An Efficient Method of Birch Ethanol Lignin Sulfation with a Sulfaic Acid-Urea Mixture

For the first time, the process of birch ethanol lignin sulfation with a sulfamic acid-urea mixture in a 1,4-dioxane medium was optimized experimentally and numerically. The high yield of the sulfated ethanol lignin (more than 96%) and containing 7.1 and 7.9 wt % of sulfur was produced at process temperatures of 80 and 90 °C for 3 h. The sample with the highest sulfur content (8.1 wt %) was obtained at a temperature of 100 °C for 2 h. The structure and molecular weight distribution of the sulfated birch ethanol lignin was established by FTIR, 2D 1H and 13C NMR spectroscopy, and gel permeation chromatography. The introduction of sulfate groups into the lignin structure was confirmed by FTIR by the appearance of absorption bands characteristic of the vibrations of sulfate group bonds. According to 2D NMR spectroscopy data, both the alcohol and phenolic hydroxyl groups of the ethanol lignin were subjected to sulfation. The sulfated birch ethanol lignin with a weight average molecular weight of 7.6 kDa and a polydispersity index of 1.81 was obtained under the optimum process conditions. Differences in the structure of the phenylpropane units of birch ethanol lignin (syringyl-type predominates) and abies ethanol lignin (guaiacyl-type predominates) was manifested in the fact that the sulfation of the former proceeds more completely at moderate temperatures than the latter. In contrast to sulfated abies ethanol lignin, the sulfated birch ethanol lignin had a bimodal and wider molecular weight distribution, as well as less thermal stability. The introduction of sulfate groups into ethanol lignin reduced its thermal stability.


Introduction
Lignin is the most widespread aromatic polymer on the earth. The lignin content in the dry mass of woody plants ranges within 15-40%, depending on the species [1,2]: 5-12% in herbaceous plants, 25-35% in coniferous wood, and 15-30% in deciduous wood. Lignin has an irregular 3D structure built from phenylpropane units (PPUs) with different numbers of methoxyl groups in the aromatic ring: p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S). The PPUs are randomly cross-linked with simple ether C-O-C and C-C bonds [3]. In addition, the propane chains of the lignin PPUs can contain different functional groups: hydroxyl (-OH), carbonyl (C=O), carboxyl (-COOH), and double bonds (-CH=CH-).
The structure, chemical composition, and physicochemical properties of lignins vary within fairly wide limits, depending on the lignocellulosic raw material and lignin isolation method used [4]. The complex heterogeneous composition of lignins complicates the development of efficient techniques for utilization to obtain valuable products [5].
Recently, there has been an increased interest in organosolv methods for extracting cellulose from lignocellulosic biomass. The use of organic solvents makes the organosolv The use of the low-toxic and non-corrosive mixture of sulfamic acid and urea fo sulfation of abies ethanol lignin was previously proposed [16]. The yield of sulf ethanol lignin of abies wood and sulfur content can be regulated by varying the sulf The use of the low-toxic and non-corrosive mixture of sulfamic acid and urea for the sulfation of abies ethanol lignin was previously proposed [16]. The yield of sulfated ethanol lignin of abies wood and sulfur content can be regulated by varying the sulfation process temperature, time, and the ratio of lignin to sulfating complex (sulfamic acid and urea mixture).
A high yield of sulfated lignin and sulfur content can be obtained at different combinations of the above-mentioned parameters of the sulfation process. Taking into account the data obtained when optimizing the process of abies ethanol lignin sulfation with a mixture of sulfamic acid and urea, the ratio of birch ethanol lignin to the sulfating complex was chosen in this study to be 1:3 mol/mol.
The sulfation process temperature ranged from 70 to 110 • C and the process time from 0.5 to 3.0 h. The data on the effect of the birch ethanol lignin sulfation conditions on the yield of sulfated lignin and sulfur content are given in Table 1. The high yield of sulfated birch ethanol lignin and the high sulfur content can be obtained at different combinations of the specified parameters of the sulfation process (temperature and time).
The sulfated ethanol lignin samples with a high yield (91.1-96.4%) and a sulfur content of 7.1-8.1 wt % were obtained at a process temperature of 90 • C and a time of 3 h or at a temperature of 100 • C and a time of 1.5-2.0 h. A further increase in the sulfation time and temperature did not significantly affect the sulfur content in the product but reduced the sulfated lignin yield. This may be due to the intensification of secondary condensation and destruction reactions under more severe conditions, which leads to the formation of products that are removed at the stage of sulfated lignin dialysis. It should be noted that the products of sulfation of birch ethanol lignin contain somewhat more sulfur than the products of sulfation of abies ethanol lignin under similar conditions [16]. This is possibly related to the lower reactivity of coniferous lignins that contain more condensed structural units than hardwood lignins [4].
The kinetics of the birch ethanol lignin sulfation with sulfamic acid-urea mixture in 1,4-dioxane medium was investigated in the temperature range of 70-100 • C ( Figure 2). The apparent rates of the birch ethanol lignin sulfation were calculated from the change in the sulfur content in sulfated ethanol lignin. The calculation was made using the firstorder equation. The activation energy of the sulfation process was determined from the temperature dependence of the rate constants in the Arrhenius coordinates ( Figure 3). time and temperature did not significantly affect the sulfur conten reduced the sulfated lignin yield. This may be due to the intensifi condensation and destruction reactions under more severe condition formation of products that are removed at the stage of sulfated ligni be noted that the products of sulfation of birch ethanol lignin cont sulfur than the products of sulfation of abies ethanol lignin under sim This is possibly related to the lower reactivity of coniferous lignin condensed structural units than hardwood lignins [4].
The kinetics of the birch ethanol lignin sulfation with sulfamic a 1,4-dioxane medium was investigated in the temperature range of 7 The apparent rates of the birch ethanol lignin sulfation were calcula in the sulfur content in sulfated ethanol lignin. The calculation w first-order equation. The activation energy of the sulfation process w the temperature dependence of the rate constants in the Arrhenius co   The calculated apparent rate constants and activation energies of the birch ethanol lignin sulfation process are given in Table 2.  The calculated apparent rate constants and activation energies of the birch ethanol lignin sulfation process are given in Table 2. The activation energies of the processes of sulfation of birch and abies ethanol lignins with sulfamic acid-urea mixture in 1,4-dioxane medium are similar: 10.7 kJ/mol for birch lignin ( Table 2) and 8.4 kJ/mol for abies lignin [16]. It should be noted that, for the process of starch sulfation in a deep eutectic solvent (the sulfamic acid-urea mixture), the activation energy was 6.4 kJ/mol [20] and, for the sulfation of arabinogalactan with sulfamic acid in DMSO it was 13.1 kJ/mol [21]. It is known that the low activation energy of the process may indicate the presence of significant diffusion restrictions [22,23]. Taking this into account, we can conclude that, under the chosen conditions, the processes of biopolymer sulfation proceed under diffusion restrictions.
The solubility of the sulfated lignin in water increased with an increase in the content of sulfate groups. The maximum sulfur content in the sulfated ethanol lignin was estimated to be 10.6 wt %, taking into account the hypothetical structure of the Berkman spruce lignin [4], in which one phenylpropane unit has 0.9 mol of free OH groups capable of sulfating. In order to find the conditions that ensure the production of sulfated birch ethanol lignin with maximum yield and sulfur content, a numerical optimization of the sulfation process was carried out.

