*Article* **Physical and Chemical Properties of** *Acacia mangium* **Lignin Isolated from Pulp Mill Byproduct for Potential Application in Wood Composites**

**Nissa Nurfajrin Solihat 1,\* , Eko Budi Santoso <sup>2</sup> , Azizatul Karimah 1,2, Elvara Windra Madyaratri <sup>2</sup> , Fahriya Puspita Sari <sup>1</sup> , Faizatul Falah <sup>1</sup> , Apri Heri Iswanto 3,4 , Maya Ismayati <sup>1</sup> , Muhammad Adly Rahandi Lubis <sup>1</sup> , Widya Fatriasari 1,\* , Petar Antov <sup>5</sup> , Viktor Savov <sup>5</sup> , Milada Gajtanska 6,\* and Wasrin Syafii <sup>2</sup>**


**Abstract:** The efficient isolation process and understanding of lignin properties are essential to determine key features and insights for more effective lignin valorization as a renewable feedstock for the production of bio-based chemicals including wood adhesives. This study successfully used dilute acid precipitation to recover lignin from black liquor (BL) through a single-step and ethanolfractionated-step, with a lignin recovery of ~35% and ~16%, respectively. The physical characteristics of lignin, i.e., its morphological structure, were evaluated by scanning electron microscopy (SEM). The chemical properties of the isolated lignin were characterized using comprehensive analytical techniques such as chemical composition, solubility test, morphological structure, Fourier-transform infrared spectroscopy (FTIR), <sup>1</sup>H and <sup>13</sup>C Nuclear Magnetic Resonance (NMR), elucidation structure by pyrolysis-gas chromatography-mass spectroscopy (Py-GCMS), and gel permeation chromatography (GPC). The fingerprint analysis by FTIR detected the unique peaks corresponding to lignin, such as C=C and C-O in aromatic rings, but no significant differences in the fingerprint result between both lignin. The <sup>1</sup>H and <sup>13</sup>C NMR showed unique signals related to functional groups in lignin molecules such as methoxy, aromatic protons, aldehyde, and carboxylic acid. The lower insoluble acid content of lignin derived from fractionated-step (69.94%) than single-step (77.45%) correlated to lignin yield, total phenolic content, solubility, thermal stability, and molecular distribution. It contradicted the syringyl/guaiacyl (S/G) units' ratio where ethanol fractionation slightly increased syringyl unit content, increasing the S/G ratio. Hence, the fractionation step affected more rupture and pores on the lignin morphological surface than the ethanol-fractionated step. The interrelationships between these chemical and physicochemical as well as different isolation methods were investigated. The results obtained could enhance the wider industrial application of lignin in manufacturing wood-based composites with improved properties and lower environmental impact.

**Keywords:** acid precipitation; single and fractionation step; kraft lignin; physical and chemical properties; *A. mangium* black liquor

**Citation:** Solihat, N.N.; Santoso, E.B.; Karimah, A.; Madyaratri, E.W.; Sari, F.P.; Falah, F.; Iswanto, A.H.; Ismayati, M.; Lubis, M.A.R.; Fatriasari, W.; et al. Physical and Chemical Properties of *Acacia mangium* Lignin Isolated from Pulp Mill Byproduct for Potential Application in Wood Composites. *Polymers* **2022**, *14*, 491. https:// doi.org/10.3390/polym14030491

Academic Editor: Nathanael Guigo

Received: 22 December 2021 Accepted: 24 January 2022 Published: 26 January 2022

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**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

#### **1. Introduction**

In 2019, Indonesia was ranked among the top 10 countries concerning pulp and paper production. In 2018, Indonesia produced 16 million tons of paper and 11 million tons of pulp [1]. For every 1 ton of pulp produced, about 7 tons of black liquor (BL) were generated as a residue at 15% solids by weight, with two-thirds of the solids consisting of organic chemicals, and the remains were inorganic chemicals [2]. In recent decades, lignin derived from BL has been considered a natural biopolymer, a viable alternative to the fossil-based chemicals due to its abundance in BL, reaching 45% dry weight [3].

