molecules
A icle
Chemical, The mal and An ioxidan P ope ies o Lignins
Solubilized du ing Soda/AQ Pulping o O ange and Oli e T ee
P uning Residues
Ma ía E. Eugenio 1, Raquel Ma ín-Samped o 1, JoséI. San os 2, Be nd Wicklein 3and Da id Iba a 1,*
Ci a ion: Eugenio, M.E.;
Ma ín-Samped o, R.; San os, J.I.;
Wicklein, B.; Iba a, D. Chemical,
The mal and An ioxidan P ope ies
o Lignins Solubilized du ing
Soda/AQ Pulping o O ange and
Oli e T ee P uning Residues.
Molecules 2021,26, 3819.
h ps://doi.o g/10.3390/
molecules26133819
Academic Edi o s: Ma gi Schulze
and Bi gi Kamm
Recei ed: 31 May 2021
Accep ed: 18 June 2021
Published: 23 June 2021
Publishe ’s No e: MDPI s ays neu al
wi h ega d o ju isdic ional claims in
published maps and ins i u ional a il-
ia ions.
Copy igh : © 2021 by he au ho s.
Licensee MDPI, Basel, Swi ze land.
This a icle is an open access a icle
dis ibu ed unde he e ms and
condi ions o he C ea i e Commons
A ibu ion (CC BY) license (h ps://
c ea i ecommons.o g/licenses/by/
4.0/).
1Fo es Resea ch Cen e (INIA, CSIC), C a. de la Co uña Km 7.5, 28040 Mad id, Spain;
[email p o ec ed] (M.E.E.); [email p o ec ed] (R.M.-S.)
2NMR o Facili y o Resea ch (SGIke ), Uni e si y o he Basque Coun y (UPV/EHU), A enida Tolosa 72,
20018 Donos ia-San Sebas ián, Spain; [email p o ec ed]
3Ma e ials Science Ins i u e o Mad id (ICMM), Consejo Supe io de In es igaciones Cien í icas (CSIC),
So Juana Inés de la C uz 3, 28049 Mad id, Spain; [email p o ec ed]
*Co espondence: [email p o ec ed]; Tel.: +34-913473948
Abs ac :
Some ag o o es y esidues such as o ange and oli e ee p uning ha e been ex ensi ely
e alua ed o hei alo iza ion due o i s high ca bohyd a es con en . Howe e , lignin-en iched
esidues gene a ed du ing ca bohyd a es alo iza ion a e no mally incine a ed o p oduce ene gy.
In o de o ind al e na i e high added- alue applica ions o hese lignins, a dep h cha ac e iza-
ion o hem is equi ed. In his s udy, lignins isola ed om he black liquo s p oduced du ing
soda/an h aquinone (soda/AQ) pulping o o ange and oli e ee p uning esidues we e analyzed by
analy ical s anda d me hods and Fou ie - ans o m in a ed spec oscopy (FTIR), nuclea magne ic
esonance (solid s a e
13
C NMR and 2D NMR) and size exclusion ch oma og aphy (SEC). The mal
analysis ( he mog a ime ic analysis (TGA), di e en ial scanning calo ime y (DSC)) and an ioxidan
capaci y (T olox equi alen an ioxidan capaci y) we e also e alua ed. Bo h lignins showed a high
OH phenolic con en as consequence o a wide b eakdown o
β
-a yl e he linkages. This ex ensi e
deg ada ion yielded lignins wi h low molecula weigh s and polydispe si y alues. Mo eo e , bo h
lignins exhibi ed an en ichmen o sy ingyl uni s oge he wi h di e en na i e as well as soda/AQ
lignin de i ed uni s. Based on hese chemical p ope ies, o ange and oli e lignins showed ela i ely
high he mal s abili y and good an ioxidan ac i i ies. These esul s make hem po en ial addi i es o
enhance he he mo-oxida ion s abili y o syn he ic polyme s.
Keywo ds: lignin pu i y and composi ion; lignin s uc u al cha ac e iza ion; lignin he mal p ope -
ies; lignin an ioxidan p ope ies; oli e ee p uning; o ange ee p uning; soda/AQ pulping
1. In oduc ion
Ag o o es y esidues a e one o he majo esou ces o unexploi ed po en ial lignocel-
lulosic eeds ocks. Mos o hese esidues a e unde u ilized o bu ned in si u, gene a ing
se ious en i onmen al pollu ions. Thus, he ci cula bioeconomy, as a way o each a sus-
ainable de elopmen , should be a chance o manage hese lignocellulosic esidues owa ds
he p oduc ion o ene gy and high added- alue p oduc s. In addi ion, he success o his
a ge should ha e a signi ican impac on he mi iga ion o bo h pe oleum consump ion
and g eenhouse gas (GHG) emissions.
An in e es ing and abundan ag o o es y esidue is o ange ee p uning, he esul o
imming away unneeded b anches o o ange ees. O ange ee cul i a ion is signi ican
in Medi e anean coun ies, including Spain, whe e 2.82
×
10
6
ons o o anges a e p o-
duced annually [
1
]. This ag icul u al ac i i y gene a es a quan i y o o ange ee p uning
esidue a ound 2.25
×
10
6
ons each yea [
1
]. In he same way, Spain is also he main
oli e oil p oduce wo ldwide, wi h abou 2.3 M ha o oli e ees cul i a ed ha gene a es
Molecules 2021,26, 3819. h ps://doi.o g/10.3390/molecules26133819 h ps://www.mdpi.com/jou nal/molecules
Molecules 2021,26, 3819 2 o 21
1.3 ons ha−1yea −1
(3.0 ons ha
−1
biennial p uning) o oli e ee p uning esidue [
2
].