Numerical Optimization of the Process of Birch Ethanol Lignin Sulfation
As independent variables, we used two factors: process temperature X 1 (70, 80, 90, 100, 110 • C) and time X 2 (0.5, 0.75, 1.0, 1.5, 2.0, 3.0 h). The result of the sulfation process was characterized by two output parameters: sulfur content Y 1 (wt %) in the sulfated ethanol lignin and sulfated ethanol lignin yield Y 2 (wt %). The fixed parameter was the ratio L:SC = 1:3. A combined multilevel experiment plan (Users Design) was used in the calculations. The designations of the variables are listed in Table 3. Table 3. Designations of independent variables (factors) and output parameters (experimental results).

Factors and Parameters Designations in the Equations
The experimental results given in Table 1 were used in the mathematical processing and optimization of the birch ethanol lignin sulfation process.
The dependences of the output parameters on the variable process factors were approximated by second-order regression equations. The results of the variance analysis are given in Table 4. The variance analysis showed that, within the limits of the experimental conditions used, the greatest contribution to the total variance of the output parameter was made by both factors: the temperature and time of the birch ethanol lignin sulfation process. This is indicated by the high variance ratios (F) for the main effects, which are called also the influence efficiencies. The data in the columns of Table 4 (P) are interpreted similarly. The influence of the variance source on the output parameter is considered to be statistically significant if its significance level is lower than a specified critical value (in our case, 0.05).
The dependence of the sulfur content Y 1 in the sulfated birch ethanol lignin on the process variables is approximated by the regression equation The predictive properties of Equation (1) are illustrated in Figure 4  The approximation quality is characterized also by the determination coefficient R 2 adj. In the case under consideration, the value is R 2 adj = 86.1%, which indicates acceptable approximation quality. This confirms the adequacy of Equation (1) for the experiment and makes it possible to use this equation as a mathematical model of the process under study.
Using the mathematical model, the dependence of the output parameter Y1 on the variables X1 and X2 was plotted in the form of a response surface ( Figure 5). The approximation quality is characterized also by the determination coefficient R 2 adj . In the case under consideration, the value is R 2 adj = 86.1%, which indicates acceptable approximation quality. This confirms the adequacy of Equation (1) for the experiment and makes it possible to use this equation as a mathematical model of the process under study.

