Acid-catalysed α-O-4 aryl-ether bond cleavage in methanol/(aqueous) ethanol: understanding depolymerisation of a lignin model compound during organosolv pretreatment

The selective lignin conversion into bio-based organic mono-aromatics is a major general challenge due to complex structure itself/additional macromolecule modifications, caused by the cleavage of the ether chemical bonds during the lignocellulosic biomass organosolv pulping in acidified aqueous ethanol. Herein, the acido-lysis of connected benzyl phenyl (BPE), being a representative model compound with α-O-4 linkage, was investigated in methanol, EtOH and 75 vol% EtOH/water mixture solutions, progressing each time with protonating sulfuric acid. The effect of the physical solvent properties, acidity of the reaction process media and temperature on rate was determined. Experiments suggested BPE following SN1 mechanism due to the formation of a stable primary carbocation/polarity. The product species distribution in non-aqueous functional alcohols was strongly affected. The addition of H2O was advantageous, especially for alkoxylation. Yield was reduced by a factor of 3, consequently preserving reactive hydroxyl group. Quantitative experimental results indicated key performance parameters to achieve optimum. Organosolv lignins were further isolated under significantly moderate conditions. Consecutive structural differences observed supported findings, obtained when using BPE. H2O presence was again found to grant a higher measured –OH content. Mechanistic pathway analysis thus represents the first step when continuing to kinetics, structure–activity relationships or bio-refining industrial resources.

Scientific RepoRtS | (2020) 10:11037 | https://doi.org/10.1038/s41598-020-67787-9 www.nature.com/scientificreports/ BPE), the solvent (ethanol, methanol) or the solvent mixture (ethanol/water) and sulfuric acid. The reactor was placed in a housing, sealed, flushed twice and pressurized with nitrogen up to 1 MPa. All trials were performed with the stirring speed of 600 min -1 . The heating was set to start at room temperature, gradually increase and kept at the set temperature for 4 h. The reaction was quenched by rapidly cooling down the reactor. Before opening the autoclave, the gaseous phase was released and the headspace was purged with nitrogen. The operating conditions such as time, temperature and pressure were automatically recorded by a Scada system. The experiments performed are summarized in Supplementary Table S1 and can be found online.
Here, BPE was used as the reactant throughout. BPE was dissolved in a solvent (ethanol, methanol) or solvent mixture (75 vol% ethanol/water) in the ratio of 1 : 70 (w/v) making 120 mL of total reaction volume. Experiments were performed at various temperatures (160 -200 °C) and with various catalyst loadings 0.5 -1.5 % of H 2 SO 4 based on dry matter (for 1 g of model compound 0.025 -0.075 g of H 2 SO 4 in form of 2 M solution were used, thus taking into account that wood contains approximately 20 % of lignin).
Samples were collected during the reaction in a following order: the first sample was taken at room temperature, the second one halfway up the heating ramp, the third one at the final reaction temperature and then every 30 min until the end of the treatment. Eleven samples of 1 mL were collected for each experiment.
GC-MS analysis. The samples were analysed using gas chromatography coupled with mass spectrometry (GC-MS; 2010 Ultra, Shimadzu, Kyoto, Japan) with an additional FID (flame ionization detector) detector, equipped with the Zebron ZB-5 (Phenomenex, Torrance, CA, USA) 60 m × 0.25 mm × 0.25 µm, or RXi-1MS (Restek, Bellefonte, PA, USA) 15 m × 0.25 mm × 0.25 µm capillary column. Before the analysis, the samples were diluted with ethanol by a factor of 10. The concentration of the obtained products in samples was evaluated based on the calibration curves for the known concentrations of external standards. The column oven temperature was programmed from 50 (5.5 min hold) to 290 °C (7.5 min hold) at 20 °C min −1 . Helium was used as a carrier gas at a constant flow of 0.8 mL min -1 . The temperature of the injector and detector was 290 °C, the injection volume has been set to 1 µL with a split ratio of 50. The separated compounds were identified using mass spectrometry. Every product was sent through the ion source and the fragments were separated by a single quadrupole in the range from 35 to 600 m/z. The mass spectra were compared to the spectra of pure compounds from commercial FFNSC and NIST17 libraries.