These ee p uning esidues, which include a main woody ac ion and a emaining ac ion
consis ing o lea es and hin b anches, ha e been ex ensi ely e alua ed o hei alo iza-
ion due o i s high ca bohyd a es (cellulose and hemicellulose) con en . Then, cellulose has
been used o p oduc ion o bioe hanol [
3
], cellulosic pulp and ad anced ma e ials such as
nanocellulose, among o he s [
1
,
4
–
6
]. Hemicelluloses ha e been exploi ed o p oduc ion o
xyli ol and xylooligosacha ides [
7
,
8
]. Howe e , lignin-en iched esidues gene a ed du ing
hese p ocesses, solubilized in black liquo s om pulp and pape indus y o as non-
e men able esidues esul ing om bioe hanol p oduc ion, a e cu en ly unde u ilized,
being no mally incine a ed o gene a e ene gy i.e., hea and elec ici y, which supplies pa
o he demands o he pulp and pape and bioe hanol indus ies.
Lignin is a complex a oma ic mac omolecule composed by p-hyd oxyphenyl (H) ( om
p-couma yl alcohol), guaiacyl (G) ( om coni e yl alcohol), and sy ingyl (S) ( om sinapyl
alcohol) phenylp opane uni s [
9
]. These uni s a e linked h ough a a ie y o in e -uni s
linkages, including a yl e he and ca bon–ca bon (C–C) bonds [
9
]. Among hem,
β
-a yl
e he linkage (
β
-O-4’) ep esen s he mos abundan bond, ollowed by esinol (
β
-
β
’) and
phenylcouma an (
β
-5’) among o he s. Depending on he lignocellulosic eeds ock, ex ac-
ion p ocess and condi ions, lignin displays a ange o p ope ies, i.e., a oma ic s uc u e,
chemical unc ionali ies, hyd ophobici y, he mal s abili y and ea abili y, an ioxidan ,
e c., which makes i in e es ing as a eeds ock o p oduce chemicals and ma e ials [
10
].
Then, lignin has been assessed o polyme and ma e ial applica ions such as ca bon ma e-
ials, esins, hyd ogels, and polyme modi ie s and/o as a sou ce o p oduce bulk and
ine chemicals such as benzene, oluene and xylene (BTX), phenols and anillin [
10
,
11
].
The e o e, in addi ion o helping o he compe i i e and sus ainable p oduc ion o ene gy
and high added- alue p oduc s om ca bohyd a es, he alo iza ion o lignin-en iched
esidues gene a ed du ing ca bohyd a es ans o ma ion will also con ibu e o he imple-
men a ion o he ci cula bioeconomy, which aims o maximize he usage and alue o all
aw ma e ials, p oduc s, and esidues.
K a pulping wi h NaOH and Na
2
S is he alkaline p ocess mos widely employed
o deligni ica ion o woods, i.e., ha dwoods and so woods, in he pulp and pape p o-
duc ion [
12
]. Yea ly, 120,000 ons o k a lignin is gene a ed [
13
]. On he o he hand,
soda/an h aquinone (soda/AQ) pulping, wi h AQ as a pulping addi i e o limi he ca -
bohyd a e deg ada ion [
12
], is gene ally used o ag icul u e esidues [
14
]. In his sense,
soda/AQ pulping has been ex ensi ely used o p oduce pape pulp om o ange and oli e
ee p uning esidues [
1
,
6
]. Al hough smalle olumes o soda lignin a e p oduced annually
(5000 ons) compa ed o k a lignin [
13
], soda lignin is sul u - ee, which makes i mo e
a ac i e o he p oduc ion o bio-based p oduc s. Ne e heless, p io o i s alo iza ion,
a ho ough knowledge o he pu i y, he ype and p opo ion o he di e en in e -uni
linkages (ei he na i e o de i ed ex ac ion p ocess linkages), he con en o sy ingyl and
guaiacyl uni s, he molecula weigh , he hyd oxyl p opo ion, and he he mal beha io is
equi ed o selec he bes way o lignin alo iza ion. In his sense, mul i ude o s udies
ha e analyzed di e en esidual lignins o elucida e i s ea u es depending on he biomass
o igin, i.e., ha dwood, so wood, and non-woody ma e ials [
15
–
17
], and he ex ac ion
echnology, i.e., k a pulping, o ganosol , acid hyd olysis and s eam explosion [
18
–
21
].
Lignins solubilized du ing soda/AQ pulping ha e also been cha ac e ized [22–24].
In his s udy, lignins solubilized du ing soda/AQ pulping o o ange and oli e ee
p uning esidues we e eco e ed and hei chemical composi ion and s uc u al ea u es
we e analyzed by analy ical s anda d me hods and Fou ie - ans o m in a ed spec oscopy
(FTIR), nuclea magne ic esonance (solid s a e
13
C NMR and 2D NMR) and size exclusion
ch oma og aphy (SEC). The mal analysis ( he mog a ime ic analysis (TGA), di e en ial
scanning calo ime y (DSC)) and an ioxidan abili ies (T olox equi alen an ioxidan capac-
i y) we e also e alua ed. Acco ding o all his in o ma ion, possible alo iza ion ways a e
discussed o hese lignins.
Molecules 2021,26, 3819 3 o 21
2. Resul s and Discussion
2.1. Chemical Composi ion o Lignins
Soda/AQ pulping was ca ied ou using o ange and oli e ee p uning esidues
as aw ma e ials. The esul ing soda/AQ pulps we e il e ed, and he black liquo s
con aining solubilized lignins eco e ed. Then, acidi ica ion a pH 2.5 o soda/AQ black
liquo s gene a ed o ange and oli e soda/AQ lignins (deno ed as soda/AQ-o ange and
soda/AQ-oli e lignins, espec i ely). O ange lignin sample showed a lignin con en o
75.1%
(64.5 ±1.6%
o acid insoluble lignin and 10.6
±
0.6% o acid soluble lignin), whe eas
oli e lignin sample exhibi ed a lignin con en o 69.9% (61.3
±
0.22% o acid insoluble
lignin and 8.6
±
0.0% o acid soluble lignin). Some ca bohyd a es impu i ies we e also
de e mined in bo h lignins, being much highe in soda/AQ-oli e lignin (11.0
±
0.0% glucan,
4.7 ±0.0%
xylan and 1.6
±
0.0% a abinan) compa ed o soda/AQ-o ange lignin (
2.7 ±0.1%
glucan, 3.1
±
0.1% xylan and 0.6
±
0.0% a abinan). Domínguez-Robles e al. [
22
] also
desc ibed he p esence o ca bohyd a es in soda lignins om ag icul u al esidues such
as whea s aw and ba ley s aw as well as soda/AQ lignins om as g owing plan s
such as Leucaena leucocephala and Chamaecy isus p oli e us. Pa o he ca bohyd a es in he
lignocellulosic ma e ials is dissol ed du ing alkaline p ocesses, s a ing immedia ely when
he lignocellulosic ib es come in con ac wi h he alkaline pulping liquo and p oceeding
apidly e en a empe a u es a ound 100
◦
C, especially he xylans. A empe a u es abou
170
◦
C, o highe , a andom alkaline hyd olysis o glucosidic bonds may also ake place [
12
].