Most BL is incinerated for boiler heating sources and energy, and only 5% of the BL is used for value-added applications [4]. The economical consideration of lignin isolation from BL includes recovery yield, purification, non-uniform structure, and unique reactivity [5]. The three main phenolic hydroxyl precursors in lignin are coniferyl alcohol (G), p-coumaryl alcohol (H), and sinapyl alcohol (S), which are linked to each other mostly by aryl ether linkage (β-O-40 ) [6,7]. The actual properties of lignin, such as thermal stability, reactivity, molecular distribution, and solubility, depend on the ratio of these aromatic units. It varies depending on the technique of extraction and the plant source. For instance, softwoods contain mostly G units; hardwoods include both S and G, while non-wood plants have all three units [8].

Precipitation by dilute acid such as sulphuric acid is a common and feasible technique to isolate lignin from BL [9]. However, using sulphuric acid can increase lignin's ash and sulfur content. Therefore, lignin for sulfur-sensitive utilizations should be restricted [5]. Haz et al. evaluated the effect of four different dilute acids (chloric, sulphuric, acetic, and nitric) on lignin properties. Lignin precipitated by nitric and chloric acid obtained high phenolic hydroxyl both in non-conjugated and conjugated forms (>2 mmol/g), suitable for phenolic polycondensates production and rubber stabilizer [10]. Handika et al. [11] reported that high free-phenolic hydroxyl in lignin increased its reactivity to produce the high-thermal stability of polyurethane resin for textile application. As a polyphenol molecule, lignin contains high free-phenolic hydroxyl groups that are favorable for modifications, such as phenolation and methylolation [12], tailored for increasing its chemical reactivity to formaldehyde in formaldehyde-based resins used in the production of wood composites such as particleboards [13], oriented strand boards [14], and flame-retardant composites [15]. Besides, lignin modification either with poly(butylene succinate) or polypropylene biocomposites increased the thermal stability of kenaf core fiber [16,17]. According to Tejado et al., kraft lignin is suitable for phenol-formaldehyde resin due to its higher amount of free phenolic content, molecular weight, and thermal properties [18]. Therefore, it is necessary to understand the specific chemical structure of lignin to achieve optimal utilization. However, the lack of understanding of the lignin structure-property– application relationship (SPARs) is a major roadblock to further development of lignin [19]. Of these, one characterization technique is insufficient to produce coherent data to identify the feature of lignin because of its complex structure and variation. Therefore, a comprehensive analytical technique is pivotal to understanding the properties of isolated lignin, allowing its large-scale utilization.

This study emphasizes the efficient isolation method of lignin from industrial residues of the pulp and paper industry. Our fundamental comprehensive analytical technique provides knowledge for industries to produce superior lignin-based, value-added products, especially for wood composites. Lignin was isolated by two different methods of dilute chloric acid precipitation. The physical characteristics of lignin, such as morphological structure, were conducted by scanning electron microscopy (SEM). Chemical features, including its total hydroxyl phenolic content and solubility in the organic and base solvent, were determined by ultraviolet-visible spectrophotometer (UV-Vis). The functional group was identified by attenuated total reflection Fourier-transform infrared (ATR-FTIR). <sup>1</sup>H and <sup>13</sup>C nuclear magnetic resonance (NMR) were employed to predict structural properties of lignin corresponding to its fingerprint signal. The elucidation aromatic precursors unit in lignin structure was analyzed by pyrolysis-gas chromatography-mass spectrometry

(PyGC/MS). Meanwhile, the thermal features of lignin were characterized by thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC). Gel permeation chromatography (GPC) was used to measure the molecular distribution of lignin.