Estimated Response Surface
Using the mathematical model, the dependence of the output parameter Y 1 on the variables X 1 and X 2 was plotted in the form of a response surface ( Figure 5). R 2 adj. In the case under consideration, the value is R 2 adj = 86.1%, which indicates acceptable approximation quality. This confirms the adequacy of Equation (1) for the experiment and makes it possible to use this equation as a mathematical model of the process under study.
Using the mathematical model, the dependence of the output parameter Y1 on the variables X1 and X2 was plotted in the form of a response surface ( Figure 5). According to the calculation using mathematical model (1), the maximum predicted sulfur content (8.4 wt %) was obtained at the point corresponding to a process temperature of 107 °C and a process time of 2.3 h.
According to the results of the variance analysis within the limits of the chosen experimental conditions, the sulfation temperature contributes significantly to the total variance of the output parameter Y2 (sulfated lignin yield, wt %). This is indicated by the high variance relation (F) corresponding to this factor and the small Р criterion.
The dependence of Y2 on the variable process factors is approximated by the regression equation The determination coefficient is fairly high, R 2 adj = 92.8%, which evidences the good approximation quality. The latter is also confirmed by the good agreement between the According to the calculation using mathematical model (1), the maximum predicted sulfur content (8.4 wt %) was obtained at the point corresponding to a process temperature of 107 • C and a process time of 2.3 h.
According to the results of the variance analysis within the limits of the chosen experimental conditions, the sulfation temperature contributes significantly to the total variance of the output parameter Y 2 (sulfated lignin yield, wt %). This is indicated by the high variance relation (F) corresponding to this factor and the small P criterion.
The dependence of Y 2 on the variable process factors is approximated by the regression equation The determination coefficient is fairly high, R 2 adj = 92.8%, which evidences the good approximation quality. The latter is also confirmed by the good agreement between the output parameters calculated using Equation (2) and those obtained in the experimental measurements. This confirms the adequacy of Equation (2) for the experiment and its use as a mathematical model of the process under study ( Figure 6). output parameters calculated using Equation (2) and those obtained in the experimental measurements. This confirms the adequacy of Equation (2) for the experiment and its use as a mathematical model of the process under study ( Figure 6).      According to the above-described model, the optimum conditions for the sulfation of birch ethanol lignin that ensure the maximum yield of sulfated birch ethanol lignin (96.1 wt %) correspond to a process temperature of 78 °C and a time of 2.9 h.

Characterization of the Sulfated Birch Ethanol Lignin
The substitution of hydroxyl groups for sulfate groups during the sulfation of the birch ethanol lignin with the sulfamic acid-urea mixture was confirmed by FTIR and NMR spectroscopy.
The FTIR spectrum of birch ethanol lignin ( Figure 8) contains absorption bands characteristic of hardwood lignins (GS) [24]. According to the above-described model, the optimum conditions for the sulfation of birch ethanol lignin that ensure the maximum yield of sulfated birch ethanol lignin (96.1 wt %) correspond to a process temperature of 78 • C and a time of 2.9 h.