These dissol ed ca bohyd a es can pa ially p ecipi a e du ing lignin p ecipi a ion due
o hei lowe solubili y unde acidic condi ions, explaining he ca bohyd a es con en
de e mined in bo h lignin samples [
18
]. Ne e heless, hey can also be a ibu ed o
lignin-ca bohyd a e complexes [25].
2.2. FTIR Spec a Analysis o Lignins
The FTIR spec a o o ange and oli e soda/AQ lignins a e showed in Figu e 1, being
he isible bands iden i ied acco ding o p e ious s udies [
21
–
23
,
26
–
28
]. Bo h spec a
displayed cha ac e is ic lignin pa e ns, wi h a b oad band a 3400–3300 cm
−1
a ibu ed
o he O–H s e ching ib a ion in a oma ic and alipha ic lignin s uc u es. The bands a
2930 cm−1
and 2850 cm
−1
a e associa ed o he symme ical and asymme ical C–H s e ch-
ing in he me hyl and me hylene g oups, espec i ely, oge he wi h he band a
1455 cm−1
associa ed o he C–H asymme ic ib a ions and de o ma ion (asymme ic in me hyl and
me hylene). The abso p ion in ensi y a 1700 cm
−1
, co esponding o he ca bonyl in he
unconjuga ed ke ones and es e g oups s e ching om lignin oxida ion [
26
], was also
clea ly isible in bo h lignin samples. Ne e heless, ca bonyl g oups in hemicelluloses ha
a e emaining in bo h lignin samples as impu i ies (Sec ion 2.1) could also be con ibu ing
o his abso p ion [22].
Bo h lignin spec a displayed bands a 1595 cm
−1
, 1507 cm
−1
, and a shoulde a
1417 cm−1
co esponding o a oma ic skele on lignin ib a ions. O he bands we e associ-
a ed o sy ingyl (S) and guaiacyl (G) uni s, including bands a 1315 cm
−1
(a oma ic ing
b ea hing, S and G condensed uni s), 1264 cm
−1
(G ing b ea hing wi h C=O s e ching),
1208 cm
−1
(G ing b ea hing wi h C–C, C–O, and C=O s e ching), 1110 cm
−1
(C–H bond
de o ma ion in S uni s), 1025 cm
−1
(C–H bond de o ma ion in G uni s) and 819 cm
−1
(C–H ou o plane de o ma ion o S uni s). These lignin bands a e ypical o lignins om
ha dwoods such as eucalyp , popla , black locus and elm ma e ials [19,20,27,29].
In addi ion o ca bonyl g oups om hemicelluloses abso bing a 1700 cm
−1
, o he
bands in bo h lignin spec a can also e lec he ca bohyd a e impu i ies de e mined by
chemical composi ion analysis (Sec ion 2.1). Thus, cellulose and hemicellulose bands
a 1110 cm
−1
(C–OH skele al ib a ion) and 1025 cm
−1
(C–O s e ching ib a ion) we e
obse ed. Finally, he signal a 617 cm
−1
is a ibu ed o C–S bending gene a ed om he
use o H2SO4du ing he p ecipi a ion o lignins om soda/AQ black liquo s [22].
Molecules 2021,26, 3819 4 o 21
Molecules 2021, 26, x FOR PEER REVIEW 4 o 23
obse ed. Finally, he signal a 617 cm−1 is a ibu ed o C–S bending gene a ed om he
use o H2SO4 du ing he p ecipi a ion o lignins om soda/AQ black liquo s [22].
Figu e 1. FTIR spec a, 4000–600 cm−1 egion, o soda/AQ-o ange lignin (discon inuous line) and
soda/AQ-oli e lignin (con inuous line).
2.3. Solid S a e 13C NMR Spec a Analysis o Lignins
The 13C NMR spec a o o ange and oli e soda/AQ lignins a e displayed in Figu e 2,
being he signals iden i ied based on hose desc ibed in bibliog aphy [22,26,29,30]. In ac-
co dance wi h FTIR pa e ns (Sec ion 2.2), bo h 13C NMR spec a exhibi ed a signal a δC
175 ppm, especially in soda/AQ-oli e lignin, assigned o ca bonyl g oups and alipha ic
COOR om lignin oxida ion du ing alkaline pulping [31]. Ne e heless, hemicelluloses
impu i ies can also con ibu e o his signal [32]. Mo eo e , he a oma ic egions (a ound
δC 152–95 ppm) o bo h spec a we e domina ed by signals co esponding o phenolic
uni s. Then, he signals a δc 147 ppm, associa ed o C3 and C5 in S uni s and C3 and C5 in
G uni s, and a δc 133 ppm, endo sed o C1 and C4 in S uni s and C1 in G uni s, showed a
g ea in ensi y. Con a y, only a small shoulde a δC 152 ppm om non-phenolic uni s
was isible in bo h spec a. This high con en o phenolic uni s obse ed by 13C NMR in
bo h lignins (sligh ly highe o soda/AQ-o ange lignin (317.6 ± 9.2 g GAE mg−1 lignin)
han soda/AQ-oli e lignin (294.3 ± 1.6 g GAE mg−1 lignin) a e eac ion wi h Folin–Cio-
cal eau eagen ) indica es an abundan deg ada ion o hem p oduced du ing soda/AQ
pulping. As i is well known, he β-O-4’ subs uc u es is he dominan linkage in na i e
lignin [9], ep esen ing a ound 50−60% o all linkages. Alkaline pulping p ocesses, ei he
k a o soda pulping, gene a e phenolic uni s by clea ing β-O-4’ e he bonds, which help
o solubilize lignin [23,24,28].