The quantitative GC-MS/FID data reported in this work are averages of three experiments. The maximum standard deviation of our results was 1.1 × 10 −3 mol L -1 , while the maximum standard error was 6.1 × 10 −4 mol L -1 .
Lignin extraction. Lignin was extracted from beech tree sawdust (25 g, < 24 mesh, dried at 105 °C for 24 h) with methanol, ethanol, 75 vol% methanol/water and 75 vol% ethanol/water in the ratio of 1 : 7 (w/v). To catalyse the reaction, 1 % of H 2 SO 4 based on dry matter was added to the reaction mixture. The extraction was carried out in a 300 mL cylindrical stainless steel slurry reactor (Autoclave Engineers) at 180 °C over 1 h. The reactor was flushed twice and pressurized with nitrogen up to 1 MPa. The experiments were performed in a batch regime with the agitation speed of 200 min -1 . The reaction was quenched by dipping the reactor in an ice-bath. The solids were filtered out and rinsed with 150 mL of the 4 : 1 (m)ethanol/water mixture heated up to 60 °C in order to remove the extracted lignin, trapped on the surface of the wood particles. The remaining solids were dried at 105 °C for 48 h. Lignin was precipitated by adding three volumes of distilled water. The precipitate was collected by centrifugation for 10 min at 4,500 min −1 , repeatedly washed with distilled water and freeze-dried. Yields of isolated lignin (%) were calculated according to Eq. 1 considering that beech wood contains 24.4 % 29 of lignin.
Here W t -weight of isolated lignin (g), W 0 -initial lignin content (g). The yield of residue (%) was calculated according to Eq. 2: Here Z t -weight of the remaining solids (g), Z 0 -weight of starting beech wood (g). ATR/FT-IR spectroscopic analysis. Fourier Transform Infra-Red (FT-IR) spectra of isolated lignin samples were recorded using a FTIR spectrophotometer equipped with a LiTaO 3 detector (PerkinElmer, FT-IR spectrophotometer, Spectrum Two, Manchester, UK), in the range between 400 and 4,000 cm −1 using diamond ATR mode of operation with 64 accumulated scans, at a resolution of 4 cm −1 . The background spectrum was collected before every measurement and was subtracted from the sample spectrum automatically.
Size-exclusion chromatography (SEC). SEC of the acetylated lignin samples was performed on a sizeexclusion chromatographic system (Thermo Scientific Ultimate 3000, ThermoFisher, Waltham, MA, USA) equipped with a UV detector set at 280 nm. The analyses were carried out at ambient temperature using THF as eluent at a flow rate of 1 cm 3 min −1 . The aliquots (100 µL) of each sample dissolved in THF (1.5 mg cm −3 ) were injected into Plgel 3 µm MIXED E 7.5 × 300 mm. The column specifications allow the separation by molecular weight up to 30 kDa. The SEC system was calibrated with polystyrene standards with the molecular weight in the range from 500 Da to 30 kDa. The chromatographic data were processed with the PSS (Polymer Standards www.nature.com/scientificreports/ Service) WinGPC Unity software. The acetylation of lignin was performed using acetic anhydride according to the procedure reported elsewhere 30 .

Acidolysis of the α-O-4 linkage in non-aqueous alcohols.
