Lignin is highly branched phenolic polymer and accounts 15–30% by weight of lignocellulosic biomass (LCBM). The acceptable molecular structure of lignin is composed with three main constituents linked by different linkages. However, the structure of lignin varies significantly according to the type of LCBM, and the composition of lignin strongly depends on the degradation process. Thus, the elucidation of structural features of lignin is important for the utilization of lignin in high efficient ways. Up to date, degradation of lignin with destructive methods is the main path for the analysis of molecular structure of lignin. Spectroscopic techniques can provide qualitative and quantitative information on functional groups and linkages of constituents in lignin as well as the degradation products. In this review, recent progresses on lignin degradation were presented and compared. Various spectroscopic methods, such as ultraviolet spectroscopy, Fourier-transformed infrared spectroscopy, Raman spectroscopy, and nuclear magnetic resonance (NMR) spectroscopy, for the characterization of structural and compositional features of lignin were summarized. Various NMR techniques, such as 1H, 13C, 19F, and 31P, as well as 2D NMR, were highlighted for the comprehensive investigation of lignin structure. Quantitative 13C NMR and various 2D NMR techniques provide both qualitative and quantitative results on the detailed lignin structure and composition produced from various processes which proved to be ideal methods in practice.
The main components of lignocellulosic biomass (LCBM) are cellulose, hemicellulose, and lignin. Cellulose is a polymer of glucose, accounting for 30–50 wt% of dry LCBM; hemicellulose is a mixture of heteropolymers containing various polysaccharides, such as xylan, glucuronoxylan, and glucomannan, accounting for 20–35 wt%; the mainly remaining portion with 15–30 wt% is lignin, which is a multisubstituted phenolic polymer. Lignin is the most abundant aromatic biopolymer accounting for up to 30% of the organic carbon on Earth and thus can be treated as a potential renewable feedstock for energy supplement and aromatic chemicals production [
Lignin is an amorphous, irregular three-dimensional, and highly branched phenolic polymer. The functions of lignin in the plant cell wall are to cover structural support, transport water and nutrients, and issue protection to prevent chemical or biological attacks, and so forth. Though the chemical structure is extremely complex, it is generally accepted that lignin is formed via irregular biosynthesis process constructed from three basic phenylpropanoid monomers,
Typical lignin contents are 24–33% in softwoods, 19–28% in hardwoods, and 15–25% in grasses, respectively. Functional groups in lignin include methoxyl, carbonyl, carboxyl, and hydroxyl linking to aromatic or aliphatic moieties, with various amounts and proportions, leading to different compositions and structures of lignin [
It is difficult to draw accurate structural diagram for entire lignin by using up-to-date techniques in situ. Although relative new methods for imaging and analyzing chemical structure of lignin, such as confocal Raman scattering microscopy [
Traditionally, there are two ways to isolate lignin from other components in LCBM, so-called degradation processes: one is to extract cellulose and hemicellulose leaving most of the lignin as solid residue, and the other one is to extract lignin by using fractionation methods leaving the other components. For the former process, dilute sulfuric acid and hot water are often used to break down cellulose and hemicellulose releasing sugars and facilitating the further enzymatic hydrolysis, while leaving lignin as the main content in solid residue. For the later process, hydroxide solution, either with sodium, potassium or calcium, is used to remove lignin from LCBM samples. The degradation processes are designed to cleave the bonds between lignin and carbohydrates, leading to more or less extensive changes compared to native lignin structure. Consequently, the chemical compositional features of the resulting technical lignins, such as the relative abundance of S/G/H units, the status of side chains, and the contents of functional groups, are highly dependent on the methods and conditions used in degradation processes [
For the structural and compositional elucidation of complex samples, various instrumental methods were used. For example, the chromatographic techniques coupled to mass spectrometers and high-resolution mass spectrometric techniques were used extensively in the analysis of the bio-oil, biomass, and lignin samples [
In this review, we focused on the recent development and interesting findings on the structural investigation of lignin with spectroscopic methods over various degradation processes. Structural and compositional characters of lignin samples produced from different degradation processes were presented and compared, and developments of spectroscopic methodologies on the qualitative and quantitative elucidation of lignin structure were also summarized. The degradation processes and instrumental methods involved in the detailed and comprehensive understanding of the lignin structure were prospected.