Characterization of the Sulfated Birch Ethanol Lignin
The substitution of hydroxyl groups for sulfate groups during the sulfation of the birch ethanol lignin with the sulfamic acid-urea mixture was confirmed by FTIR and NMR spectroscopy.
The FTIR spectrum of birch ethanol lignin ( Figure 8) contains absorption bands characteristic of hardwood lignins (GS) [24]. The band at 1123 cm −1 corresponds to planar bending vibrations of the C-H syringyl aromatic rings, and the C-O stretching vibrations in secondary alcohols are dominant in the spectrum. The pronounced band with a maximum at 1327 cm −1 belongs to the skeletal vibrations of the syringyl ring with the C-O stretching vibrations. In addition, the spectrum includes medium-intensity absorption bands around 1271 and 1034 cm −1 , characteristic of the vibrations of the guaiacyl units of lignin [4].  In contrast to the spectrum of the initial ethanol lignin, the FTIR spectrum of the ammonium salts of the ethanol lignin sulfates contained a new absorption band at 798 cm −1 (Figure 8), which corresponds to the stretching vibrations of the C-O-S bond of the sulfate group and a broad absorption band with the maximum at 1218 cm −1 corresponding to the asymmetric stretching vibrations υas(O=S=O).
The FTIR spectra of birch and abies ethanol lignin sulfates [16] obtained in a similar way are almost identical, except for the presence in the spectrum of the sulfated birch ethanol lignin of the adsorption band at 1331 cm −1 characteristic of the syringyl structures.
The 2D HSQC NMR spectra of the initial and sulfated birch ethanol lignins are shown in Figures 9 and 10, respectively. The main 1 H-13 C peaks in the HSQC spectra identified using the literature data [25][26][27] are given in Table 5, together with the chemi- In contrast to the spectrum of the initial ethanol lignin, the FTIR spectrum of the ammonium salts of the ethanol lignin sulfates contained a new absorption band at 798 cm −1 (Figure 8), which corresponds to the stretching vibrations of the C-O-S bond of the sulfate group and a broad absorption band with the maximum at 1218 cm −1 corresponding to the asymmetric stretching vibrations υ as (O=S=O).
The FTIR spectra of birch and abies ethanol lignin sulfates [16] obtained in a similar way are almost identical, except for the presence in the spectrum of the sulfated birch ethanol lignin of the adsorption band at 1331 cm −1 characteristic of the syringyl structures. The 2D HSQC NMR spectra of the initial and sulfated birch ethanol lignins are shown in Figures 9 and 10, respectively. The main 1 H-13 C peaks in the HSQC spectra identified using the literature data [25][26][27] are given in Table 5, together with the chemical shifts of some low-intensity peaks (not shown in Figures 9 and 10). The main structural units and fragments of the initial and sulfated birch ethanol lignins are presented in Figure 11. The assignment of signals is given in Table 5, and the main identified structural units and fragments are shown in Figure 11. The assignment of signals is given in Table 5, and the main identified structural units and fragments are shown in Figure 11. 022, 27, x 11 of 23 Figure 10. HSQCed spectrum of sulfated ethanol lignin: aliphatic oxygenated region 1 and aromatic region 2. The assignment of signals is given in Table 5, and the main identified structural units and fragments are shown in Figure 11.  Table 5, and the main identified structural units and fragments are shown in Figure 11.    The HSQC spectra of the initial and sulfated ethanol lignin samples were compared in the regions of the chemical shifts of atoms from the lignin side chains (δC/δH 50-90/2.9-5.7 ppm) and aromatic rings (δC/δH 100-150/5.5-8.0 ppm).
Considering the region of the 1 H-13 C side chain signals in the HSQC spectrum of the birch ethanol lignin (Figure 9), we can see that it contains the intense correlation peaks of β-aryl ethers (A), pinoresinol (B), and phenylcoumaran fragments (C) (see Figure 11). A part of β-aryl ethers is ethoxylated in the α-position, judging by the fact that the spectra contain signals of the methylene group in the α-ethoxylated β-O-4′ bonds (δC/δH 64.4/3.33) and the α-position of the α-acylated β-O-4′ bonds (δC/δH 81.2/4.56). This assumption is confirmed by the presence of a correlation signal of the methyl group at δC/δH 14.3/1.00 ppm.
In the spectrum of the sulfated ethanol lignin (Figure 10), the group of peaks assigned to the phenylcoumaran fragments (C) is characterized by a change in the position of the correlation signals Cγ-Hγ and Cβ-Hβ (Figure 10), which is related to the effect of sulfation of OH groups in the γ position (Cγ-Hγ: a shift of ΔδC ~ 4 ppm toward weak fields; Cβ-Hβ: a shift of ΔδC ~ 3 ppm toward strong fields).
A similar change in the peak positions along the carbon atom axis in the sulfated ethanol lignin spectrum is observed for β-aryl ethers β-O-4′ (A) and α-ethoxylated β-aryl ethers (A′). The Cγ-Hγ correlation signals of the sulfated lignin are shifted relative to the initial lignin signals toward weak fields by ~5 ppm and the Cβ-Hβ signals toward strong fields by ~4 ppm. Such shifts of the signals in the spectra are most likely due to the sul- The HSQC spectra of the initial and sulfated ethanol lignin samples were compared in the regions of the chemical shifts of atoms from the lignin side chains (δ C /δ H 50-90/2.9-5.7 ppm) and aromatic rings (δ C /δ H 100-150/5.5-8.0 ppm).
Considering the region of the 1 H-13 C side chain signals in the HSQC spectrum of the birch ethanol lignin (Figure 9), we can see that it contains the intense correlation peaks of β-aryl ethers (A), pinoresinol (B), and phenylcoumaran fragments (C) (see Figure 11). A part of β-aryl ethers is ethoxylated in the α-position, judging by the fact that the spectra contain signals of the methylene group in the α-ethoxylated β-O-4 bonds (δ C /δ H 64.4/3.33) and the α-position of the α-acylated β-O-4 bonds (δ C /δ H 81.2/4.56). This assumption is confirmed by the presence of a correlation signal of the methyl group at δ C /δ H 14.3/1.00 ppm.
In the spectrum of the sulfated ethanol lignin (Figure 10), the group of peaks assigned to the phenylcoumaran fragments (C) is characterized by a change in the position of the correlation signals Cγ-H γ and Cβ-H β (Figure 10), which is related to the effect of sulfation of OH groups in the γ position (Cγ-H γ : a shift of ∆δ C~4 ppm toward weak fields; Cβ-H β : a shift of ∆δ C~3 ppm toward strong fields).
A similar change in the peak positions along the carbon atom axis in the sulfated ethanol lignin spectrum is observed for β-aryl ethers β-O-4 (A) and α-ethoxylated β-aryl ethers (A ). The Cγ-H γ correlation signals of the sulfated lignin are shifted relative to the initial lignin signals toward weak fields by~5 ppm and the Cβ-H β signals toward strong fields by~4 ppm. Such shifts of the signals in the spectra are most likely due to the sulfation of OH groups of β-aryl ethers of the lignin macromolecule in the γ position. In addition, free OH groups in the α position of β-aryl ethers (A) are probably subjected to the sulfation, since the Cα-H α peak at δ C /δ H 72.5/4.88 ppm is missing in the sulfated lignin spectrum.
The shift of signals in the aliphatic region of the spectrum of the sulfated lignin sample as compared with the spectrum of the initial lignin was also found for the C γ -H γ correlations of the cinnamyl alcohol end groups (I). This is also indicative of the sulfation of OH groups bonded with carbon atoms C γ of this lignin fragment.