O he signals in he a oma ic egion o bo h spec a we e also isible a δC 128 ppm,
C2 and C6 in p-hyd oxyphenyl (H) uni s; δC 120 ppm, C6 in G uni s; δC 115 ppm, C5 in G
1100
16002100
2600
31003600
cm-1 600
3330
2930
2850
1700
1595
1507
1452
1417
1315
1264
1208
1110
1025
818
3400
617
Figu e 1.
FTIR spec a, 4000–600 cm
−1
egion, o soda/AQ-o ange lignin (discon inuous line) and
soda/AQ-oli e lignin (con inuous line).
2.3. Solid S a e 13C NMR Spec a Analysis o Lignins
The
13
C NMR spec a o o ange and oli e soda/AQ lignins a e displayed in
Figu e 2
,
being he signals iden i ied based on hose desc ibed in bibliog aphy [
22
,
26
,
29
,
30
]. In
acco dance wi h FTIR pa e ns (Sec ion 2.2), bo h
13
C NMR spec a exhibi ed a signal a
δC
175 ppm, especially in soda/AQ-oli e lignin, assigned o ca bonyl g oups and alipha ic
COOR om lignin oxida ion du ing alkaline pulping [
31
]. Ne e heless, hemicelluloses
impu i ies can also con ibu e o his signal [
32
]. Mo eo e , he a oma ic egions (a ound
δC
152–95 ppm) o bo h spec a we e domina ed by signals co esponding o phenolic
uni s. Then, he signals a
δ
c 147 ppm, associa ed o C
3
and C
5
in S uni s and C
3
and
C
5
in G uni s, and a
δ
c 133 ppm, endo sed o C
1
and C
4
in S uni s and C
1
in G uni s,
showed a g ea in ensi y. Con a y, only a small shoulde a
δC
152 ppm om non-phenolic
uni s was isible in bo h spec a. This high con en o phenolic uni s obse ed by
13
C
NMR in bo h lignins (sligh ly highe o soda/AQ-o ange lignin (317.6
±
9.2 g GAE mg
−1
lignin) han soda/AQ-oli e lignin (294.3
±
1.6 g GAE mg
−1
lignin) a e eac ion wi h
Folin–Ciocal eau eagen ) indica es an abundan deg ada ion o hem p oduced du ing
soda/AQ pulping. As i is well known, he
β
-O-4’ subs uc u es is he dominan linkage in
na i e lignin [
9
], ep esen ing a ound 50
−
60% o all linkages. Alkaline pulping p ocesses,
ei he k a o soda pulping, gene a e phenolic uni s by clea ing
β
-O-4’ e he bonds, which
help o solubilize lignin [23,24,28].
O he signals in he a oma ic egion o bo h spec a we e also isible a
δC
128 ppm,
C
2
and C
6
in p-hyd oxyphenyl (H) uni s;
δC
120 ppm, C
6
in G uni s;
δC
115 ppm, C
5
in
G uni s; and
δC
102 ppm, C
2
and C
6
in S uni s. Ne e heless his signal is o e lapped by
C
1
in hemicellulose, o shi ed o
δC
105 ppm o C
1
in cellulose [
32
,
33
], acco ding o he
ca bohyd a es con amina ion de e mined by chemical composi ion analysis (Sec ion 2.1).
Rega ding o oxygena ed alipha ic egion (a ound
δC
95–50 ppm), lignin signals om
na i e
β
-O-4’ subs uc u e (A), including signals a
δC
81 ppm o C
β
in
β
-O-4’,
δC73 ppm
o C
α
in
β
-O-4’ and
δC
62 ppm o C
γ
in
β
-O-4’ we e de ec ed in bo h spec a. A signal
a δC71 ppm o Cγin na i e β-β’ esinol subs uc u e (B) was also isible, oge he wi h
Molecules 2021,26, 3819 5 o 21
a signal a
δC
56 ppm co esponding o me hoxyl g oups (–OCH
3
). Ne e heless, he
exis ence o ca bohyd a es signals in his egion complica es he in e p e a ion o lignin
subs uc u es [
32
,
33
]. Then, in ag eemen wi h he ca bohyd a es impu i ies de e mined
in bo h soda/AQ lignin samples (Sec ion 2.1), cellulose signals a
δC
81 ppm o C
4
(amo -
phous),
δC
73 ppm o C
2
, C
3
, C
5
and
δC
62 ppm o C
6
(amo phous), and hemicelluloses
signals a
δC
73 ppm o C
2
, C
3
, C
5
and
δC
62 ppm o C
6
can in e e e wi h he signals
iden i ied o lignin subs uc u es. 2D NMR analysis was applied o soda/AQ lignins
analysis (Sec ion 2.4) o esol e he lignin and ca bohyd a es signals o e lapping.
Finally, he non-oxygena ed alypha ic egion (a ound
δC
50–0 ppm) showed a b oad
signal a
δC
30 ppm, gene ally a ibu ed o alkyl ca bons such as he
γ
-me hyl, as well as he
α
- and
β
- me hylene g oups in n-p opyl side chains o lignins, he ace yl o hemicelluloses
and sa u a ed alipha ic moie ies associa ed wi h lipid ex ac i es [32].
Molecules 2021, 26, x FOR PEER REVIEW 5 o 23
uni s; and δC 102 ppm, C2 and C6 in S uni s. Ne e heless his signal is o e lapped by C1
in hemicellulose, o shi ed o δC 105 ppm o C1 in cellulose [32,33], acco ding o he ca -
bohyd a es con amina ion de e mined by chemical composi ion analysis (Sec ion 2.1).