#### **2. Materials and Methods**

#### *2.1. Material*

A derived BL from *Acacia mangium* was collected from a pulp and paper mill factory in Sumatra, Indonesia. Hydrochloric acid (HCl), dioxane, sodium hydroxide (NaOH), acetic acid anhydride, dimethyl sulfoxide (DMSO), and tetrahydrofuran (THF) were purchased from Merck (Darmstadt, Germany), while pyridine was obtained from Wako Pure Chemical Industries (Osaka, Japan). Lignin alkali (kraft) from Sigma-Aldrich (Saint Louis, MO, USA) was used for the lignin standard. All chemical materials used in this study were analytical grade without any purification.

#### *2.2. Lignin Isolation*

Dilute acid precipitation was used to isolate lignin from BL through a single-step and fractionated-step based on Hermiati et al. [20] with one major modification. For the single step, HCl 1 M was poured into BL until pH 2. The solution was kept overnight and the residue was separated by decantation. The deionized (DI) water was added to the residue with similar acid volume, and the decantation process was conducted again after 24 h. This process was repeated six times. The residue was kept in the refrigerator overnight and separated by vacuum filtration. Wet lignin on the filter paper was washed with DI water and dried in an oven at 45 ◦C for 24 h. The lignin yield percentage was measured by dividing the dry weight of lignin (g) by the dry weight of BL. Dried lignin was kept in sealed plastic for further analysis.

For fractionated-step, HCl 1 M was added into BL until pH 7. Ethanol, as much as four times the volume of the acid, was added to the solution. Non-lignin components such as sugar and carbohydrates were filtrated as a residue. The filtrate was evaporated until the ethanol completely dried up. The acidification was continued by adding acid until pH 2. Lignin precipitate was separated without decantation six times by water, unlike the single-step. The following step is similar to the procedure from the single-step method.

#### *2.3. Chemical Features Measurement: Chemical Component, Total Phenolic Hydroxyl, and Solubility*

The water content of lignin was determined according to TAPPI T 264 cm-97 [21], and ash content was calculated following the TAPPI T211 om-02 method [22]. Contents of acid-insoluble lignin (AIL) and acid-soluble lignin (ASL) were analyzed based on the method of Sluiter et al. (NREL/TP-510-42618) [23]. Triplicate analysis was performed for all chemical features measurements. Lignin alkali (kraft) from Sigma-Aldrich (370958) (Saint Louis, MO, USA) was used as a reference.

Total phenolic hydroxyl (phOH) was determined by the UV-Vis method [24]. A total of 1 mg/mL of lignin was diluted in dioxane: 0.2 M NaOH (1:1), and the mixture was filtered by microfiltration (0.45 µm). The filtrate was diluted in 0.2 M NaOH until the 0.08 mg/mL concentration was reached. The UV spectrum was recorded in a 200–600 nm range by Shimadzu UV vis-1800 spectrophotometer, where lignin in pH6 was used as reference. The absorbance of maximum spectra at 300 and 350 nm was used to calculate total phOH by the following equation:

$$\text{Total phOH (mmol/g)} = (0.425 \times A\_{300 \text{ nm}}) + (0.812 \times A\_{350 \text{ nm}}) \times \frac{1}{c \times a} \times \frac{10}{17}$$

where *A* is absorbance, *c* stands for lignin concentration, and *a* is path length (1 cm) [24].

The solubility test of lignin in the base and the organic solvent was conducted according to the method by Hermiati et al. Lignin 7 mg/5 mL was dissolved in NaOH pH 12 as the alkali solvent and mixture of dioxane water (9:1). Each solution was diluted 50 times by DI water. The UV spectrum was measured in the range of wavelength 200–400 nm [20].

## *2.4. Morphological Assessment by SEM*

A scanning electron microscope (JSM-IT200, JEOL, Tokyo, Japan) was used to observe morphological surfaces and particle size of the reference lignin and the isolated lignins. Lignin samples were placed on the carbon tube, and the surface was coated with gold using Ion Coater iB2. The micrograph of the sample was recorded at 200 and 5000 magnifications under a high vacuum and working distance of 11 mm with 5.0 kV accelerating voltage.