Despite the presence of the fairly intense Cα-H α , Cβ-H β , and Cγ-H γ signals of pinoresinol (β-β ) fragments (B) in the spectrum of the initial ethanol lignin, the intensity of these signals in the spectrum of the sulfated sample drops dramatically. The peaks assigned to the Cγ-H γ correlations (δ C /δ H 71.6/3.80 and 4.18 ppm) disappear almost completely.
It is important to note the appearance of a peak at δ C /δ H 53.6/5.00 ppm assigned to the CH 3 or CH groups in the aliphatic region of the sulfated ethanol lignin spectrum. We failed to establish an unambiguous correspondence of this peak to any structural fragment.
The 1 H-13 C aromatic region in the HSQC spectrum of the birch ethanol lignin (Figure 9) contains characteristic correlation peaks of the syringyl (S) and guaiacyl (G) units, p-coumarates (pCA), and cinnamyl aldehyde end groups (J). The syringyl units are of several types. In particular, using the assignments made in [28][29][30], we found S units with a substituent in the 4-position (δ C /δ H 104.5/6.70), S units with a free hydroxyl group in the 4-position (δ C /δ H 106.2/6.51), S units with a carbonyl group in the α-position (δ C /δ H 107.1/7.35), and S units with a carboxyl group in the α position (δ C /δ H 107.2/7.23).
In addition, there are high-intensity signals at δ C /δ H = 129.1/7.73 and 131.9/7.67 ppm corresponding to the CH or CH 3 groups. Some researchers believe that the signals located at these chemical shifts can be attributed to both C α,β −H α,β in stilbenes [31] and C2,6-H 2,6 in p-benzoates [32].
In the aromatic region of the sulfated lignin spectrum containing the main part of the peaks of the syringyl (S 2,6 eth, S 2,6 , S" 2,6 ) and guaiacyl units (G 2 , G 5 , G 6 ), p-coumarates (pCA 3,5 ), and cinnamyl aldehyde end groups (J 2,6 ) (see Table 6), the signal of the C2,6-H 2,6 syringyl units with a free hydroxyl group in the 4-position disappears almost completely. This is apparently due to the replacement of this hydroxyl group by the sulfate one. In addition, in this region of the spectrum, a new peak at δ C /δ H = 120.8/7.38 and 7.32 ppm appears, which is most likely a signal of the C5-H 5 guaiacyl (Gs) and C3,5-H 3,5 p-coumarate (pCAs) structural units with sulfate groups attached to the 4-position. These changes in the chemical shifts of adjacent positions 3 and 5 of the aromatic ring caused by esterification correspond to the expected change of the substituent in phenol [33]. The possible substitution of the sulfate group of the phenolic hydroxyls for the guaiacyl (G) units in the 4-position is evidenced also by the almost complete disappearance of the peak at δ C /δ H = 115.7/6.97 ppm characteristic of the C5-H 5 guaiacyl units (G 5 ) with unsubstituted hydroxyl in the 4-position [34]. Based on the data obtained, it can be concluded that sulfation affects the acceptable aliphatic hydroxyl groups of lignin at the γ-positions of β-aryl ethers, α-ethoxylated β-aryl ethers, phenylcoumaran substructures, and cinnamyl alcohol end groups, as well as the unsubstituted hydroxyl groups in the α-position of β-aryl ethers. In addition, free phenolic hydroxyl groups in the 4-position of syringyl and guaiacyl units and p-coumarates can be subjected to sulfation.
The comparison of the 2D NMR spectroscopy data on birch and abies ethanol lignin sulfates [16] revealed higher structural diversity of the sulfated birch ethanol lignin. A significant difference between the HSQC spectra of birch ethanol lignin and abies ethanol lignin in the area of correlations of the aromatic fragments is the presence of several types of syringyl units in the former. However, both samples are built from the phenylpropane structural units linked by simple ether (β-O-4 ) and C-C (β-β , β-5 ) bonds, while the sulfate groups are localized mainly in the γ and α positions of the side chains and, probably, in the 4-position of aromatic rings.
Data on the molecular weight distribution of the initial and sulfated birch ethanol lignin were obtained by the GPC method. The molecular weight distribution curves for the initial and sulfated birch ethanol lignin samples are shown in Figure 12. The average molecular weights and polydispersity of the initial and sulfated ethanol lignins are indicated in Table 6. dicated in Table 6.
The birch ethanol lignin obtained in [35] had a low molecular weight (Mw = 1800 Da) and a monomodal distribution (PD = 2.02), which evidences higher homogeneity of the sample as compared to Alcell birch lignin (Mw = 3470 and PD = 4.1) [17].
As a result of birch ethanol lignin sulfation, the weight average molecular weight Mw of the samples increased from ~1800 to ~7600 Da. Such a significant growth is related to an increase in the weight of lignin macromolecules due to the introduction of sulfate groups and the removal of the low molecular weight fraction of the sulfated lignin along with inorganic impurities at the dialysis stage. A feature distinguishing sulfated birch ethanol lignin from abies ethanol lignin sulfated under similar conditions [16] is the bimodal molecular weight distribution ( Figure 12). The molecular weight distribution curve of birch ethanol lignin has two pronounced peaks with molecular weights of ~5000 and ~12,000 Da. These peaks can be attributed to the heterogeneity of the initial ethanol lignin molecules, which enter into the sulfation reaction in different ways. The low molecular weight lignin fraction is possibly less sulfated than the high molecular weight fraction, which is reflected in the separation of the peaks in the molecular weight distribution curve. Sulfated birch ethanol lignin has a higher polydispersity and a higher average molecular weight than sulfated abies ethanol lignin (Mw ~ 5300 Da, PD = 1.63) [16]. The birch ethanol lignin obtained in [35] had a low molecular weight (M w = 1800 Da) and a monomodal distribution (PD = 2.02), which evidences higher homogeneity of the sample as compared to Alcell birch lignin (M w = 3470 and PD = 4.1) [17].
As a result of birch ethanol lignin sulfation, the weight average molecular weight M w of the samples increased from~1800 to~7600 Da. Such a significant growth is related to an increase in the weight of lignin macromolecules due to the introduction of sulfate groups and the removal of the low molecular weight fraction of the sulfated lignin along with inorganic impurities at the dialysis stage. A feature distinguishing sulfated birch ethanol lignin from abies ethanol lignin sulfated under similar conditions [16] is the bimodal molecular weight distribution ( Figure 12). The molecular weight distribution curve of birch ethanol lignin has two pronounced peaks with molecular weights of~5000 and~12,000 Da. These peaks can be attributed to the heterogeneity of the initial ethanol lignin molecules, which enter into the sulfation reaction in different ways. The low molecular weight lignin fraction is possibly less sulfated than the high molecular weight fraction, which is reflected in the separation of the peaks in the molecular weight distribution curve. Sulfated birch ethanol lignin has a higher polydispersity and a higher average molecular weight than sulfated abies ethanol lignin (M w~5 300 Da, PD = 1.63) [16].