It is well-known that during organosolv pretreatment lignin undergoes initial depolymerisation due to the scission of the α-ether bonds, which are cleaved more easily than the β-ether bonds because of a lower bond dissociation energy (BDE(α-O-4) = 215 kJ mol -1 ; BDE(β-O-4) = 290 kJ mol -1 ) 27 . Acidolysis of the α-ether linkage was found to predominate during the initial phase of organosolv pretreatment and was considered to be the rate-determining step. Subsequently, alkoxylation of a lignin macromolecule caused by alcohols acting as external nucleophiles determines the final structure of the isolated lignin as shown in Fig. 1 31 . Therefore, to elucidate the effect of the alcohol used, acidolysis of BPE (α-O-4 model compound) was examined in ethanol and methanol. Based on the GC-MS analyses, the main identified products were phenol (Ph), 2-benzyl phenol (2BPh) and benzyl ethyl ether (BEE) in the case of acidolysis in ethanol, and benzyl methyl ether (BME) in the case of acidolysis in methanol.
Reaction mechanism. The mechanism of acidolysis of benzyl phenyl ether (BPE) in the aqueous and nonaqueous alcohols was proposed based on the GC-MS analyses of the liquid samples. The main products of the BPE acidolysis in ethanol were identified to be phenol (Ph) and benzyl ethyl ether (BEE), while minor amounts of 2-benzyl phenol (2BPh) were also detected. Acidolysis in methanol proceeded in a similar manner, yielding benzyl methyl ether (BME) instead of BEE. The acid-catalysed conversion of BPE in ethanol with water as a co-solvent led to the formation of benzyl alcohol (BA). The product distribution was strongly affected by the solvent, acidity and temperature. Ethers can be cleaved via the S N 1 or S N 2 mechanisms. We argue that BPE follows the S N 1 mechanism due to the intermittent formation of a stable primary benzyl carbocation (stabilized by the resonance effect as presented in Supplementary Fig. S1) and due to the polarity of the medium. As shown below, increasing the polarity of the medium (substituting ethanol with methanol) increased the reaction rate.
In Fig. 2, the mechanism of acidolysis is shown. In the first step, BPE is protonated by sulphuric acid. Subsequently, phenol and a primary benzylic carbocation are formed in a S N 1 reaction. This carbocation reacts with either alcohol or water, forming BME/BEE or BA, respectively 32,33 .
Small quantities of 2BPh form when phenol reacts with the carbocation. If phenol reacts with its oxygen atom, this is a self-exchange reaction (the phenolic group in BPE is replaced by another phenol). However, the hydroxyl group in phenol activates the carbon atom in the ortho position, which can also take part in the acidolysis. This results in the formation of 2BPh (after keto-enol isomerization) 32,33,34 .
The effect of solvent. The difference in the intensity of the alkoxylation reactions is most obvious when comparing the BPE, Ph, BME and BEE concentration profiles for the experiments performed with 1 % of H 2 SO 4 at 160 °C (runs 10, 11), shown in Fig. 3a,b.
During the four-hour reaction, the BPE conversion of 77.5 % in methanol and 42.1 % in ethanol was achieved. We ascribe the greater activity in methanol to the difference in acidity (methanol pK a = 15.5, ethanol pK a = 15.9) 35 and polarity (methanol > ethanol) 36 . Firstly, the O-H bond is more polarized in methanol (no stabilizing inductive effect), making it a better nucleophile to attack the benzyl carbocation. Moreover, greater polarity of methanol stabilizes the carbocation intermediate, which is well known to accelerate the S N 1 reactions 32,33 .  The concentration profiles of 2-benzyl phenyl (2BPh) are similar for all temperatures and both alcohols. However, slightly higher concentrations of 2BPh were observed in methanol. The more pronounced formation of 2BPh is the consequence of the overall greater reactivity in methanol.
The influence of temperature on the BPE conversion is presented in Fig. 5a, while Fig. 5b shows the effect of acid concentration. The temperature effect is most obvious. Increasing the temperature from 160 to 200 °C increased the conversion in methanol from 77.5 to 98.8 % and in ethanol from 42.1 to 70.0 %. With methanol, the conversion was nearly complete (96.4 %) already at 180 °C and only negligible improvement was observed with a further increase of the reaction temperature to 200 °C. A similar effect was seen when using ethanol, only the conversions were not as high (vide infra).