#### *2.5. Functional Group Analysis by UATR-FTIR*

Attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopic equipped with UATR unit cell from PerkinElmer (spectrum two) (PerkinElmer Corporation, Waltham, MA, USA) was employed to investigate the functional group of lignin. The sample was placed on the diamond crystal, and the spectrum at a wavelength of 400–500 cm−<sup>1</sup> was taken by pressing the torque knob with the same pressure. An average of 32 scans with 4 cm−<sup>1</sup> resolution were used to acquire the spectrum. The same average scanning was carried out for background correction and scanning before analysis.

#### *2.6. Fingerprint Observation by 1H and 1C NMR*

Lignin samples were acetylated before undergoing nuclear magnetic resonance (NMR) and molecular weight distribution test based on Wen et al. [25] method with a minor adjustment. A total of 200 mg lignin was dissolved in an 8 mL mixture of acetic acid anhydride: pyridine (1:1) for 72 h in the dark bottle. Ethanol was added until the mixture was concentrated. Acetylated lignin was obtained by slowly dropping the mixture into ice acid (pH 2) and separated through centrifugation. The wet acetylated lignin was washed with 50 mL DI water three times and freeze-dried until dry acetylated-lignin (AL) was obtained.

Each 20 mg AL sample was diluted in 0.7 mL DMSO. The solution was transferred to a 3 mm tube. <sup>1</sup>H NMR (JEOL JNM-ECZR 500, Tokyo, Japan) data points were acquired with an acquisition time of 1.75 s, a relaxation time of 5.0 s, and 24 scans. For typical <sup>13</sup>C NMR, 20,480 spectra scanning were averaged to increase the signal-to-noise ratio with 2.0 s delayed relaxation and 0.9 s acquisition time.

### *2.7. Thermal Investigation by TGA and DSC*

The thermal investigation of the isolated lignin was conducted using a thermogravimetric analyzer (TGA 4000, PerkinElmer, Waltham, MA, USA) and differential scanning calorimetry (DSC) (DSC 4000, PerkinElmer, Waltham, MA, USA). For TGA analysis, about 4 mg lignin sample was placed on the crucible ceramics sample holder, and the measurement was conducted under argon atmosphere with the flow of 20 mL/min. The sample was heated from 25 ◦C to 750 ◦C at a 10 ◦C/min rate. The automatic curve of weight loss versus temperature was generated from the instrument.

DSC analysis was carried out with ~4 mg lignin samples on a standard aluminium pan to determine the glass transition temperature (Tg) and curing properties of lignin. Each sample was heated until 300 ◦C with a 10 ◦C/min heating rate under a nitrogen atmosphere (flowrate = 20 mL/min). Tg value was automatically calculated by DSC 4000 pyris 1 PerkinElmer software (Pyris 11 software Version 11.1.1.0492, PerkinElmer, Shelton, CT, USA).

#### *2.8. Chemical Elucidation by Py-GCMS*

Chemical elucidation analysis of lignin was studied by pyrolysis-gas chromatographymass spectrometry (PyGC/MS) (Shimadzu GC/MS system QP-2020 NX, Shimadzu, Kyoto, Japan) equipped with multi-shot pyrolyzer EGA/PY-3030D. Between 500–600 µg of lignin was placed in eco-cup SF PY1-EC50F, and the cup was sealed by glass wool. The ecocup was pyrolyzed at 500 ◦C for 0.1 min using helium as carrier gas and SH-Rxi-5Sil MS column (30 m × 0.25 mm i.d. film thickness. 0.25 µm). The PyGC/MS temperature was programmed as follows: 50 ◦C for 1 min, 5 ◦C/min to 280 ◦C, and 13 min at 280 ◦C. The

mass spectrum was taken at 70 eV with a pressure of 20.0 kPa (15.9 mL/min, column flow 0.61 mL/min). The obtained pyrolysis product was identified by approaching mass spectra and retention times using the data library in NIST LIBRARY 2017.