Thermochemical Properties of the Birch Ethanol Lignin
The thermochemical properties of the birch ethanol lignin were studied using the nonisothermal TG/DTG analysis in an argon medium in the temperature range of 30-900 • C.
The thermal decomposition of the ethanol lignin occurred over a wide temperature range, since its structure contains various functional groups with different thermal stabilities ( Figure 13). This is explained by the loss of moisture and adsorbed gases. The main thermal decomposition of the ethanol lignin started after 200 °C and practically ended at 600 °C. The solid residue yield gradually decreased with an increase in temperature to 700 °C and then remained constant invariable. At a pyrolysis temperature of 900 °C, the solid residue yield was 34.6 wt %, which is somewhat less than in the case of pyrolysis of the abies ethanol lignin under similar conditions (36.2 wt %) [36]. As is known [37], coniferous lignins consist mainly of the guaiacyl structures, while in hardwood lignins, the syringyl structures dominate. The high yield of the carbon residue during the thermal decomposition of abies ethanol lignin is probably due to the tendency of the guaiacyl propane units to condensation reactions [38]. The DTG curve has a broad peak corresponding to the main thermal decomposition of ethanol lignin and an implicit peak. The maximum rate of thermal degradation of the birch ethanol lignin (4.1%/min) was reached at 372 °C. In the temperature range of 350-400 °C, the main lignin structural moieties (guaiacyl and syringyl) underwent cracking with the formation of phenol-type compounds of different molecular weights, the yield of which increased with temperature [39].
At this temperature range, the pyrolysis products represent a complex mixture of organic compounds containing the aromatic, hydroxyl, and alkyl groups and reflecting the composition and structural features of the initial lignin [40]. During the thermal decomposition of the lignin, the competing depolymerization reactions with the formation of lower molecular weight aromatic products and cross-linking reactions of aromatic compounds and their carbonization occurred [40]. In the temperature range of 450-600 °C, the birch ethanol lignin weight loss rate significantly decreased and the thermal decomposition was mainly completed at 600 °C. In this case, some of the aromatic rings in the lignin probably decomposed and condensed into carbon products [41].
The sulfation of the birch ethanol lignin noticeably changed the nature of its thermal transformation ( Figure 14). The sample weight loss at temperatures of 30-180 • C was found to be less than 1%. This is explained by the loss of moisture and adsorbed gases. The main thermal decomposition of the ethanol lignin started after 200 • C and practically ended at 600 • C. The solid residue yield gradually decreased with an increase in temperature to 700 • C and then remained constant invariable. At a pyrolysis temperature of 900 • C, the solid residue yield was 34.6 wt %, which is somewhat less than in the case of pyrolysis of the abies ethanol lignin under similar conditions (36.2 wt %) [36]. As is known [37], coniferous lignins consist mainly of the guaiacyl structures, while in hardwood lignins, the syringyl structures dominate. The high yield of the carbon residue during the thermal decomposition of abies ethanol lignin is probably due to the tendency of the guaiacyl propane units to condensation reactions [38].
The DTG curve has a broad peak corresponding to the main thermal decomposition of ethanol lignin and an implicit peak. The maximum rate of thermal degradation of the birch ethanol lignin (4.1%/min) was reached at 372 • C. In the temperature range of 350-400 • C, the main lignin structural moieties (guaiacyl and syringyl) underwent cracking with the formation of phenol-type compounds of different molecular weights, the yield of which increased with temperature [39].
At this temperature range, the pyrolysis products represent a complex mixture of organic compounds containing the aromatic, hydroxyl, and alkyl groups and reflecting the composition and structural features of the initial lignin [40]. During the thermal decomposition of the lignin, the competing depolymerization reactions with the formation of lower molecular weight aromatic products and cross-linking reactions of aromatic compounds and their carbonization occurred [40]. In the temperature range of 450-600 • C, the birch ethanol lignin weight loss rate significantly decreased and the thermal decomposition was mainly completed at 600 • C. In this case, some of the aromatic rings in the lignin probably decomposed and condensed into carbon products [41].
The sulfation of the birch ethanol lignin noticeably changed the nature of its thermal transformation ( Figure 14). According to the data presented in Figure 14a, the sulfation of the birch ethanol lignin reduced its thermal stability. At temperature 300 °C, the sulfated ethanol lignin lost 26.4% of its initial weight, while the initial lignin lost only 15.7%. This tendency continued until the completion of the pyrolysis process.
The sulfation of the birch ethanol lignin also changed its thermal transformation profile (Figure 14b). In the temperature range of 100-150 °C, the sulfated ethanol lignin weight loss rate was much higher than in the case of initial ethanol lignin. As was shown in [42], in this temperature range, the aliphatic hydroxyl groups, carbonyl groups, and C-C bonds in the lignin side chains are broken.
In the temperature range of 200-350 °C, an intense narrow peak appeared in the DTG curve of the sulfated birch ethanol lignin, with a maximum weight loss rate of 6.7%/min at 317 °C, which is attributed to the thermal decomposition of sulfate groups [43].
Thus, the TG/DTG study showed that the syringyl structure of hardwood (birch) ethanol lignin was thermally less stable than the guaiacyl structure dominating in softwood (abies) ethanol lignin. Additionally, the introduction of sulfate groups into the structure of birch ethanol lignin reduced its thermal stability.

