Characterization of Lignin Extracted from Willow by Deep Eutectic Solvent Treatments

Purity, morphology, and structural characterization of synthesized deep eutectic solvent (DES)-lignins (D6h, D9h, D12h, D18h, D24h) extracted from willow (Salix matsudana cv. Zhuliu) after treatment with a 1:10 molar ratio of choline chloride and lactic acid at 120 °C for 6, 9, 12, 18, and 24 h were carried out. The purity of DES-lignin was ~95.4%. The proportion of hydrogen (H) in DES-lignin samples increased from 4.22% to 6.90% with lignin extraction time. The DES-lignin samples had low number/weight average molecular weights (1348.1/1806.7 to 920.2/1042.5 g/mol, from D6h to D24h) and low particle sizes (702–400 nm). Atomic force microscopy (AFM) analysis demonstrated that DES-lignin nanoparticles had smooth surfaces and diameters of 200–420 nm. Syringyl (S) units were dominant, and total phenolic hydroxyl content and total hydroxyl content reached their highest values of 2.05 and 3.42 mmol·g−1 in D12h and D6h, respectively. β-Aryl ether (β-O-4) linkages were eliminated during DES treatment.


Introduction
As a substitute for fossil energy, biomass-based energy has received significant attention over the last decade [1,2]. Willow (Salix matsudana cv. Zhuliu), among the various biomass resources of interest, is a new hybrid plant variety (Salicaceae Salix spp.) that displays strong resistance and high survival rate. It is widely planted in desert areas for wind prevention and sand fixation. Moreover, particularly in China with scarce timber resources, it has great potential in pulp and papermaking operations, due to its fast-growing nature [3].
Lignin is one of the three components (cellulose, hemicellulose, and lignin) that make up biomass, and the only fully aromatic polymer found in nature. It is a bio-based raw material attracting intense research in conversion and modification, despite its complex structure [4][5][6]. The effective separation of lignin from lignocellulosic biomass, nonetheless, is key to subsequent utilization. A variety of isolation technologies have been developed to effect its isolation such as alkaline, enzymatic, and dilute acid hydrolysis, organosolv, ionic liquids, and deep eutectic solvent extraction [7][8][9][10][11][12][13].
Deep eutectic solvents (DESs) are a fundamentally new paradigm in the chemical sciences. Because of their highly tunable nature and exceptional properties, DESs have become essential tools within the fields of extraction, analytics, electrochemistry, biotechnology, catalysis, and synthesis amongst various disciplines [14][15][16]. Over the last decade, global research efforts have explored the A deep eutectic solvent (DES) composed of choline chloride and lactic acid (molar ratio 1:10) was applied for isolating lignin from willow. Raw material was first extracted with organic solvents (v/v, benzene to 95% ethanol = 2/1) for 8 h to remove extracts. The extracted sample (2.5 g) was added to a round-bottomed flask containing DES (m/m of raw material to DES = 1 to 30). The mixture of willow sample and DES was heated to 120 • C by an oil bath with magnetic stirring, and treatment temperatures (6-24 h) effects were studied.
After treatment, the DES soluble fractions and solid residues were separated by filtration using a G2 glass crucible. The solid residues were thoroughly washed with anhydrous ethanol. The filtrate was combined and concentrated to recover ethanol, and was then added to deionized water (2000 mL) to precipitate lignin. After 24 h, the precipitate was collected after centrifugation, and washed with an ethanol/water mixture (1:9, v/v). The DES-lignin or the lignin extracted from willow by DES treatment, was obtained by vacuum freeze-drying [25]. The isolated DES-lignin samples were labeled D 6h , D 9h , D 12h , D 18h , D 24h (the DES-lignin extracted from willow by DES treatment for 6,9,12,18, and 24 h).

Willow Enzymatic/Mild Acidolysis Lignin (EMAL) Preparation
The 40 to 60 mesh fractions of willow after acetone extraction for 48 h were used as raw material for the lignin separation. The dried raw material was placed in a roller ball mill (PM200, Retsch, Düsseldorf, Germany) for 72 h at a rotation frequency of 500 rpm. The ball-milled raw material was used for the preparation of EMAL after being subjected to ethanol and benzene extraction for 8 h. The preparation of EMAL was based on past research [3].

Purity Analysis of DES-lignin
The chemical composition of the DES-lignin samples was analyzed by NREL methods [27], and the purity of DES-lignin was calculated from the sum of acid insoluble lignin (%) and acid soluble lignin (%).