#### *2.9. Molecular Weight by GPC*

Gel permeation chromatography (GPC) is a rapid and versatile tool to provide information on the molecular weight of lignin. Lignin was dissolved in the THF, and Shimadzu LC-20 (Shimadzu, Kyoto, Japan) equipped with a UV-RID detector was used to quantify the molecular weight distribution acetylated lignin. The analysis was employed using the LF-800 column with an injection volume of 20 uL. Polystyrene standard was used to create a calibration curve and GPC system calibration.

#### **3. Result and Discussion**

#### *3.1. Chemical Composition and Lignin Solubility*

Lignin recovery was one critical factor for selecting the lignin isolation method that is economically feasible. Lignin yield recovery from BL by single and fractionated-step (oven dry based) was 35.39% and 16.34%. Both isolation methods reported lignin recovery yield at the expected range of 20–40% [5]. Ethanol fractionation resulted in lignin depolymerization in the liquid solution, decreasing the solid lignin residue by acid. Lignin yield recovery is related to the larger size of the fractionated-step lignin based on the SEM micrograph. Large lignin particle size results in a smaller reaction surface area for precipitation, reducing the amount of lignin recovered after acid precipitation. This suggestion was in correlation with the ASL content. This finding agreed with Hamzah et al. (2020), where lignin recovery from *Miscanthus x giganteus* decreased from 75% to 25% with the increased ethanol concentration from 0% to 50% [26].

The chemical composition of lignin Is presented in Table 1, where the data is the average value from a triplicate experiment with a deviation standard less than ±5%. Ethanol was added in fractionated-step to precipitate non-lignin components such as sugar and carbohydrates, theoretically increasing lignin purity. However, the total lignin from single-step lignin (~99%) was slightly higher than from the fractionated step. Besides, the impurities component in the single-step lignin, represented as ash content, was lower than the fractionated step. It suggests that the single-step isolation method effectively isolates lignin with high purity.


**Table 1.** Chemical composition of lignin.

Interestingly, lignin isolation from BL by dilute hydrochloric acid obtained high ASL content while reference lignin had low ASL content. Different isolation methods likely obtained the different proportions of AIL and ASL content. Similar results were reported by Sameni et al. [8] where isolation lignin from BL by using dilute sulfuric acid resulted in low ASL (<4%) and high AIL ~91. Sulfuric acid is popular acid to isolate lignin and obtain a high concentration of AIL. Consequently, it will increase sulfuric and ash content [5].

In this study, the highest free phOH content was obtained from the isolated lignin, where the lowest was from the reference lignin. This finding agreed with the previous report where Kraft pulping process and precipitation lignin by HCl enhanced condensed structure and the phenolic hydroxyl group [10,27]. Unfortunately, we could not find the source and isolation process of reference lignin from Sigma-Aldrich. The total phOH content is correlated to the Tg value because a higher condensed structure in polymer

created a high char amount in high temperature. Eventually, the combustion rate can be reduced by the presence of char [11,28,29]. UV spectroscopy is used to monitor the lignin purity and molecular distribution. A similar pattern of UV-Vis spectra from both commercial and isolated lignin is observed in

**ASL (%)** 

**Total phOH (mmol/g)** 

UV spectroscopy is used to monitor the lignin purity and molecular distribution. A similar pattern of UV-Vis spectra from both commercial and isolated lignin is observed in Figure 1a. The distinct absorption at 215–222 nm corresponded to non-conjugated phenolic groups (excitation of π-π\*) that appear due to shifting band is an effect of hypsochromic NaOH. The spectrum of single-step lignin is slightly higher than others which may correlate to the total phOH (Table 1). Another intensive peak was observed in the range of 296–303 nm, originating from the conjugated phenolic group due to n-π\* excitation [30]. Figure 1a. The distinct absorption at 215–222 nm corresponded to non-conjugated phenolic groups (excitation of π-π\*) that appear due to shifting band is an effect of hypsochromic NaOH. The spectrum of single-step lignin is slightly higher than others which may correlate to the total phOH (Table 1). Another intensive peak was observed in the range of 296–303 nm, originating from the conjugated phenolic group due to n-π\* excita-

*Polymers* **2022**, *14*, x FOR PEER REVIEW 6 of 20

by the presence of char [11,28,29].