Materials
The silver birch (Betula pendula Roth) wood harvested in the vicinity of Krasnoyarsk city was used as a feedstock for the isolation of ethanol lignin. The contents of the main birch wood components (% of the absolutely dry wood weight) were 47.3 cellulose, 28.5 hemicelluloses, 19.0 lignin, 4.9 extractives, and 0.3 ash.

Ethanol Lignin Isolation
The ethanol lignin was separated from birch wood by extraction with an ethanolwater (60:40) mixture in a Rexo Engineering autoclave reactor (Rexo Engineering Co., Ltd., Seoul, Korea) with a capacity of 3000 mL at a temperature of 185 °C under a working pressure of 0.75 MPa for 3 h and subsequent precipitation with cold water using the technique described in [35].

Ethanol Lignin Sulfation
The obtained ethanol lignin was sulfated with sulfamic acid in 1,4-dioxane in the According to the data presented in Figure 14a, the sulfation of the birch ethanol lignin reduced its thermal stability. At temperature 300 • C, the sulfated ethanol lignin lost 26.4% of its initial weight, while the initial lignin lost only 15.7%. This tendency continued until the completion of the pyrolysis process.
The sulfation of the birch ethanol lignin also changed its thermal transformation profile (Figure 14b). In the temperature range of 100-150 • C, the sulfated ethanol lignin weight loss rate was much higher than in the case of initial ethanol lignin. As was shown in [42], in this temperature range, the aliphatic hydroxyl groups, carbonyl groups, and C-C bonds in the lignin side chains are broken.
In the temperature range of 200-350 • C, an intense narrow peak appeared in the DTG curve of the sulfated birch ethanol lignin, with a maximum weight loss rate of 6.7%/min at 317 • C, which is attributed to the thermal decomposition of sulfate groups [43].
Thus, the TG/DTG study showed that the syringyl structure of hardwood (birch) ethanol lignin was thermally less stable than the guaiacyl structure dominating in softwood (abies) ethanol lignin. Additionally, the introduction of sulfate groups into the structure of birch ethanol lignin reduced its thermal stability.

Materials
The silver birch (Betula pendula Roth) wood harvested in the vicinity of Krasnoyarsk city was used as a feedstock for the isolation of ethanol lignin. The contents of the main birch wood components (% of the absolutely dry wood weight) were 47.3 cellulose, 28.5 hemicelluloses, 19.0 lignin, 4.9 extractives, and 0.3 ash.

Ethanol Lignin Isolation
The ethanol lignin was separated from birch wood by extraction with an ethanol-water (60:40) mixture in a Rexo Engineering autoclave reactor (Rexo Engineering Co., Ltd., Seoul, Korea) with a capacity of 3000 mL at a temperature of 185 • C under a working pressure of 0.75 MPa for 3 h and subsequent precipitation with cold water using the technique described in [35].

Ethanol Lignin Sulfation
The obtained ethanol lignin was sulfated with sulfamic acid in 1,4-dioxane in the presence of urea using the procedure described in [16,44].
The sulfation of ethanol lignin was carried out in a three-neck flask (150 mL) equipped with a reflux condenser, a thermometer, and a mechanical stirrer at temperatures 70-100 • C.
The ethanol lignin (1.25 g) was added to a mixture of sulfamic acid and urea (mol. ratio 1:1) in 15 mL of 1,4-dioxane. The mixture was intensively stirred for 30-180 min and cooled to room temperature. The solvent was decanted, and the remaining solid product was dissolved in a small amount of water and neutralized with aqueous ammonia to pH 8. To remove the excess reactants, the product was dialyzed against water in a plastic bag of MF-503-46 MFPI brand (USA) with a pore size of 3.5 kDa for 8-10 h. After dialysis, the aqueous solution of sulfated lignin was evaporated with the use of a rotary evaporator to obtain a solid residue-sulfated lignin in the form of an ammonium salt.
The sulfation process of the birch ethanol lignin with sulfamic acid was numerically optimized using the Statgraphics Centurion XVI software, DOE (Design of Experiment) block by the method described in [16].
The estimated sulfated ethanol lignin weight was calculated on the basis of the sulfur content [21] as The sulfated product Yield (%) was determined as where m calc is the calculated sulfated ethanol lignin weight (g), m is the weight of the initial ethanol lignin sample (g), m actual is the sulfated ethanol lignin weight (g), and S is the sulfur content in the sulfated ethanol lignin (%).