The concentration of acid had a small effect on the conversion. Upon comparing the experiments at 180 °C with 0.5 %, 1.0 % and 1.5 % of H 2 SO 4 (Fig. 4a,c,e), no noticeable difference in the conversion is seen (93 %, 96.4 % and 97.4 %, respectively).
The effect on the initial reaction rate was more distinct, however not directly proportional to the amount of the used catalyst. For instance, 70 % of the BPE conversion during the acidolysis in methanol at 180 °C acidified  www.nature.com/scientificreports/ with 0.5 %, 1.0 % and 1.5 % of H 2 SO 4 was achieved after 130 min, 75 min and 55 min, respectively (Fig. 5b). Based on the obtained results, we assume that the alcohol acidity (pK a ) and polarity are the key parameter, determining the intensity of the α-ether bond acidolysis in non-aqueous alcohols. The rate of ether bond acidolysis is more strongly dependent on the reaction temperature than on the concentration of hydronium ions.

The effect of temperature and acid concentration in ethanol.
A similar temperature effect as in methanol has been observed also in ethanol, acidified with 1.0 % of H 2 SO 4 (Fig. 3b,d,f). Here, increasing the reaction temperature from 160 to 180 °C caused an analogous increase in the α-ether bond cleavage. After four hours of the reaction, the conversion rose from 42.1 to 70.0 %. Analogously, any further rise of the reaction temperature (up to 200 °C) had a negligible effect on the conversion. This suggests that the optimal temperature for the cleavage of the α-O-4 linkage is 180 °C. The product distribution in ethanol was significantly affected by the acidity of the reaction media. As shown in the concentration profiles in Fig. 4b,d,f, the effect of the acid concentration in ethanol has a much more pronounced effect than in the case of methanol. When running the reaction in ethanol with 0.5 %, 1.0 % and 1.5 % of H 2 SO 4 at 180 °C, the conversions were 47.9 %, 70.13 % and 82.3 %, respectively. This is a consequence of the different polarity of methanol and ethanol, as discussed (vide supra) 36 .
From the obtained results we conclude that the extent and rate of the α-O-4 bond cleavage is strongly dependent on the type of the solvent, specifically the acidity and polarity of the solvent itself. A greater solvent polarity can be beneficial as it reduces the required amount of sulfuric acid, which is the most frequently used catalyst for organosolv pretreatment. Expectedly, the reaction is also influenced by the concentration of the hydronium ion (a catalyst) and temperature.

Acidolysis of the α-O-4 linkage in aqueous ethanol. The effect of water.
Water is the most environmentally friendly solvent and is usually used in combination with organic solvents for organosolv pretreatment 37,38 . The highest lignin solubility in ethanol-water mixtures has been achieved in 70 % ethanol/ water 39 . According to the Hildebrand solubility parameter theory, the materials having similar δ-values show good solubility or miscibility. It has been determined that good solvents for lignin have the δ-value around 11 (cal cm -3 ) 1/240 . An efficient dissolution of high and low molecular weight lignin fractions has been demonstrated in a mixture of 75 % ethanol (δ = 12.9 (cal cm -3 ) 1/2 ) and 25 % of water (δ = 23.5 (cal cm -3 ) 1/2 ) with the δ-value close to the one of lignin (δ = 12 − 15.5 (cal cm −3 ) 1/2 ) 41,42,43 .
Hence, 75 vol% ethanol/water was examined as a solvent in our study. We sought to determine the effect of water on the α-ether bond acidolysis. Additionally, the influence of the reaction temperature and acidity of the reaction media was investigated. A methanol/water mixture was not studied due to poor solubility of the model compound (BPE).
Based on the GC-MS analyses, the main identified products were phenol (Ph), 2-benzyl phenol (2BPh), benzyl ethyl ether (BEE) and benzylic alcohol (BA). As shown in Fig. 2, water or ethanol participate in the S N 1 reaction after the protonation step, yielding BA or BEE, respectively.