**Water Content** 

tion [30].

purity.

*3.2. SEM Micrograph of Lignin* 

**Table 1.** Chemical composition of lignin.

**(%) Ash Content (%) AIL** 

Lignin reference 2.60 ± 0.27 2.44 ± 0.00 96.02 ± 0.50 1.54 ± 0.06 6.00 ± 0.50

Lignin fraction method 15.79 ± 0.74 1.94 ± 0.08 69.94 ± 5.55 28.12 ± 0.94 7.31 ± 0.78

In this study, the highest free phOH content was obtained from the isolated lignin, where the lowest was from the reference lignin. This finding agreed with the previous report where Kraft pulping process and precipitation lignin by HCl enhanced condensed structure and the phenolic hydroxyl group [10,27]. Unfortunately, we could not find the source and isolation process of reference lignin from Sigma-Aldrich. The total phOH content is correlated to the Tg value because a higher condensed structure in polymer created a high char amount in high temperature. Eventually, the combustion rate can be reduced

**(%)** 

(**b**)

**Figure 1.** Lignin solubility in base (**a**) and organic solvent () determined by UV-Vis.

A glance at Figure 1b reveals an identical UV-Vis spectrum among three lignin samples when lignin is diluted in dioxane/water. Unlike the lignin in the base solution, solubilization lignin in dioxane/water is limited to wavelengths above 250 nm seen in the spectra Figure 1b. This finding is similar to lignin Alfa grass kraft from industrial waste [31] and Kraft-anthraquinone (AQ) lignin [32]. According to Ammar et al.'s report, the large absorbance of lignin in dioxane/water at 280 nm corresponded to non-conjugated phenolic hydroxyl groups. In comparison, the presence of both ferulic acids and p-coumaric acids could be attributed to the presence of the second type region of lignin absorption at about 300 nm [31]. Lignin reference has slightly higher absorbance than isolated lignin regarding and pores on the lignin surface were observed in more concentrated ethanol [26]. **Figure 1.** Lignin solubility in base (**a**) and organic solvent (**b**) determined by UV-Vis. A glance at Figure 1b reveals an identical UV-Vis spectrum among three lignin samples when lignin is diluted in dioxane/water. Unlike the lignin in the base solution, solubilization lignin in dioxane/water is limited to wavelengths above 250 nm seen in the spectra Figure 1b. This finding is similar to lignin Alfa grass kraft from industrial waste [31] and Kraftanthraquinone (AQ) lignin [32]. According to Ammar et al.'s report, the large absorbance of lignin in dioxane/water at 280 nm corresponded to non-conjugated phenolic hydroxyl groups. In comparison, the presence of both ferulic acids and p-coumaric acids could be attributed to the presence of the second type region of lignin absorption at about 300 nm [31]. Lignin reference has slightly higher absorbance than isolated lignin regarding purity.

had a smaller particle of 59 µm than lignin from fractionated-step isolation (~72 µm). Interestingly, the single-step lignin had the lowest particle size, ~51 µm, indicating that ethanol impacted the behavior of lignin aggregates. At 5000-times magnification (Figure 2d– f), the morphological image of reference lignin (Figure 2d) and lignin from a single-step (Figure 2e) showed more rupture and pores on the lignin surface. Lignin from fractionated-step (Figure 2f) depicted smooth and rigid surfaces. This finding was following lignin isolation from *Miscanthus x giganteus* where the particle size of lignin increased with higher ethanol concentration, from 306 to 2050 nm. Besides, more crystalline structures