Rega ding o oxygena ed alipha ic egion (a ound δC 95–50 ppm), lignin signals om
na i e β-O-4’ subs uc u e (A), including signals a δC 81 ppm o Cβ in β-O-4’, δC 73 ppm
o Cα in β-O-4’ and δC 62 ppm o Cγ in β-O-4’ we e de ec ed in bo h spec a. A signal a
δC 71 ppm o Cγ in na i e β-β’ esinol subs uc u e (B) was also isible, oge he wi h a
signal a δC 56 ppm co esponding o me hoxyl g oups (–OCH3). Ne e heless, he exis -
ence o ca bohyd a es signals in his egion complica es he in e p e a ion o lignin sub-
s uc u es [32,33]. Then, in ag eemen wi h he ca bohyd a es impu i ies de e mined in
bo h soda/AQ lignin samples (Sec ion 2.1), cellulose signals a δC 81 ppm o C4 (amo -
phous), δC 73 ppm o C2, C3, C5 and δC 62 ppm o C6 (amo phous), and hemicelluloses
signals a δC 73 ppm o C2, C3, C5 and δC 62 ppm o C6 can in e e e wi h he signals iden-
i ied o lignin subs uc u es. 2D NMR analysis was applied o soda/AQ lignins analysis
(Sec ion 2.4) o esol e he lignin and ca bohyd a es signals o e lapping.
Finally, he non-oxygena ed alypha ic egion (a ound δC 50–0 ppm) showed a b oad
signal a δC 30 ppm, gene ally a ibu ed o alkyl ca bons such as he γ-me hyl, as well as
he α- and β- me hylene g oups in n-p opyl side chains o lignins, he ace yl o hemicel-
luloses and sa u a ed alipha ic moie ies associa ed wi h lipid ex ac i es [32].
Figu e 2. 13C NMR spec a, δC 200.0–0.0 ppm, o soda/AQ-o ange lignin (discon inuous line) and
soda/AQ-oli e lignin (con inuous line).
2.4. 2D NMR Spec a Analysis o Lignins
The 13C–1H wo dimensional nuclea magne ic esonance (2D NMR) spec a o o ange
and oli e soda/AQ lignins a e showed in Figu es 3 and 4, espec i ely, including he
whole spec a (δC/δH 0.0–150.0/0.0–9.0) and he spec a co esponding o he oxygena ed
alipha ic (δC/δH 45.0–95.0/2.5–6.0 ppm) and he a oma ic (δC/δH 90.0–150.0/5.0–9.0 ppm)
egions. The main 13C–1H lignin co ela ion signals iden i ied in HSQC spec a a e lis ed
in Table 1, assigned acco ding o hose epo ed by di e en s udies [19,20,22–24,29,34–
150 100 50
ppm
175 175
152
147
152
133
128
133
128
115
115
102 105
81
73
81
71
62
62
55
30
120
Figu e 2. 13
C NMR spec a,
δC
200.0–0.0 ppm, o soda/AQ-o ange lignin (discon inuous line) and
soda/AQ-oli e lignin (con inuous line).
2.4. 2D NMR Spec a Analysis o Lignins
The
13
C–
1
H wo dimensional nuclea magne ic esonance (2D NMR) spec a o o ange
and oli e soda/AQ lignins a e showed in Figu es 3and 4, espec i ely, including he
whole spec a (
δC
/
δH
0.0–150.0/0.0–9.0) and he spec a co esponding o he oxygena ed
alipha ic (
δC
/
δH
45.0–95.0/2.5–6.0 ppm) and he a oma ic (
δC
/
δH
90.0–150.0/5.0–9.0 ppm)
egions. The main
13
C–
1
H lignin co ela ion signals iden i ied in HSQC spec a a e lis ed
in Table 1, assigned acco ding o hose epo ed by di e en s udies [
19
,
20
,
22
–
24
,
29
,
34
–
37
].
The lignin subs uc u es and ca bohyd a es iden i ied a e ep esen ed in Figu es 5and 6.
The non-oxygena ed alipha ic egion (a ound
δC
/
δH
0.0–50.0/0.0–5.0 ppm) exhib-
i ed a a ie y o sa u a ed alipha ic moie ies wi h qui e high in ensi ies, especially in
soda/AQ-oli e lignin spec um (Figu e 4a). Some o hese signals could be associa ed
o ex ac i es [
38
], whe eas o he s could be assigned o g oups neighbou ing alkene and
oxygen-con aining g oups such as e he s, ca bonyl and alcohol, which could o igina e
om lignin deg ada ion [39].
The oxygena ed alipha ic egion o bo h spec a displayed he in o ma ion abou he
di e en in e -uni linkages o lignin samples, including hose om na i e and soda/AQ
lignin de i ed linkages (Figu es 3b and 4b). The p edominan signals co esponding o na-
i e lignin linkages obse ed in bo h spec a we e assigned o
β
-
β
’ esinol subs uc u es, in-
cluding co ela ions o C
α
–H
α
(B
α
), C
β
–H
β
(B
β
) and he double C
γ
–H
γ
(B
γ
). The esinol
Molecules 2021,26, 3819 6 o 21
subs uc u es wi h C–C bonds a e usually s able o alkaline pulping
p ocesses [24,40]
.
O he signals om na i e lignin linkages we e also isible, al hough in a lesse ex en p ob-
ably due o i s deg ada ion du ing alkaline pulping. Then, C
β
–H
β
co ela ion (C
β
) om
β
-5’ phenylcouma an subs uc u es was obse ed in soda/AQ-o ange lignin spec um
(Figu e 3b), whe eas C
γ
–H
γ
co ela ion (B
γ
) was ound in bo h o ange and oli e soda/AQ
lignins spec a (Figu es 3b and 4b, espec i ely). Signals o spi odienones we e clea ly
obse ed in soda/AQ-o ange lignin (Figu e 3b), con aining C
α
–H
α
(E
α
) and C
α’
–H
α’
(E
α’
) co ela ions, and in a lesse ex en in soda/AQ-oli e lignin (Figu e 4b), whe eas
C
γ
–H
γ
co ela ion signal o cinnamyl alcohol end-g oups (I
γ
) was de ec ed in bo h lignin
samples. Rega ding
β
-O-4’ subs uc u es, a weak in ensi y signal o C
α
–H
α
o
β
-O-4’
subs uc u es (A
α
) was no iced in bo h spec a, in ol ing S uni s in soda/AQ-o ange and
soda/AQ-oli e lignins (Figu es 3b and 4b, espec i ely) and also G uni s in soda/AQ-oli e
lignin (Figu e 4b). C
γ
–H
γ
(A
γ
) co ela ions om
β
-O-4’ subs uc u es we e also obse ed
in bo h lignin samples, which in pa a e o e lapped wi h o he signals. This sca ce p es-
ence o signals a ibu ed o
β
-O-4’ subs uc u es is explained by he p e e en ial
β
-O-4’
linkage b eakdown unde alkaline condi ions [
23
,
24
,
28
], which suppo s he high phenolic
con en p e iously desc ibed by 13C NMR o bo h lignins (Sec ion 2.3).