The intensity of the reaction is most evident in the BPE, Ph, BEE and BA concentration profiles for the experiments performed with 1.5 % of H 2 SO 4 at 180 °C (runs 7, 9), shown in Fig. 6e and f. After four hours, the conversion was 82.3 % in ethanol and 65.3 % in 75 % ethanol/water was achieved.
The lower activity in the ethanol/water mixture compared to ethanol might be explained by its acidity. Water itself (pK a = − log (K w /(55.56 mol L -1 )) = 15.7) is more acidic than ethanol (pK a = 15.9) 35 . This means that H 2 SO 4 more easily protonates ethanol than water, causing the reaction to be slower in the ethanol/water mixture.
However, one of the main challenges is an efficient lignin isolation with as few structural changes (condensation reactions, scission of β-ether linkages and alkoxylation reactions) as possible. During organosolv pretreatment it is important to preserve the reactive functional groups (aliphatic and phenolic hydroxyl groups). We show that the intensity of the ethoxylation reactions can be reduced by a factor of 3 by adding 25 % of water to ethanol. Water evidently represents one of the key parameters and has to be taken into account for eventual lignin isolation by the organosolv fractionation.
The effect of temperature. The effect of temperature on the α-ether scission in acidified aqueous ethanol was tested at 160 °C, 180 °C and 200 °C (runs 12, 6, 15) with 1.0 % of H 2 SO 4 . The product distribution in aqueous ethanol at different temperatures is shown in Fig. 6.
The effect is most evident in the concentration profiles of BPE. As the temperature rises from 160 to 200 °C, α-ether bond cleavage is significantly promoted and the conversion increases from 23.7 to 92.0 %. The temperature increase significantly increased the reaction rate and strongly affected the product distribution.
The reactant conversion in aqueous ethanol as a function of the reaction time is shown in Supplementary  Fig. S2. We see that a 15 % conversion of BPE in aqueous ethanol with 1 % of H 2 SO 4 at 160 °C, 180 °C, 200 °C was achieved after 150 min, 75 min, 50 min, respectively. The highest catalyst activity and conversion (up to 92.0%) was demonstrated at 200 °C with 1.0 % of H 2 SO 4 , yielding the highest concentrations of Ph, BEE and BA. Moreover, the BEE : BA ratio at the end of the experiment was 3 : 1, which corresponds to the initial ratio of ethanol and water. This additionally confirms the S N 1 reaction mechanism shown in Fig. 2.
The  Supplementary Fig. S2. For instance, a 30 % conversion in aqueous ethanol with 0.5 %, 1.0 % and 1.5 % of H 2 SO 4 at 180 °C was achieved after 200 min, 125 min and 100 min, respectively. This shows that after a certain threshold (in our case 1.0%), the increased acidity does not translate into higher reaction rate.
However, when carrying out the reaction in ethanol, the effect was more pronounced. Increasing the concentration of H 2 SO 4 from 0.5 to 1.5 % improved the final conversion from 47.9 to 82.3 %. This is consistent with the overall decrease of the reaction rate when water is added to ethanol. A greater amount of the catalyst is required, meaning that the threshold is moved above 1.0 %. Due to the higher acidity of water compared to ethanol, H 2 SO 4 is less likely to dissociate and protonate it, results in the overall lower concentration of protonated species (hydronium ions or alcohols).
Overall, the addition of water has a desired effect on the reaction. Firstly, the addition of 25 % of water to ethanol decreased the extent of alkoxylation reaction by a factor of 3, preserving the reactive hydroxyl group. Specifically, concentration of benzyl ethyl ether (BEE) has been reduced from 0.03 to 0.01 mol L −1 (Figs. 3d and  6c). Secondly, a more pronounced temperature-acidity dependence in aqueous ethanol is a key parameter to be considered for attaining the optimal extent of α-ether bond cleavage. Thirdly, by comparing the patterns in the concentration profiles in Figs. 3 and 4 with Fig. 6, the addition of water has reduced experimental variations, resulting in a more consistent process.