#### *3.2. SEM Micrograph of Lignin*

SEM micrograph in Figure 2a–c, shown in 200-times magnification, described irregular and not uniform particles in terms of size from lignin samples. The reference lignin had a smaller particle of 59 µm than lignin from fractionated-step isolation (~72 µm). Interestingly, the single-step lignin had the lowest particle size, ~51 µm, indicating that ethanol impacted the behavior of lignin aggregates. At 5000-times magnification (Figure 2d–f), the morphological image of reference lignin (Figure 2d) and lignin from a single-step (Figure 2e) showed more rupture and pores on the lignin surface. Lignin from fractionatedstep (Figure 2f) depicted smooth and rigid surfaces. This finding was following lignin isolation from *Miscanthus x giganteus* where the particle size of lignin increased with higher ethanol concentration, from 306 to 2050 nm. Besides, more crystalline structures and pores on the lignin surface were observed in more concentrated ethanol [26]. *Polymers* **2022**, *14*, x FOR PEER REVIEW 8 of 20

**Figure 2.** SEM micrographs show the morphological surface of reference lignin (**a**), single-step (**b**), fractionated-step (**c**) at 200× magnification and reference lignin (**d**), single-step (**e**), and fractionatedstep (**f**), at 5000× magnification. **Figure 2.** SEM micrographs show the morphological surface of reference lignin (**a**), single-step (**b**), fractionated-step (**c**) at 200× magnification and reference lignin (**d**), single-step (**e**), and fractionatedstep (**f**), at 5000× magnification.

#### *3.3. Functional Group of Lignin 3.3. Functional Group of Lignin*

H asymmetric in a methyl group [4].

described in the Py-GCMS section.

FTIR is a versatile analytical tool to investigate functional groups and the general structure in lignin. The functional group of lignin may vary depending on the source of lignin. The main functional group in lignin is hydroxyl, methoxyl, carboxylic acid, and carbonyl. Figure 3 shows the FTIR spectra of reference lignin, and two isolated lignin, while a summary of peaks interpretation is available in Table 2. As shown in Figure 3, most of the peaks were similar between three samples, such as the broadband corresponding to hydroxyl group stretching (O-H) from aliphatic and aromatic in lignin structure detected at the wavelength 3500–3400 cm−1 (a), while the sharp peak at 2918 (b) and 2854 (c) cm−1, respectively, were attributed to C-H stretching in methylene from side chain and aromatic methoxyl groups [33]. A small band assigned to carbonyl (C=O) stretching in unconjugated aldehyde and ketone in the ester group at 1716–1704 cm−1 (d) was found in the reference lignin. Still, a pronounced peak was obtained from both isolated lignin due to different lignin structures. Noticeable peaks attributed to vibration of the aromatic skeleton in all types of lignin appeared in the range of 1590–1460 cm−1 (e–g) [34]. An intense FTIR is a versatile analytical tool to investigate functional groups and the general structure in lignin. The functional group of lignin may vary depending on the source of lignin. The main functional group in lignin is hydroxyl, methoxyl, carboxylic acid, and carbonyl. Figure 3 shows the FTIR spectra of reference lignin, and two isolated lignin, while a summary of peaks interpretation is available in Table 2. As shown in Figure 3, most of the peaks were similar between three samples, such as the broadband corresponding to hydroxyl group stretching (O-H) from aliphatic and aromatic in lignin structure detected at the wavelength 3500–3400 cm−<sup>1</sup> (a), while the sharp peak at 2918 (b) and 2854 (c) cm−<sup>1</sup> , respectively, were attributed to C-H stretching in methylene from side chain and aromatic methoxyl groups [33]. A small band assigned to carbonyl (C=O) stretching in unconjugated aldehyde and ketone in the ester group at 1716–1704 cm−<sup>1</sup> (d) was found in the reference lignin. Still, a pronounced peak was obtained from both isolated lignin due to different lignin structures. Noticeable peaks attributed to vibration of the aromatic skeleton in all types of lignin appeared in the range of 1590–1460 cm−<sup>1</sup> (e–g) [34]. An intense peak at 1430–1420 cm−<sup>1</sup> (h) referred to aromatic skeletal vibration with deformation of C-H asymmetric in a methyl group [4].

