The concentration of the OFR in the samples analyzed is higher in inner (older) rings. Although outer rings are exposed to light that induces formation of free radicals, the continuous metabolism reactions reduces the number of radicals. The free radicals amount had an inverse relationship with the carbon content in the inner and outer rings. This suggests that the EPR spectroscopy, using specific quality control equipment such as Bruker e-scan EPR systems, could be a fast method for a qualitative determination of carbon content in wood. But further analysis should be carried out with a larger number of specimens in order to establish the exact relationship between these two parameters.

Regarding the recently cultivated and the Pleistocene Araucaria angustifolia samples, the concentration of free radicals along the rings is more disperse. However, the inner (older) rings have a higher amount of radicals than outer rings, similar to the profile found in G. arborea. The concentration of free radicals in this sample of A. angustifolia was one order of magnitude higher than the values found in the recently cultivated specimen. Finally, the amount of radicals in the recent specimen fell in the range of free radicals concentration found in the Araucaria sample studied along different rings, suggesting that EPR spectroscopy could be used to qualitatively estimate the age of the tree from which a wood sample is taken.

ACKNOWLEDGEMENTS

To Ronny Rocha Ribeiro for providing assistance for the EPR measurements.

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