The effect of solvent on lignin.
To estimate the effect of solvents tested with the model compound BPE, lignin was isolated from beech wood with MeOH, EtOH, MeOH/H 2 O, EtOH/H 2 O under moderate conditions at 180 °C with 1.0 % of H 2 SO 4 according to the procedure described in Section "Material and methods. " ATR/FT-IR spectroscopic analysis. Preliminary information on the amount of the total hydroxyl groups was obtained using a FTIR analysis. The FTIR spectra were collected in ATR mode, which enables a simple and fast comparison of lignin samples. The assignment of lignin FTIR spectra was reported in detail by Faix 44 and allows the identification of characteristic bands for total hydroxyl groups (O-H stretch) at 3,412-3,460 cm -1 as well as for secondary alcohols and aliphatic ethers (C-O deformation) at 1,086 cm -1 . Figure 7 shows the FTIR spectra Intensity of the absorption band corresponding to the total OH groups increases in the following sequence: EtOH < MeOH < MeOH/H 2 O < EtOH/H 2 O and thus confirms that the addition of water to alcohol reduces the alkoxylation reactions preserving more OH groups in lignin. While there are minor differences between the spectra of lignin isolated using non-aqueous alcohols, the addition of water into the system causes observable changes. Lignin, isolated in aqueous methanol, exhibits a lower absorption band intensity corresponding to the O-H stretch and consequently a lower OH group content compared to the one isolated in aqueous ethanol. This difference could be explained by the different acidity of the solvents. As already mentioned, acidity increases in the following order EtOH (pK a = 15.9) < H 2 O (pK a = 15.7) < MeOH (pK a = 15.5) 35 . When aqueous methanol is used for lignin isolation, H 2 SO 4 more easily protonates methanol than water, affecting the predominance of the transetherification reactions at the Cα position (alkoxylation). Moreover, the aliphatic α-ether formation is confirmed by a notably more intensive shoulder at 1,086 cm -1 compared to the one of lignin isolated in aqueous ethanol. In contrast to the aqueous methanol, the easier protonation of water than ethanol in aqueous ethanol, makes the α-ether bond hydrolysis the preferential reaction resulting in the formation of the aliphatic OH groups at the Cα position and accordingly increases overall OH group content. Correspondingly, the FTIR spectrum of lignin isolated in aqueous ethanol displays the highest band intensity corresponding to the O-H stretch (3,412-3,460 cm -1 ) along with the less intensive shoulder at 1,086 cm -1 revealing a reduced extent of the transetherification of α-ethers (alkoxylation).
Fractionation. The beech wood fractionation results are summarized in Supplementary Table S2. The highest yields of lignin were obtained using aqueous alcohols. Despite the comparable yields of residue, significant differences between the lignin yields (76.0 % and 83.4 %) were achieved using MeOH/H 2 O and EtOH/H 2 O, respectively. As expected, EtOH/H 2 O showed the most desired results. The residue obtained after the fractionation in MeOH/H 2 O had a notably darker colour and apparently a certain amount of lignin remained trapped on the surface of the particles. MeOH/H 2 O seems to be a less efficient solvent for lignin which is also in agreement with the Hildebrand solubility parameter theory, were the lignin (δ = 12 -15.5 (cal cm -3 ) 1/2 ) solubility in EtOH/ H 2 O (δ = 15.5 (cal cm -3 ) 1/2 ) should be better compared to MeOH/H 2 O (δ = 16.6 (cal cm −3 ) 1/2 ) 41,42,43 . In addition, a higher degree of methylation in aqueous methanol limited a sufficient lignin isolation by precipitation in water and in the end, a yield of 76 % was obtained. The effect of more pronounced (m)ethylation is evident in the www.nature.com/scientificreports/ case of fractionation performed using MeOH and EtOH. In those two cases, the precipitated lignin formed a milky suspension that made its separation extremely complicated. Therefore, despite the beneficial δ-values that describe MeOH (δ = 14.3 (cal cm -3 ) 1/2 ) and EtOH (δ = 12.9 (cal cm -3 ) 1/2 ) as suitable solvents, notably lower lignin yields (70.6 % and 64.4 %, respectively) were attained. The different degree of lignin (m)ethylation which is also confirmed using FTIR analysis, accordingly affected lignin self-aggregation intensity. Self-aggregation of the dissolved lignin depends on the balance of electrostatic repulsion and van der Waals attraction in a solvent as well as on the isolation process which directly affects the number of cross-linking sites (methoxyl and hydroxyl groups) 45 . Accordingly, due to the relatively low hydroxyl group content in lignin isolated using EtOH and MeOH formed a milky suspension during the precipitation. While the higher degree of lignin methylation in MeOH/H 2 O than the degree of ethylation in EtOH/H 2 O was confirmed by the notably reduced lignin self-aggregation intensity.