Elemental Analysis of DES-lignin
The carbon (C), hydrogen (H), nitrogen (N) contents (wt %) in the DES-lignin samples were analyzed by the elemental analyzer (Vario EL III, Elementar, Frankfurt, Hesse, Germany). The oxygen content (wt %) was determined by difference.

Molecular Weight Distribution Analysis of DES-lignin and EMAL
The molecular weight distribution of the DES-lignin and EMAL samples were identified using liquid gel permeation chromatography (GPC) 1200 (Agilent Technologies Inc., Palo Alto, CA, USA). Ten milligrams of each sample were completely dissolved in 10 mL high-performance liquid chromatography-grade THF, and 20 µL of resulting solutions were injected into the column. The eluent (THF) flow rate was kept as 1.0 mL/min, and the temperature was maintained at 30 • C. A set of polystyrene standards (200 to 30,000 Da, Aldrich, Shanghai, China) were used for calibration.

Particle Size Analysis of DES-lignin
A nanoparticle size analyzer (Zetasizer Nano S90, Malvern Instruments Ltd., London, UK) was used to measure the particle size of DES-lignin samples. Samples (10 mg) were dissolved in 10 mL THF with ultrasound, from which a 1 mL solution was added to a quartz cuvette for analysis. The sample was circulated three times for a total of 10 min to identify the average size for each sample.

Atomic Force Microscope (AFM) Analysis of DES-lignin
The DES-lignin sample was completely dissolved in acetone at a concentration of 0.1 g per liter, and a drop of diluted DES-lignin suspension was deposited onto a clean mica surface and air-dried overnight in ambient environment before examining the DES-lignin morphologies using atomic force microscope (AFM) imaging (AFM Workshop). The morphologies of DES-lignin sample were analyzed using the NanoScope analysis software (Bruker, Karlsruhe, Germany) with the AFM system. The resolution in the X-axis and Y-axis direction is 1 nm, and the resolution in the Z-axis direction is 0.1 nm.

2D-HSQC NMR Analysis of DES-Lignin and EMAL
Approximately 80 mg samples and 500 µL of dimethyl sulfoxide (DMSO)-d 6 were stirred at 25 • C for 30 min to obtain a solution. The solution was then analyzed by using an ADVANCE III 600 MHz spectrometer (Bruker, Karlsruhe, Germany) for 16 h. Matrices of 1024 data points for the 13 C dimension and 2048 data points for the 1 H dimension were collected from 160 to 0 ppm and 13 to −1 ppm for the 13 C and 1 H dimensions, respectively. Relaxation delay was set at 6 s.

Purity Analysis
To investigate the purity of DES-lignin samples (D 6h , D 9h , D 12h , D 18h , D 24h ), the acid soluble lignin (ASL), acid insoluble lignin (AIL), sugars, and ash content in DES-lignin samples were measured ( Table 1). The purity of all lignin samples was >90%, and with extension of treatment time, the purity increased from 90.0% to 95.4%. This showed that 1.12% of glucose, 0.96% of xylose, 0.12% arabinose and 0.05% mannose were present in D 6h , which may be attributed to the presence of LCCs (lignin-carbohydrate complexes) from the plant cell wall [28][29][30]. It was interesting that only 0.21% of glucose and 0.15% of xylose were present in D 12h , suggesting that less carbohydrate remained in the DES-lignin at an extraction time of 12 h relative to 6 h, which may be the reason for the increased purity. The monosaccharides were not detected in the acid hydrolysis of D 24h , indicating that the residual carbohydrates in the separated lignin were negligible when extraction time was >24 h.

Element Analysis
The elemental compositions of the DES-lignin samples (D 6h , D 9h , D 12h , D 18h , D 24h ) from DES treatments are listed in Table 2. DES-lignin had a high carbon content (58.4-60.0%), and negligible amount of nitrogen (0.39-0.45%). With extraction time, the content of oxygen in the separated DES-lignin samples gradually decreased from 35.87% in D 6h to 33.26% in D 24h . The hydrogen content increased from 4.22% in D 6h to 6.90% in D 24 , which may mean that the oxygen-containing functional groups or linkages, such as methoxy group and aryl ether bond in willow lignin, were eliminated during DES treatment. It has been previously proposed that both the cleavage of ether bonds and the solvent properties of DES played an important role on the depolymerization of lignin, which facilitated its separation from wood [23]. DES may provide a mild acid-base catalysis mechanism that would favorable for cleavage of labile ether linkages, and thus lead to generation of a low molecular weight lignin fragment [31]. This chemistry may affect the elemental composition of the isolated lignin.

