3.1. Ultimate and Proximate Analysis
The ultimate and proximate analysis of fractionated lignin was carried out and the results are shown in
Table 1. Organosolv lignin fractions were free from sulphur, confirming previous results [
30].
Table 1 shows carbon (C), oxygen (O), hydrogen (H), nitrogen (N) and sulphur (S) contents of all lignins according to CHNS analyzer. The carbon content of lignin from sweet sorghum bagasse was slightly lower than that of lignin samples from other feedstocks. Nitrogen content found in lignin reflects contamination by residues of proteins or industrial fertilisers, especially in the case of the annual plant-derived sorghum and cotton lignins. In most lignins except pine sawdust lignin, nitrogen amount is in the range of 0.4–1.1 wt.% [
31,
32].
Table 2 shows the results of analyses of impurities, cellulose and hemicellulose sugars and ash in the various lignins, determined according to the NREL technical report [
33]. The results indicate that lignin samples are of high purity, exhibiting only low ash and carbohydrate contents. Specifically, the highest carbohydrate impurities were observed in SSL and reached 7%
w/
w, whereas for the rest of lignins it was less than 2.3%
w/
w. The ash content in all lignin samples was less than 0.6%
w/
w, with most of the samples to be below 0.3%
w/
w (
Table 2).
3.3. FTIR Analysis
FTIR analysis was conducted to investigate the differences in main functional groups and monomer composition in the tested lignin samples, as shown in
Figure 1; band assignments are summarized in the
Appendix A (
Table A1).
The bands at 1514 and 1597 cm
were significantly pronounced in all lignin samples and represent the aromatic skeletal vibrations [
38,
39]. All organosolv lignins from the different feedstocks presented the vibration of C-H stretching in -CH
- and -CH
group at 2960–2933 and 2853 cm
, respectively, as well as the C=O group stretching of carbonyl groups of
-oxidized structural motifs with bands at 1694–1701 cm
. Aromatic skeletal vibrations at 1514 cm
, C-H deformations in CH
and CH
group (1450–1460 cm
), aliphatic C-H stretch in CH
, not in OCH
(1372 cm
), G ring breathing (1267–1272 cm
), and aromatic C-H in plane deformation (G > S) (1028 cm
) can be observed in all lignin samples with different intensities. The intensity of the guaiacyl unit in CL and SSL was represented by the peak at C-C and C-O stretch. FTIR absorptions indicative of syringyl units were found to be stronger in CL and SSL, as one would expect based on the type of feedstock.
3.4. P NMR and H-C HSQC Analysis
Quantitative
P NMR analyses of the lignins under study reveal structural differences and dominating types of aromatic structures given the starting biomasses. Results are summarized in
Table 3.
Noteworthy, only the two bark lignin samples exhibit more phenolic OH than aliphatic OH, with a significant difference in the SBAL sample. The positive effect of the presence of the acid catalyst with respect to generating free phenolics is obvious in this case. The higher amounts of S-type-phenolics found for CL and SSL reflect the presence of the syringyl monomers in the structure, while overall notable presence of condensed units suggests that the treatment might cause, to a low extend, intramolecular condensations.
HSQC spectra of lignins were acquired using fixed, standardised conditions in terms of concentrations of samples and acquisition parameters to allow a comparative analysis without quantification. Non-acetylated samples were dissolved in DMSO for analysis. Results are summarized in
Table 4. HSQC-analyses essentially confirm structural differences of the lignins as it can be expected based on the starting biomasses. In terms of monomer composition, PL which has been chosen as a base for its essentially ’pure G’ character compared to the other samples, shows the typical G-units as essentially only monomer type present, and the presence of typical interunit binding motifs. Cross peaks indicative for
-oxidized
-
O-4’ motifs are present. Cinnamyl alcohol and aldehyde are detectable end motifs in PL. The overall intensities of cross-peaks in the lower aliphatic region, eventually attributable to the presence of further extractable aliphatic impurities and aliphatic end groups of various nature are comparable to those found for CL and SSL, but inferior to those of the bark samples. Traces of hemicellulose residues, especially xylan residues, in the lignin are detectable, signs indicative of lignin-carbohydrate complexes (LCCs) in PL are de facto absent. CL shows the typical distribution of monomer types for herbaceous lignins, and standard interunit motifs are present as well as typical termination motifs. Cross peaks indicative for
-oxidized
-
O-4’ motifs are less intense than for PL. Traces of hemicellulose residues in the lignin are detectable, signs indicative of lignin-carbohydrate complexes (LCCs) are absent here as well. The sample contains a significant amount of para-coumarates as one could expect compared to PL.