Signals om soda/AQ de i ed lignin linkages we e also obse ed in he oxygena ed
alipha ic egion o bo h spec a. A yl-glyce ol subs uc u e (AG), wi h co ela ions o
C
α
–H
α
(AG
α
), C
β
–H
β
(AG
β
) and C
γ
–H
γ
(AG
γ
), could be en a i ely iden i ied in bo h
spec a (Figu es 3b and 4b), especially in soda/AQ-o ange lignin. This subs uc u e is
p oduced om he non-phenolic
β
-a yl e he linkage unde alkaline pulping p ocesses,
especially in soda pulping a he han k a pulping [
24
]. C
α
–H
α
co ela ion signal o lignin
e minal s uc u es wi h a ca boxyl g oup in C
β
(A –CHOH–COOH; F
α
), which o e laps
wi h C
α
–H
α
co ela ion signal o a yl-glyce ol subs uc u e, could also be assigned in
bo h spec a. This kind o lignin e minal s uc u es has ecen ly been desc ibed du ing
alkaline p ocesses such as k a pulping o popla , elm and sp uce [
19
,
24
,
29
]. Finally, signals
om epi esinol (B’), a dias e eome esul ing om he con e sion o na i e esinol (
β
-
β
’)
subs uc u e du ing k a p ocess [
35
], we e also ound in bo h spec a (Figu es 3b and 4b).
C
α
–H
α
(B’
α
), C
β
–H
β
(B’
β
) and C
γ
–H
γ
(B’
γ
) co ela ion signals we e de ec ed in he case
o soda/AQ-o ange lignin (Figu e 3b), whe eas C
γ
–H
γ
(B’
γ
) in he case o soda/AQ-oli e
lignin (Figu e 4b).
Ca bohyd a es signals we e also obse ed in he oxygena ed alipha ic egion o
o ange and oli e soda/AQ-lignin spec a (Figu es 3b and 4b, espec i ely). These signals
comp ised mainly co ela ions o xylan chain o C
2
–H
2
(X
2
), C
3
–H
3
(X
3
), C
4
–H
4
(X
4
), and
C
5
–H
5
(X
5
). Mo eo e , bo h spec a showed he C-1 c oss peak o (1-4)
β
-D-Xylp o xylan
(Figu es 3a and 4a).
In he a oma ic egion o bo h spec a (Figu es 3c and 4c), he cha ac e is ic co ela ion
signals o S, G, and H lignin uni s we e seen, in he same way ha o he ha dwoods
such as eucalyp , popla , elm and black locus [
19
,
20
,
29
,
40
]. The S lignin uni s p esen ed
co ela ion signals o C
2,6
–H
2,6
(S
2,6
). C
2,6
–H
2,6
in oxidized S uni s (S’
2,6
) wi h a ke one
g oup (ace osy ingone) o aldehyde end-g oup (sy ingaldehyde) in C
α
was also obse ed.
The G lignin uni s showed co ela ion signals o C
2
–H
2
(G
2
), C
5
–H
5
(G
5
), and C
6
–H
6
(G
6
). Signals om oxidized G uni s we e also isible, including co ela ions in soda/AQ-
o ange lignin a ibu ed o C
2
–H
2
(G’
2
) and C
6
–H
6
(G’
6
) in C
α
oxidized G uni s bea ing
an aldehyde-end g oup ( anillin), C
2
–H
2
(G”
2
) and C
6
–H
6
(G”
6
) in C
α
oxidized G uni s
bea ing a ke one g oup (ace o anillone) and C
2
–H
2
(G”’
2
) and C
6
–H
6
(G”’
6
) in C
α
oxidized
G uni s bea ing a ca boxylic g oup ( anillic acid) (Figu e 3c). Some o hese oxidized
G signals we e also obse ed in soda/AQ-oli e lignin spec um (Figu e 4c), con i ming
he lignin oxida ion obse ed by FTIR (Sec ion 2.2) and
13
C NMR (Sec ion 3.3) p oduced
du ing soda/AQ pulping p ocess. In his sense, P insen e al. [
23
] epo ed a highe lignin
oxida ion, wi h an inc emen be ween 200
−
400% o ca boxylic g oups, du ing soda/AQ
pulping o eucalyp . Las ly, he H lignin uni s showed co ela ion signals o C
2,6
–H
2,6
(H
2,6
)
and C3,5–H3,5 (H3,5).
Molecules 2021,26, 3819 7 o 21
Molecules 2021, 26, x FOR PEER REVIEW 8 o 23
Figu e 3. HSQC 2D-NMR spec a o soda/AQ-o ange lignin. (a) whole spec um, δC/δH 0.0–
150.0/0.0–9.0; (b) alipha ic oxygena ed egion, δC/δH 45.0–95.0/2.5–6.0 ppm; (c) a oma ic egion, δC/δH
90.0–150.0/5.0–9.0 ppm.
50.0
60.0
70.0
80.0
90.0
ppm
B
γ
A
α
(S) / F
α
AG
α
E
α
/B'
α
B
α
E
α´
B'
α
X
4
X
3
X
2
/AG
β
X
5
X
5
A
γ
and o he s
B
β
I
γ
B'
β
B'
γ
3.03.54.0
4.5
5.05.5 ppm
B'
γ
V
ans α
V
cis α
S
2/6
S
'
2/6
SB1
α
H
2/6
SB5
α
SB5
β
G'
2
G''
2
G''
6
G'
6
G
2
G
5
+ H
3/5
G
6
100.0
110.0
120.0
130.0
6.0 ppm
140.0
7.0
8.0
ppm
MeO
B'
γ
FA
2
FA
α
FA
β
C
γ
50.0
100.0
1.02.0
3.0
4.0
5.0
6.0 ppm
ppm
7.0
8.0
G'''
6
G'''
2
β-D-X
C1-H1
DMSO
/ AG
γ
a
b
c
Figu e 3.