Molecular Weight Distribution
The change of number average molecular weight (Mn) and weight average molecular weight Mw distribution of DES-lignin samples (D 6h , D 9h , D 12h , D 18h , D 24h ) are shown in Figure 1. In addition, the values of the Mn and Mw from the GPC curves and the polydispersity index (Mw/Mn) are listed in Table 3. Compared with EMAL and lignin extracted by other methods, the number average and weight average molecular weight of DES-lignin were significantly reduced [3,32,33]. The Mw and Mn of DES-lignin samples extracted from willow by DES treatment at 120 • C at different times decreased from 1806.7 and 1348.1 g/mol of D 6h to 1042.5 and 920.2 g/mol of D 24h , which indicated that the average molecular weight of DES-lignin gradually decreased with increase of DES treatment time. The above results may be due to elimination of intermolecular linkages of lignin under DES, and with increase of time, the degradation of the lignin macromolecule was more significant, similar to traditional ionic liquids [10,34].

Molecular Weight Distribution
The change of number average molecular weight ( n and weight average molecular weight w distribution of DES-lignin samples (D6h, D9h, D12h, D18h, D24h) are shown in Figure 1. In addition, the values of the n and w from the GPC curves and the polydispersity index ( w / n ) are listed in Table 3. Compared with EMAL and lignin extracted by other methods, the number average and weight average molecular weight of DES-lignin were significantly reduced [3,32,33]. The w and n of DES-lignin samples extracted from willow by DES treatment at 120 °C at different times decreased from 1806.7 and 1348.1 g/mol of D6h to 1042.5 and 920.2 g/mol of D24h, which indicated that the average molecular weight of DES-lignin gradually decreased with increase of DES treatment time.
The above results may be due to elimination of intermolecular linkages of lignin under DES, and with increase of time, the degradation of the lignin macromolecule was more significant, similar to traditional ionic liquids [10,34]. The polydispersity of DES-lignin samples and EMAL was calculated. It can be seen from Table  3 that the polydispersity value of DES-lignin samples decreased from 1.34 of D6h to 1.13 of D24h, which indicated that the distribution of DES-lignin gradually concentrated with treatment time. The polydispersity of the lignin extracted from willow by DES treatment was lower than EMAL when the DES treatment time was >9 h.    The polydispersity of DES-lignin samples and EMAL was calculated. It can be seen from Table 3 that the polydispersity value of DES-lignin samples decreased from 1.34 of D 6h to 1.13 of D 24h , which indicated that the distribution of DES-lignin gradually concentrated with treatment time. The polydispersity of the lignin extracted from willow by DES treatment was lower than EMAL when the DES treatment time was >9 h.

Particle Size Analysis
The lignin particle sizes by DES treatment are shown in Table 4. The particle size of all the DES-lignin samples were lower than 1 µm and the particle size of DES-lignin samples decreased substantially from 704 to 400 nm with extraction time from 6 to 18 h. However, D 18h and D 24h had nearly the same particle size (~400 nm), which suggested that it was difficult to further reduce lignin particle size with more treatment time.

AFM Analysis of DES-lignin
The topic of lignin nanoparticles for green biobased functional materials is becoming more and more appealing [35,36]. Morphological regularity and small particle sizes are the key criteria for the successful preparation of lignin-based functional materials. To investigate the surface morphology of DES-lignin, the DES-lignin sample (D 12h ) was dissolved in acetone at a concentration of 0.1 g per liter, after which a drop of liquid was air-dried and examined on clean mica by atomic force microscopy (AFM). Figure 2 confirmed that nanoscale lignin particles formed by self-assembly of dissolved lignin after precipitation via dilution. Aggregated particle indicated by multiple peaks in profile in Figure 2B, corresponding to the line in Figure 2A with diameters between 200 nm to 420 nm, were observed. There were broad peaks in the smooth curve of Figure 2B, which indicated that the surface of DES-lignin nanoparticles was relatively smooth.

Particle Size Analysis
The lignin particle sizes by DES treatment are shown in Table 4. The particle size of all the DESlignin samples were lower than 1 μm and the particle size of DES-lignin samples decreased substantially from 704 to 400 nm with extraction time from 6 to 18 h. However, D18h and D24h had nearly the same particle size (~400 nm), which suggested that it was difficult to further reduce lignin particle size with more treatment time.