A more densely populated aliphatic region indicates the presence of larger amounts of extractives in the sample compared to PL. Moreover, SSL contains still some extractives/aliphatics but in overall lower concentrations when normalizing abundancies to PL. Apart from standard monomer units for this lignin, the sweet sorghum bagasse sample contains the standard bonding motifs, seemingly in less abundance than PL and CL. However, given the fact that the sample due to the presence of the S-units, contains significantly more methoxy groups per C9-unit, differences as highlighted in
Table 4 must not be overinterpreted. Nevertheless, coumarate residues can be seen as significantly enhanced also with respect to CL. The sample contains significantly higher concentrations of hemicellulose residues than PL and CL.
Both bark extracts, i.e., SBAL and SBNL, give rather different HSQC spectra compared to the other lignin samples as is expectable. The two samples are overall very similar, showing mainly -O-4’ and -O-5’ interunit bondings. The clear presence of LCC-indicating cross-peaks in SBNL vs. traces of these peaks in SBAL is in agreement with the P NMR findings discussed above, hinting at the effectiveness of the acid treatment for eventually cleaving LCC bonding motifs and facilitating thus removal of carbohydrates. Both bark samples are characterized by high intensities of cross-peaks in the lower aliphatic region, eventually attributable to the presence of higher amounts of polar extractable impurities.
3.5. Headspace Gas Chromatography-Mass Spectrometry
The formation of main compounds during HSGC-MS treatment of lignin was investigated at treatment temperatures of 110, 180 and 270
C. Only compounds with a spectral match quality greater than 85% and an abundance of greater than 0.5% are listed in the
Table 5.
The HSGC-MS analysis at 110
C indicated only a few compounds in the vapor of lignin samples: Isovanilline in CL, PL, SSL, vanillin in SBNL and SBAL, eugenol in PL, coumaran,
-curcumene,
o-guaiacol and succinic acid in CL, cadelene in SBAL, CL and PL. The presence of coumaran in CL agrees with the literature as lignin in grasses generally contains significant greater amount of coumaryl (H) (5–35%) than in softwoods [
40]. The detected vanillin-based compounds represent the G-group unit [
41]. The increase of vanillin concentration during the heating is the result of the oxidative degradation of guaiacyl structures [
42]. The other released aromatic compounds, i.e., isovanilline, etc., have other aromatic origins or can be seen as products of more complex degradation/migration and rearrangements occuring upon heating under air. The relative amounts of vanillin were less abundant in CL and SSL for the reason of smaller proportion of guaiacyl units in herbaceous biomass than in softwood. Both bark lignin samples retain greater concentrations of extractives bonded to their structure than other samples, as the results listed in
Table 5 show. Even using the additional ethanol-water or water-diethyl ether washing of lignin indicated that extractives can remain partially linked to lignin fibers due to their affinity [
43]. In addition, previous results showed that
-curcumene and cadelene could be formed during low temperature heating of resins [
44]. The most abundant fragment released during HSGC-MS analysis was calamenene in SBNL and SBAL and PL samples, which were previously found in the released compound vapor of coniferous wood [
45]. This result agrees with the thermogravimetric analysis of lignin samples, whereas lignins fractionated from softwood showed a pronounced DTG peak at low temperatures, as discussed in
Section 3.6. Overall, cotton stalks lignin showed the broadest distribution of released compounds during headspace analysis at 110
C.