Various physical/chemical methods were carried out for the degradation and isolation of lignin. Optimization or modification of these methods was conducted on various LCBMs due to the difference in the structure of lignin. In order to facilitate further structural and/or compositional analyses or to produce high purity lignin, modified or multistep processes were usually carried out.
MWL is produced via the extraction of milled sample particles from LCBM with a neutral organic solvent (e.g., 1,4-dioxane) under mild conditions to remove other components. In the extraction process, only minor changes may occur with respect to the milled sample; hence, the obtained lignin has similar property with the milled sample. Nevertheless, MWL is not considered to be a representative of the original lignin in the LCBM due to its relative low yield (based on Klason lignin).
In order to improve the yield, CEL was developed from the extraction of enzymatically hydrolyzed MWL residue. Typically, the residual carbohydrate contents in CEL account 10–15 wt% of initial MWL sample. The structure of CEL is similar to MWL, and it is more representative of total lignin in LCBM than in MWL. CEL has commonly been used for the structural analysis of lignin in the cell wall of plants. In a recent study, cellulolytic enzyme hydrolysis was carried out prior to water/dioxane extraction of MWL to remove carbohydrates. The lignin was obtained with high yield and purity [
Sulfite, soda, and kraft lignins are by far the main technical lignins produced via industrial processes. Among them, sulfite and kraft methods are sulfur-involving processes, accounting more than 90% of the chemical pulp production worldwide [
In the organosolv process, high purity lignin and cellulose are produced at the same time with various solvents; however, no technical lignins are commercially available from this process up to now. Organosolv process typically results in more than 50% lignin removal from LCBM through cleavage of lignin-carbohydrate bonds and
The extraction conditions affect the structure of organosolv lignin, that is, severity factor (H-factor). The molecular weight of the ethanol organosolv lignin decreased within a 36–56% range with respect to the MWL with the increase of the severity. Moreover, an obvious decrease in the content of aliphatic hydroxyl groups and an increase of syringyl phenolic units and condensed phenolic structures with the increase in severity of the organosolv treatment were also observed [
Traditionally, in the acidolysis process, lignin is extracted from LCBM sample with 1,4-dioxane containing hydrochloric acid under room temperature. The obtained lignin with high purity is considered to be a representative of the original lignin. However, a limitation of this process is that the same conditions used to hydrolyze polysaccharides also degrade the liberated monosaccharides, leading to overestimate monosaccharide degradation and introducing bias between polysaccharides of different liability. Modifications were introduced to reduce these errors [
Modified acidolysis processes were carried out to produce lignin with high yield and purity. Thioacidolysis process, in which ethanethiol is used instead of water, produced more lignin and less complex monomer mixtures. In this process, thioethylated H, G, and S monomers by the cleavage of
IL provides an alternative path for lignin removal to classic organosolv pretreatment for enhancing subsequent enzymatic hydrolysis and isolation. Some ILs, such as 1-ethyl-3-methylimidazolium acetate, can extract lignin from poplar and birch with most structural features retained [
The ILs containing 1-butyl-3-methylimidazolium (bmim), 1-ethyl-3-methylimidazolium (emim), and 1-allyl-3-methylimidazolium (amim) cations either with acetate or chloride as the anions are commonly used in the lignin dissolution [
Cholinium ILs are novel bio-ILs used in the lignin valorization, in which different chemical reactions take place during the lignin dissolution from imidazolium ILs [
Other ILs, such as 1-ethyl-3-methylimidazolium xylenesulfonate [emim][ABS] and 1-butyl-3-methylimidazolium methylsulfate [bmim][MeSO4], could promote depolymerization of organosolv lignin and Klason lignin under the oxidative conditions using a Cu/EDTA complex in the presence of a monomeric phenol (4-tert-butyl-2,6-dimethylphenol) [
An acidic IL, called 1-(4-sulfobutyl)-3-methyl imidazolium hydrosulfate ([C4H8SO3Hmim]HSO4), was proven to be an efficient catalyst for direct liquefaction of bagasse lignin, where more than 65% degree of liquefaction and 13.5% yield of phenolic monomer without any char formation [
A switchable ionic liquid (SIL), synthesized from 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), monoethanol amine (MEA), and CO2, named CO2-switched [DBU][MEASIL], was demonstrated to have high ability to extract the interlinked polysaccharide impurities from the sodium lignosulfonate while the linkages and aromatic subunits remain unaffected during the dissolution-recovery cycle. This SIL can be used as an affordable solvent medium to obtain carbohydrate-free lignin from an impure lignin source [
Future developments on the IL degradation of lignin will focus on selective lignin extraction/degradation and functionalization as well as minimization of process costs for recovery and recycling of ILs.