AFM Analysis of DES-lignin
The topic of lignin nanoparticles for green biobased functional materials is becoming more and more appealing [35,36]. Morphological regularity and small particle sizes are the key criteria for the successful preparation of lignin-based functional materials. To investigate the surface morphology of DES-lignin, the DES-lignin sample (D12h) was dissolved in acetone at a concentration of 0.1 g per liter, after which a drop of liquid was air-dried and examined on clean mica by atomic force microscopy (AFM). Figure 2 confirmed that nanoscale lignin particles formed by self-assembly of dissolved lignin after precipitation via dilution. Aggregated particle indicated by multiple peaks in profile in Figure  2B, corresponding to the line in Figure 2A with diameters between 200 nm to 420 nm, were observed. There were broad peaks in the smooth curve of Figure 2B, which indicated that the surface of DESlignin nanoparticles was relatively smooth.  31 P NMR can be used to quantitate hydroxyl groups on lignin [37,38]. In Figure 3, lignin extracted from willow by DES treatment is GSH (containing guaiacyl, syringyl, and p-hydroxyphenyl units) type lignin, and the syringyl units were dominant, consistent with the structure of hardwood lignin [39,40]. The content of the guaiacyl phenol hydroxyls (G-OH) and p-phenol hydroxyl (H-OH) of D12h was highest compared to D6h, D18h, and D24h. The content of the syringyl phenol hydroxyl (S-OH) decreased from 0.82 mmol·g −1 in D6h to 0.65 mmol·g −1 in D24h, indicating the content of S-OH in DESlignin extracted by DES gradually decreased with treatment time (see Table 5). Generally, the content of total phenol hydroxyl reached its highest value for D12h, and the content of total hydroxy groups in D6h was the highest mainly due to the presence of aliphatic hydroxy (A-OH) in D6h relative to other DES-lignin samples. Interestingly, the content of A-OH in the extracted DES-lignin samples decreased from 1.46 mmol·g −1 in D6h to 0.93 mmol·g −1 in D12h when the extraction times of DES-lignin 3.6. 31 P NMR Analysis of DES-lignin 31 P NMR can be used to quantitate hydroxyl groups on lignin [37,38]. In Figure 3, lignin extracted from willow by DES treatment is GSH (containing guaiacyl, syringyl, and p-hydroxyphenyl units) type lignin, and the syringyl units were dominant, consistent with the structure of hardwood lignin [39,40]. The content of the guaiacyl phenol hydroxyls (G-OH) and p-phenol hydroxyl (H-OH) of D 12h was highest compared to D 6h , D 18h , and D 24h . The content of the syringyl phenol hydroxyl (S-OH) decreased from 0.82 mmol·g −1 in D 6h to 0.65 mmol·g −1 in D 24h , indicating the content of S-OH in DES-lignin extracted by DES gradually decreased with treatment time (see Table 5). Generally, the content of total phenol hydroxyl reached its highest value for D 12h , and the content of total hydroxy groups in D 6h was the highest mainly due to the presence of aliphatic hydroxy (A-OH) in D 6h relative to other DES-lignin samples. Interestingly, the content of A-OH in the extracted DES-lignin samples decreased from 1.46 mmol·g −1 in D 6h to 0.93 mmol·g −1 in D 12h when the extraction times of DES-lignin increased from 6 to 24 h, which may be due to gradual decrease of the long carbon chains in the separated lignin and the carbohydrates attached to the lignin under the treatment of DES for a longer period of time [41,42]. It can also be seen from Figure 3 that the content of the carboxyl (COOH) in the extracted lignin was significantly reduced when the extraction time was extended to 18 h.

31 P NMR Analysis of DES-lignin
Polymers 2018, 10, x FOR PEER REVIEW 7 of 11 separated lignin and the carbohydrates attached to the lignin under the treatment of DES for a longer period of time [41,42]. It can also be seen from Figure 3 that the content of the carboxyl (COOH) in the extracted lignin was significantly reduced when the extraction time was extended to 18 h.

2D-HSQC NMR Spectra of DES-lignin and EMAL
To investigate the structural characteristics of DES-lignin, 2D-HSQC NMR for DES-lignin sample (D12h) extracted from willow by DES-treatment at 120 °C for 12 h and the EMAL of willow were obtained (Figure 4). Assignment of the peaks in the 2D-HSQC NMR spectra was conducted based on previous reports [32,[43][44][45]. The peaks corresponding to methoxyl (3.81/56.7 parts per million (ppm)) and β-aryl ether are shown for Aα (

Conclusions
High purity (90-95.4%) lignin can be obtained from willow (Salix matsudana cv. Zhuliu) after DES (the molar ratio of choline chloride to lactic acid was 1 to 10) treatment. The elemental composition of DES-lignin varies with the extraction time. The DES-lignin had low molecular weights and low particle sizes, and were reduced with extension of the extraction time. Atomic force microscopy (AFM) analysis results demonstrated that the lignin nanoparticles extracted from willow by DES treatment had smooth surfaces and diameters of 200-420 nm. DES-lignin is discernible as