Elemental Analysis
The elemental analysis of the sulfated ethanol lignin was carried out using a Thermo-Quest FlashEA-1112 analyzer (Milan, Italy).

FTIR Analysis
The FTIR spectra of the initial and sulfated ethanol lignin were recorded using a Shimadzu IRTracer-100 Fourier transform IR spectrophotometer (Tokyo, Japan) in the wavelength range of 400-4000 cm −1 . The spectral data were processed using the OPUS software (version 5.0). The solid samples for the analysis were tablets in a KBr matrix (2 mg of the sample/1000 mg of KBr).

NMR Analysis
The 2D NMR spectra were recorded at 25 • C in 5-mm ampoules using a Bruker Avance III 600 NMR spectrometer (Billerica, MA, USA) at working frequencies of 600 ( 1 H) and 150 MHz ( 13 C). Approximately 80 mg of lignin was dissolved in 0.6 mL of deuterated dimethyl sulfoxide, and then the spectra were recorded in the heteronuclear single quantum correlation (HSQC) experiments with editing (HSQCed) using the Bruker standard sequence library. The solvent signal was used as an internal standard (δ C 40.1 and δ H 2.5).

Gel Permeation Chromatography
The number average molecular weight M n , weight average molecular weight M w , and polydispersity index of the initial and sulfated ethanol lignin samples were determined by gel permeation chromatography (GPC) using an Agilent 1260 Infinity II Multi-Detector GPC/SEC System with triple detection: refractometer (RI), viscometer (VS), and light scattering (LS). The water-soluble samples were separated on two combined PL Aquagel-OH Mixed-M columns using 0.1 M NaNO 3 as the mobile phase. The tetrahydrofuran (THF)soluble samples were separated on a PLgel 10 µm MIXED-E column with the THF mobile phase stabilized with 250 ppm of butylhydroxytoluene. The columns were calibrated using the polyethylene glycol and polystyrene polydisperse standards (Agilent, Santa Clara, CA, USA), respectively. The eluent flow rate was 1 mL/min, and the injected sample volume was 100 µL. Before the analysis, the ethanol lignin and sulfated ethanol lignin samples were dissolved in THF and water (5 mg/mL), respectively; after that, they were filtered through a 0.45-µm Millipore PTFE membrane filter. The data were collected and processed using the Agilent GPC/SEC MDS software.

Thermogravimetric Analysis
The thermogravimetry analysis was carried out using a Netzsch STA 449 F1 Jupiter instrument (Waldkraiburg, Germany). The thermal degradation of the lignin samples was studied in argon in the temperature range from 298 to 1173 K. The samples were heated in the dynamic mode at a heating rate of 10 • C/min in corundum crucibles. The measured data were processed using the Netzsch Proteus Thermal Analysis.5.1.0 software package supplied with the instrument.

Kinetic Calculations
The kinetics of the process of birch ethanol lignin sulfation was studied in the temperature range of 70-100 • C. The apparent initial rates and rate constants of the sulfation reaction were calculated from the change in the sulfur content in the sulfated ethanol lignin. The calculation was carried out according to the first-order equation: where V is the rate of the sulfation reaction, wt %/s; k is the rate constant of the sulfation reaction, 1/s; dS is the change in the sulfur content in birch ethanol lignin sulfate, wt %; and dt is the change in time, s. The activation energy was found by the tangent of the slope of the dependence of ln k on 11/T.

Conclusions
As a result of the accomplished study, the main regularities of the process of birch ethanol lignin sulfation with a sulfamic acid-urea mixture in a 1,4-dioxane medium at temperatures of 70-110 • C were established and the sulfating products were characterized using chemical and physical analysis methods.
It was found that similar to the sulfation of abies ethanol lignin under the same conditions [16], the process was complicated by diffusion restrictions due to the increased viscosity of the reaction medium. In the case of excess sulfating agent, the main factors affecting the yield of the sulfated product and sulfur content were the temperature and the duration of the process. Using experimental and computational methods, the optimal conditions for the process of birch ethanol lignin sulfation with a sulfamic acid-urea mixture to provide a high yield of sulfated product (more than 96 wt %) with a sulfur content of 8.1 wt % were established. As in the case of abies ethanol lignin, the sulfation increased the molecular weight of the birch ethanol lignin from 1800 Da to 7600 Da and decreased the polydispersity from 2.02 to 1.81. Moreover, aliphatic hydroxyl groups were more easily sulfated.
Some differences in the sulfation of ethanol lignins of birch and abies were established due to the presence of phenylpropane units of different compositions within these lignins. The sulfation of birch ethanol lignin in which syringyl structures predominate proceeds more completely at moderate temperatures than the abies ethanol lignin with guaiacyl structure. Additionally, in contrast to sulfated abies ethanol lignin, the sulfated birch ethanol lignin had a bimodal and wider molecular weight distribution, as well as less thermal stability.
The sulfated birch ethanol lignin has prospects for use in the production of new sorbents, biocomposites, and nanomaterials, as well as in the development of new anticoagulant and antiviral drugs [12,13,45].