Multistep processes were used to enhance the removal of lignin. A two-step process was carried out in which anhydrous ammonia pretreatment was followed by mild NaOH extraction on corn stover to solubilize and fractionate lignin [
Alkaline lignins were found to have higher carbohydrate content (up to 30 wt%) with higher molecular weights around 3000 Da; on the other hand, organosolv lignins had considerable high purity (better than 93 wt%) with molecular weights in the range of 600–1600 Da [
Yang et al. [
Clearly, different degradation processes or pretreatments have significant influence on the compositional and structural features of lignin. The selectivity and efficiency of these processes are the main consideration. To elucidate the original structure of lignin, relatively undestroyed and effective degradation methods are feasible, such as IL extraction and organosolv process. To produce value-added chemical from lignin, more aggressive methods aiming at the cleavage of the weak linkages in lignin (i.e
Structural investigation of lignin with spectroscopic techniques has been considered to be promising high-throughput and routine methods, which can provide detailed qualitative and quantitative information on structural features including functional groups, types of chemical bonds, and states of atoms.
The content of acid-soluble lignin, the purity, and the components of isolated lignin, can be determined by using UV spectroscopy [
UV spectroscopic absorptions of typical structures in lignin [
Absorption maxima/nm | Electronic transition style | Chromophores and structures |
---|---|---|
200 |
|
Conjugated bonds/aromatic ring |
240 |
|
Free -OH |
282 |
|
Conjugated bonds/aromatic ring |
320 |
|
Aromatic ring conjugated bond with C = C |
320 |
|
C = O groups conjugated to aromatic ring |
325 |
|
Etherified ferulic acid |
Basing on the Lambert-Beer’s Law, UV spectroscopy can be used for the semiquantitative determination of the purity of lignin and its degradation products by using extinction coefficient (EC) [
FTIR spectroscopy is the most widely used technique in the functional group determination basing on the substances with chromophores. It can be treated as a nondestructive, noninvasive, highly sensitive, and rapid technique. Typical functional groups contained in lignin, such as hydroxyl, carbonyl, methoxyl, carboxyl, and aromatic and aliphatic C-H, can be assigned well in the FTIR spectrum. Figure
Assignments of signals in FTIR spectrum to functional groups in lignin [
Wavenumbers/cm−1 | Assignments | Functional groups and structures in lignin |
---|---|---|
3400–3600 |
|
Free -OH |
3100–3400 |
|
Associated -OH |
2820–2960 |
|
-CH2, -CH3 |
2920 |
|
Carboxylic -OH |
2650–2890 |
|
Methyl group in methoxyl |
1771 |
|
Aromatic |
1700–1750 |
|
Unconjugated ketones, carbonyls, and ester groups |
1722 |
|
Aliphatic |
1650–1680 |
|
Conjugated |
1500–1600, 1420–1430 |
|
Benzene ring |
1450–1470, 1360–1370 |
|
-CH2, -CH3 |
1325–1330, 1230–1235 |
|
Syringyl ring |
1270–1275 |
|
Guaiacyl ring |
1215 |
|
Ether |
1140–1145 |
|
Guaiacyl |
1130 |
|
Syringyl |
1085–1090 |
|
Secondary alcohol and aliphatic ether |
1025–1035 |
|
Aromatic ring and primary alcohol |
750–860 |
|
Aromatic ring |
Attenuated total reflectance- (ATR-) FTIR could be used for the evaluation of kraft lignin in acylation with different acyl chlorides [
It is known that functional properties of oxyethylated lignins (OELs) and the resulting substances are strongly affected by the degree of oxyethylation (DOE) of phenolic hydroxyl groups (OHphen). Passauer et al. [
Raman spectroscopy, as the sister spectroscopic technique of FTIR, can provide complementary information on the structural features even for the samples containing water. Furthermore, more absorption bands were detected with Raman spectroscopy than FTIR [