HSQC 2D-NMR spec a o soda/AQ-o ange lignin. (
a
) whole spec um,
δC
/
δH
0.0–150.0/0.0–9.0
; (
b
) alipha ic oxygena ed egion,
δC
/
δH
45.0–95.0/2.5–6.0 ppm; (
c
) a oma ic egion,
δC/δH90.0–150.0/5.0–9.0 ppm.
Molecules 2021,26, 3819 8 o 21
Molecules 2021, 26, x FOR PEER REVIEW 9 o 23
Figu e 4. HSQC 2D-NMR spec a o soda/AQ-oli e lignin. (a) whole spec um, δC/δH 0.0–150.0/0.0–
9.0; (b) alipha ic oxygena ed egion, δC/δH 45.0–95.0/2.5–6.0 ppm; (c) a oma ic egion, δC/δH 90.0–
150.0/5.0–9.0 ppm.
50.0
60.0
70.0
80.0
90.0
ppm
Bγ
Aα (S)
Bα
X4
X3
X2
/AGβ
Bβ
Iγ
B'γ
B'γ
Aγ
and o he s
Aα (G)
Cβ
MeO
B'γ
Cγ
S2/6
S
'
2/6
SB1α
H2/6
SB5α
G''6
G'2
G2
G5+ H3/5
G6
100.0
110.0
120.0
130.0
6.0 ppm
140.0
7.0
8.0
ppm
3.03.54.0
4.5
5.05.5 ppm
50.0
100.0
1.02.0
3.0
4.0
5.0
6.0 ppm
ppm
7.0
8.0
X5
Jβ
J2,6(S)
G'''6
β-D-XC1-H1
DMSO
G'6
/ Fα
AGα
Eα´
a
b
c
Figu e 4.
HSQC 2D-NMR spec a o soda/AQ-oli e lignin. (
a
) whole spec um,
δC
/
δH
0.0–150.0/0.0–9.0
; (
b
) alipha ic oxygena ed egion,
δC
/
δH
45.0–95.0/2.5–6.0 ppm; (
c
) a oma ic egion,
δC/δH90.0–150.0/5.0–9.0 ppm.
Molecules 2021,26, 3819 9 o 21
O he na i e lignin uni s could also be iden i ied in he a oma ic egion o bo h lignin
spec a. Then, low in ensi y signals co esponding o e ula es, including co ela ions
o C
2
–H
2
(FA
2
), C
α
–H
α
(FA
α
) and C
β
–H
β
(FA
β
), we e obse ed in soda/AQ-o ange
lignin spec um (Figu e 3c). Fe ula es can be ound in he s uc u e o non-woody plan s
lignins such as whea s aw and elephan g ass [
41
,
42
], acyla ing cell wall ca bohyd a es
and con ibu ing o lignin-ca bohyd a es c oss-coupling eac ions, becoming in eg ally
bound in o he lignin molecule [
43
]. Ne e heless, e ula es ha e been also epo ed in
Que cus sube L. [
44
], and mo e ecen ly in Ulmus mino Mill. [
29
]. On he o he hand,
soda/AQ-oli e lignin spec um showed weak co ela ion signals co esponding o C
β
–H
β
(Jβ) and C2,6–H2,6 (J2,6) o cinnamaldehyde end-g oups (Figu e 4c).
Molecules 2021, 26, x FOR PEER REVIEW 10 o 23
Figu e 5. Main lignin and ca bohyd a e subs uc u es iden i ied in alipha ic oxygena ed egion o
o ange and oli e soda/AQ lignins: (A), β-O-4’ alkyl-a yl e he ; (AG), a yl-glyce ol; (B), esinols; (B’),
epi esinol; (C), phenylcouma ans; (E), spi odienones; (F), A –CHOH–COOH; (I), cinnamyl alcohol
end-g oups; (X), xylopy anose (R, OH).
A
HO
O
OCH3
H3COHO
OCH3
O
H3CO
αβ
γ
1
2
3
4
5
6
1'
6'
5'
4'
3' 2'
B
O
OCH3
OCH3
O
O
O
OCH3
H3CO
α
β
γ
1
2
3
4
5
6
β'
γ'
α'
1'
2'
3'
4'
5'
6'
O
HO
OCH3
O
OCH3
H3CO
C
α
β
γ
1
2
3
4
5
6
1'
2'
3'
4
'
5'
6'
O
2
6
53
4
O
1
O
OCH3
α
β
γ
O
H
OA
α
´β´
γ´
OH
OCH3
H3CO
H3C
O
E
O
β
1
2
3
4
5
6
OCH3
α
H3CO
OH
AG
OH
HO
γ
F
O
OH
HO
O
OCH3
H3CO
αβ
1
2
3
4
5
6
H3CO
O
OCH3
OH
αβ
γ
1
2
3
4
5
6
I
1
2
3
4
5
OH
O
R
O
OH
X
O
OCH3
OCH3
O
O
O
OCH3
H3CO
B'
α
β
γ
1
2
3
4
5
6
β'
γ'
1'
2'
3'
4'
5'
6'
α'
Figu e 5.
Main lignin and ca bohyd a e subs uc u es iden i ied in alipha ic oxygena ed egion o
o ange and oli e soda/AQ lignins: (
A
),
β
-O-4’ alkyl-a yl e he ; (
AG
), a yl-glyce ol; (
B
), esinols;
(B’), epi esinol;
(
C
), phenylcouma ans; (
E
), spi odienones; (
F
), A –CHOH–COOH; (
I
), cinnamyl
alcohol end-g oups; (X), xylopy anose (R, OH).