An increase in heat treatment temperature from 110
C to 180
C and then 270
C during HSGC-MS analysis led to an increase in aromatic compounds and esters, and decrease in aldehydes which mainly originated from remaining impurities of hemicellulose and cellulose, as previously discussed [
46]. The ethyl levulinate found in SSL, SBNL, SBAL and CL and dehydroabietal detected in SSL were released at 180
C, emphasizing the presence of cellulose-related compounds, which were, however, not observed any more at 270
C. Moreover, HSGC-MS treatment at 180
C led to the formation of 5-formylfurfural in SBNL, SBAL, and SSL, confirming the presence of hemicellulose-related species, in accordance with HSQC data. This result agrees with the thermogravimetric analysis (see
Section 3.6), whereas both bark lignins showed a pronounced DTG peak at low temperatures that indicates the presence of remaining extractives and carbohydrates. Results further indicate that compounds in the vapor phase at 270
C analysis were mainly oxygenated aromatic chemicals. The main products in the HSGC-MS analysis of SSL, PL, SBAL, SBNL and CL at 270
C were vanillin, acetovanillone and
o-guaiacol, stemming from G-based structural units. Isoeugenol, 4-ethylguaiacol, vanillin and methyl vanillate were present in all lignin samples, with noteworthy exception of SBAL, whereas 4-vinylguaiacol was detected in all lignin samples with the exception of both spruce bark lignins. These results are in agreement with data of thermogravimetric analyses indicating an increased release of guaiacol and its derivatives with higher heating temperatures. At 180 and 270
C, all lignin samples, except PL at both temperatures and CL at 270
C, formed succinic acid, stemming from oxidative cleavage under the severe conditions.
The major difference of CL and SSL to other samples lies in the presence of acetylsyringol and ferulic acid ethyl ester in their structure confirming the typical presence of dimethoxyphenols for these lignins. Ferulate and coumarate esters present the major part of LCC linkages in herbaceous biomass which can produce ester linkage with polysaccharides and proteins due to the presence of carboxylic acid groups at the end of propenyl groups [
47]. In comparison to previous results, esterified fatty alcohols were detected in significant smaller numbers in this study, hinting at the effectiveness of the treatment in terms of removing these impurities [
48,
49].
No p-cresol was detected in cotton and spruce bark lignins. Free fatty acids were present in the range from palmitic to 13-octadecadienoic acid. The esterified fatty acids were detected in all lignin samples as impurities. In addition, the HSGC-MS analysis of all lignin samples at 180 and 270 C showed the presence of retene in the vapor phase, indicating the presence of such lipid components as impurities in all lignin samples, including the lignin from herbaceous cotton stalks (CL). The presence of dihydroabietal at 180 C in both spruce bark lignin samples and absence at 270 C emphasizes the impact of temperature on the released products during HSGC-MS analysis as well as their potential stability limits.
3.6. Thermogravimetric Analysis
The thermogravimetric analysis showed that the conversion of all lignin samples was similar. The main difference in O
and CO
reactivity of lignin samples was observed in the higher maximum temperature of PL compared to other lignin samples due to the shift of the DTG peak to the higher temperatures. The DTG curves show a double broad peak that indicates a heterogeneous lignocellulosic mixture with respect to O
and CO
reactivity [
50,
51].
The DTG peaks of lignin degradation can be interpreted on the basis of the above discussed structural differences between various lignin samples, taking into account also impurities [
52]. In agreement with HSQC-analyses, the first DTG peak during pyrolysis ranging from 200 to 300
C can be referred to as the degradation of carbohydrates and contained extractives, i.e., fatty acids, pheromones, etc., as observed in the GC-MS headspace analysis, respectively. The second DTG peak located between 300 and 380
C can be associated with the presence of mixed HGS structures as common in CL and SSL, followed by the third DTG peak that can be seen as reflecting G-only, or G-dominant lignin structures as typical in PL, SBAL and SBNL between 375 and 500
C. The three DTG peaks indicate the development of three main components: A reactive carbon constituent, a carbon constituent with intermediate reactivity, and a less reactive carbon structure with reactivity that approaches that of commercial guaiacol [
53]. The reactivities of lignin isolated from spruce bark with or without the use of the acid catalyst during the organosolv process were similar in terms of thermal characteristics, reflecting the basic structural similarity.
Data further underline the fact that both bark lignins have a similar composition. The first DTG peak was in absolute terms more visible for the bark lignins compared to other samples. This eventually reflects the greater amount of extractives remaining in the lignin after fractionation, as reported by the HSQC-analyses. SSL showed only a shoulder of the DTG peak with the maximum temperature shifted by 10
C to higher temperatures compared to both bark lignins. Accumulative analysis of the curves indicate that lignins fractionated from herbaceous species are thermally less stable than those of softwood, which is in line with previous results [
54,
55]. Interestingly, the thermal data do not seem to reflect the ratio of aliphatic to aromatic OH-groups, or the impact of phenolic OH-group content. Both bark lignin, i.e., SBAL and SBNL, exhibiting significantly more phenolic than aliphatic OH-groups, show similar DTG curves compared to the other lignins. SBAL and SSL, representing the two lignins with the highest amount of phenolic OH, do not show drastic differences to the other samples.