Raman spectroscopy is suitable for the investigation of the chemical structure of lignin, because it can provide in situ determination on the cell wall of plants even with no sample preparation. However, when analyzing a lignin sample in solutions with various solvents, one should consider the environmental effects of the solvents [
NMR spectroscopy provides more precise and comprehensive information on qualitative and quantitative assays for the frequencies of linkages and the composition of H/G/S units in the lignin analysis. The first discovery of dibenzodioxocine and spirodienone structures in lignin was carried out by Ralph et al. [
Compared with the spectroscopic methods mentioned above, NMR spectroscopic methods possess much higher resolution and enable a larger amount of information to be obtained. One-dimensional (1D) NMR methods, including 1H, 13C, 19F, and 31P NMR, and two-dimensional (2D) NMR methods, such as 2D HSQC NMR, were applied for the analysis of lignin samples with both solid and liquid states. The distribution of functional groups and amount of linkages and H/G/S units as well as other components in lignin can be qualitatively and quantitatively determined. The chemical shifts of functional groups in the spectra have been established.
1H NMR is the method routinely used in the structural investigation of lignin, because of the simple preparation of samples and fast scanning speed. Almost all the compositional investigations of lignins use 1H NMR for the detection of the chemical environment of proton. In the spectra, the signal observed around 7.5 ppm can be assigned to aromatic protons of H units and the other two chemical shifts around 7.0 ppm and 6.5 ppm are attributed to aromatic protons in G and S units, respectively [
Assignments of signals in 1H NMR spectrum to typical functional groups in lignin (in CD3Cl) [
Chemical shift/ppm | Assignments |
---|---|
9.7–9.9 | Cinamaldehydes and benzaldehydes |
6.7–7.1 | Aromatic-H in guaiacyl |
6.2–6.7 | Aromatic-H in syringyl |
5.8–6.2 | Benzylic OH in |
4.9–5.1 | Carbohydrates |
3.3-4.0 | Methoxyl |
3.0–3.1 | H |
2.2–2.4 | Phenolic OH |
1.6–2.2 | Aliphatic OH |
13C NMR can be carried out to overcome the overlapping resonances of some structures in 1H NMR spectra, providing qualitative and quantitative results with nondestructive detection of solid or solution samples. Although with a higher resolution, it is recommended that relative pure lignin sample is necessary in the 13C NMR analysis, since the unexpected overlapping of spectra was due to the complexity of sample. Typical 13C NMR spectra are shown in Figure
Assignments of signals in 13C NMR spectrum to functional groups in lignin [
Chemical shift/ppm | Assignments |
---|---|
167–178 | Unconjugated -COOH |
162–168 | Conjugated -COOH |
140–155 | C3, C4 aromatic ether or hydroxyl |
127–140 | C1, aromatic C-C |
123–127 | C5, aromatic C-C |
117–123 | C6, aromatic C-H |
114–117 | C5, aromatic C-H |
106–114 | C2, aromatic C-H |
78–90 | Aliphatic C |
67–78 | Aliphatic Cα-O |
54–57.5 | Methoxyl |
Constant et al. [
31P NMR has also been widely used to quantitatively determine the amount of aliphatic and phenolic hydroxyl groups as well as carboxyl groups in lignin after phosphitylation with 2-chloro-4,4,5,5-tetramethyl-1,3,2-dioxaphospholane (TMDP) [
Assignments of signals in 31P NMR spectrum to hydroxyl groups in lignin [
Chemical shift/ppm | Structural assignments |
---|---|
145.5–150.0 | Aliphatic -OH |
136.5–144.7 | Phenols |
140.0–144.5 | C5 substituted |
143.5 |
|
142.7 | Syringyl |
142.3 | 4-O-5 |
141.2 | 5–5 |
139.0–140.0 | Guaiacyl |
138.2–139.0 | Catechol |
137.3–138.2 |
|
133.6–136.6 | Carboxylic acid -OH |