peak at 1430–1420 cm−1 (h) referred to aromatic skeletal vibration with deformation of C-

lignin was observed in adsorption at 1326 cm−1 (i) and 1111 cm−1 (l) as the breathing of C-O and deformation C-H in syringyl rings. The band was absent in the reference lignin, but it appeared in two isolated lignins. Conversely, stronger C-O stretching in the guaicyl unit at 1266 cm−1 (j) and 1213 cm−1 (k) was recorded in reference lignin but not in isolated lignin since the lignin was extracted from *Acacia mangium* (hardwood). This finding suggests that the reference lignin may be derived from softwood. This result correlates with Sameni et al. [35] finding where syringyl unit portion was absent in lignin from softwood and abundant in lignin from hardwood. Furthermore, higher peak absorption of unconjugated C-O (at 1030 cm−1 (m)) and CH out-of-plane bending (at 855 cm−1 (n)) in the guaicyl ring in reference lignin suggests a higher concentration of guiacyl in softwood than hardwood. Besides, these peaks were also slightly sharper in the lignin fractionated-step than in the single-step. Further semi-quantitative analysis of syringyl versus guaicyl percentage is

**Figure 3.** Functional group peaks of reference lignin, isolated lignin from single-step and fractionated-step by UATR‒FTIR. **Figure 3.** Functional group peaks of reference lignin, isolated lignin from single-step and fractionatedstep by UATR-FTIR.


**Table 2.** Interpretation bands of UATR-FTIR spectra. **Table 2.** Interpretation bands of UATR-FTIR spectra.

*3.4. 1H and 13C NMR*  NMR analysis is frequently used to predict lignin's structural details concerning its molecular characteristics, reactivity, and composition. The acetylation of lignin before NMR analysis aims to decrease the impurities in lignin that may interfere with the spectrum [25]. Due to the complex structure of polymer lignin, typically simple proton 1H NMR resulted in overlapping spectra which is difficult to justify the structure. Hence, 13C NMR is needed to support the hypothesis of 1H NMR. The presence of condensed and uncondensed aliphatic and aromatic carbon and aryl ethers can be detected by natural 13C isotope NMR. However, longer scanning and acquisition times are required to improve signal sensitivity due to the low abundance of carbon isotope in the lignin molecule [4]. Still, quantitative 13C-NMR can be a useful technique for lignin structural investigation, particularly in determining molecular alterations caused by different isolation procedures and biomass sources [8,25,35]. However, an obvious difference in lignin structure between reference and isolated lignin was observed in adsorption at 1326 cm−<sup>1</sup> (i) and 1111 cm−<sup>1</sup> (l) as the breathing of C-O and deformation C-H in syringyl rings. The band was absent in the reference lignin, but it appeared in two isolated lignins. Conversely, stronger C-O stretching in the guaicyl unit at 1266 cm−<sup>1</sup> (j) and 1213 cm−<sup>1</sup> (k) was recorded in reference lignin but not in isolated lignin since the lignin was extracted from *Acacia mangium* (hardwood). This finding suggests that the reference lignin may be derived from softwood. This result correlates with Sameni et al. [35] finding where syringyl unit portion was absent in lignin from softwood and abundant in lignin from hardwood. Furthermore, higher peak absorption of unconjugated C-O (at 1030 cm−<sup>1</sup> (m)) and CH out-of-plane bending (at 855 cm−<sup>1</sup> (n)) in the guaicyl ring in reference lignin suggests a higher concentration of guiacyl in softwood than hardwood. Besides, these peaks were also slightly sharper in the lignin fractionatedstep than in the single-step. Further semi-quantitative analysis of syringyl versus guaicyl percentage is described in the Py-GCMS section.