SEC analysis. The effect of the used solvents is also evident from the SEC analysis data shown in Supplementary Table S2. The formation of α-etherified moieties within the lignin structure during the fractionation in MeOH and EtOH prevents any further condensation reactions resulting lignin with a lower average molecular weight (M w ) 2,150 Da and 2,020 Da, respectively. The presence of water in MeOH/H 2 O and EtOH/H 2 O creates reactive secondary OH groups that could be further etherified or involved in condensation with the other lignin functional groups, forming slightly larger fragments with an average M w of 2,300 Da and 2,700 Da, respectively. However, based on the results of the previously investigated BPE acidolysis in EtOH/H 2 O, the presence of water is more likely to reduce the rate of the reaction or lignin depolymerisation in this case. Thus, higher M w values for the lignin isolated in aqueous alcohols imply a lower degree of depolymerisation. The effect of water in terms of weaker depolymerisation is obvious from the SEC chromatogram profiles shown in Fig. 8. Here, the main peak of EtOH/H 2 O-lignin is shifted towards the higher molecular weight region pointing towards a presence of a less intact lignin structure. The minor difference between the average M w values of MeOH-lignin (2,150 Da) and MeOH/H 2 O-lignin (2,300 Da) could be explained by the acidity of the used solvents and is consistent with the previously discussed results from the FTIR analyses. When MeOH/H 2 O is used for lignin isolation, H 2 SO 4 more easily protonates methanol than water, affecting the predominance of the α-etherification reactions. Consequently, as a majority of the solvent mixture consists of MeOH (75 vol%), only a minor difference between the M w values has been observed. www.nature.com/scientificreports/ conclusions In this study, benzyl phenyl ether (BPE) was used as a model compound for the α-O-4 ether linkage in lignin to understand the intricacies of the α-ether bond cleavage in acidified methanol, ethanol and in aqueous ethanol. When using BPE, the S N 1 mechanism was postulated based on several findings. Firstly, the primary benzyl carbocation is sufficiently stable to intermittently form after the protonation of BPE. Secondly, the reaction rate was found to be accelerated in methanol, which is more polar than ethanol, thus stabilizing the carbocation. Thirdly, when using the ethanol/water mixture, the ratio of the corresponding products (BEE:BA) matched the ratio of ethanol and water in the solvent, meaning that they compete for the carbocation.
The product distribution in non-aqueous alcohols was strongly affected by the solvent acidity and polarity, and temperature while the acidity of the reaction media had a less significant effect. Adding water to ethanol had a beneficial effect on the α-ether bond cleavage, especially in terms of alkoxylation reactions. Specifically, the extent of alkoxylation was reduced by a factor of 3, thus preserving the reactive hydroxyl group.
The structural differences between the lignins, isolated with (aqueous) alcohols, were consistent with the results obtained from the BPE acidolysis. Specifically, a reduced extent of the alkoxylation reactions reflected in a less depolymerized lignin molecule thus specifying 75 vol% EtOH/H 2 O as the most favourable solvent among the ones considered in this study.