Solid-/solution-state 13C NMR spectroscopes are powerful in lignin structural elucidation either in their solid or solution state. However, solid-state 13C NMR spectroscopy is only suitable for the analysis of lignin samples that have restricted solubility and can observe some structural features of lignin due to its low resolution; and lignin is subjected to acetylation by anhydride/pyridine solution before the solution-state 13C NMR spectrum collection [
Various 2D NMR methods were carried out to overcome the overlapping of resonances in 1D NMR with higher resolution and providing more reliability to the assignments of the signals, especially in the determination of lignin [
The combination of quantitative 13C NMR and 2D HSQC NMR has been proven to be a powerful way in structural elucidation of complex samples since it takes advantage of the spectral dispersion afforded by the 2D spectrum to serve as an internal standard to measure the integral values obtained from the quantitative 13C spectrum [
The comprehensive understanding of the lignin structure relies greatly on the developments of analytical strategies used, which is extremely important for the value-added utilization of biomass. Although significant progresses have been made in the degradation and isolation of the lignin from other components in LCBM, only a fraction of lignin can be identified and analyzed. Structure and composition of lignins from different LCBMs vary significantly according to both issue and age. Furthermore, the analytical results are strongly dependent on the degradation processes and instrumental equipment used.
For the structural investigation of lignin, undestroyed, selective, and efficient isolation methods should be built to preserve the initial structure of lignin and obtain as much sample to be analyzed. Among the wet-chemistry techniques used, IL extraction and organosolv process are the promising methods. They are treated as environmentally friendly methods since relatively mild conditions used and the reagents can be recycled. Biological degradation might be another possible pathway for the oriented isolation of lignin since the outstanding selectivity and rate of conversion.
Various spectroscopic methods are routinely used for the investigation of lignin structures. These methods can provide both qualitative and quantitative information on functional groups and linkages in lignin as well as degradation products of lignin. Among these spectroscopic techniques, UV spectroscopy is less likely to be used since it can provide relatively less information on the structural features of lignin. Generally, FTIR spectroscopy is much more frequently used than Raman spectroscopy. FTIR, 1H NMR, and 13C NMR are commonly used in most of the investigations for the characterization of structure of lignins. Recently, 31P NMR is more adopted in this area. Significant progresses for structural elucidation of lignin rely on the application of quantitative 13C NMR and various 2D NMRs. They are robust techniques by providing detailed qualitative and quantitative results with high resolution and precision and can be treated as ideal methods. Rapid, accurate, and nondestructive spectroscopic techniques can be combined to overcome their individual intrinsic limitations for better elucidation of lignin structure. The data collected from these methods contributes to the understanding of LCBM structure and facilitates the design of effective processes to obtain lignin-based value-added chemicals.
The authors declare that there is no conflict of interest regarding the publication of this paper.
This work was supported by the Fundamental Research Funds for the Central Universities (Grant 2015XKMS100), the National Natural Science Foundation of China (Grant nos. 21506250 and 21676293), and the Qing Lan Project of Jiangsu Province (awarded in 2017).