Signals om soda/AQ de i ed lignin linkages we e also obse ed in he a oma ic
egion o bo h lignin spec a. Co ela ion signals a ibu ed o C
α
–H
α
in
β
1 s ilbene (SB1
α
),
and C
α
–H
α
and C
β
–H
β
in
β
5 s ilbene (SB5
α
and SB5
β
, espec i ely) we e de ec ed in
soda/AQ-o ange lignin (Figu e 3c), some o hem also obse ed in soda/AQ-oli e lignin
Molecules 2021,26, 3819 16 o 21
compa able wi h hus showed by soda/AQ lignin in ou wo k (35–47%, o 12.5mg L
−1
o lignin). Finally, Ga cía e al. [
57
] epo ed an ioxidan capaci ies be ween 16.1% and
46.8% (pe cen age wi h espec o he inhibi ion obse ed o he DPPH wi h T olox a e
60 min o eac ion) o lignin wi h mola weigh (Mw) be ween 1.1 KDa and 5.6 KDa,
ex ac ed by di e en me hods om Miscan hus sinensis. They obse ed a clea ela ion
be ween an ioxidan capaci y and he mola weigh and polydispe si y, apa om phenolic
con en . Mo eo e , among hese lignins, hey s udy a soda lignin wi h simila mola weigh
han hose p esen ed in ou wo k (Mw 5.7 KDa) which showed an an ioxidan capaci y o
16.1%, simila o hose obse ed o soda/AQ-o ange and -oli e lignins: 15.3% and 20.5%
(pe cen age wi h espec o he inhibi ion obse ed o he ABTS
•+
wi h T olox a e 6 min
o eac ion).
Table 4. T olox equi alen an ioxidan capaci y o soda/AQ lignins.
Soda/AQ-O ange Soda/AQ-Oli e
mg TE g−1lignin 149.7 ±1.2 598.2 ±4.7
mM TE g−1lignin 202.4 ±5.1 808.9 ±20.3
Soda/AQ-o ange, solubilized o ange lignin eco e ed om soda/AQ black liquo ; soda/AQ-oli e, solubilized
oli e lignin eco e ed om soda/AQ black liquo .
This an ioxidan p ope y could be exploi ed in mix u es wi h o he syn he ic poly-
me s (e.g., polye hylene, polyp opylene, poly ( inyl alcohol)) wi h he objec i e o educe
hei oxida i e deg ada ion [
58
–
60
], pa icula ly hose go e ned by adical mechanisms.
Then, comme cial an ioxidan s such as hinde ed phenols and amines adi ionally applied
o polyme s could be pa ially subs i u ed by hese na u al soda/AQ lignins. Fu he mo e,
lignin could be used also in pha maceu ical, cosme ic, ood and packaging indus ies as a
subs i u e o commonly used cy o oxic syn he ic an ioxidan like bu yla ed hid oxy oluene
(BHT) o bu yla ed hyd oxyanisole (BHA) since i has shown highe an ioxidan powe
acco ding o di e en au ho s [51,55,56,61,62].
Toge he wi h he an ioxidan ac i i y, he ela i ely high he mal s abili y desc ibed
o bo h soda/AQ lignins (Sec ion 2.6) make hem po en ial addi i es o imp o e he
he mo-oxida ion s abili y o syn he ic polyme s [
63
]. This could pa icula i y imp o e
he lame esis ance pe o mance o syn he ic polyme s [
64
], whe ein hese soda/AQ
lignins migh become po en ial subs i u es o o ganic lame e a dan s like halogena ed
bisphenol A o polyb omina ed diphenyl e he s [
65
,
66
]. Ne e heless, a his s age only
conjec u es can be made abou he applicabili y o hese o ange and oli e soda/AQ lignins
as en i onmen ally benign, lame e a dan an ioxidan s, while hese p ope ies need o be
e i ied expe imen ally in he men ioned polyme mix u es.
3. Ma e ials and Me hods
3.1. Raw Ma e ials and Chemicals
O ange ee (Ci us sinensis) and oli e ee (Olea eu opea) p uning esidues we e kindly
p o ided by Uni e sidad de Có doba and Uni e sidad de Jaén, espec i ely. The samples
we e chipped, homogenized, and s o ed un il hei use. On a e age, he o ange ee
p uning esidue p esen ed he ollowing composi ion: 3.6%, ex ac i es; 3.4%, ash; 19.9%,
lignin; and 73.2% holocellulose [
1
]; whe eas he composi ion o oli e ee p uning esidue
was: 8.0% ex ac i es; 1.4%, ash; 20.7% lignin; 89.2% and holocellulose [6].
All chemicals we e eagen -g ade and we e pu chased om Me ck (Ba celona, Spain),
Pan eac (Ba celona, Spain) o Sigma-Ald ich (Mad id, Spain).
3.2. Pulps and Lignins P oduc ion
Pulp om o ange ee p uning esidue was ob ained acco ding o Filla e al. [
1
]. The
ma e ial was cooked wi h soda/an h aquinone (soda/AQ) unde he ollowing condi ions:
185
◦
C, 60 min, 20% (w/w) NaOH, 1% (w/w) AQ (bo h o en-d ied ma e ial) and 8:1
liquid/solid a io. Rega ding oli e ee p uning esidue he cooking condi ions employed
Molecules 2021,26, 3819 17 o 21
o pulp p oduc ion we e: 175
◦
C, 120 min, 15% (w/w) NaOH, 1% (w/w) AQ (bo h o en-
d ied ma e ial) and 8:1 liquid/solid a io [
6
]. Resul ing soda/AQ pulps we e il e ed,
collec ing he black liquo s o lignin p ecipi a ion. O ange soda/AQ pulp showed a kappa
numbe , iscosi y, and b igh ness alues o 22.0, 430 mL g
−1
and 33.5% ISO, espec i ely,
whe eas oli e soda/AQ pulp showed a kappa numbe and iscosi y alues o 38.7 and
794 mL g−1and a b igh ness o 18.4% ISO.
O ange and oli e soda/AQ lignins ( e e enced as soda/AQ-o ange and soda/AQ-
oli e, espec i ely) we e ex ac ed om eco e ed black liquo s by acid p ecipi a ion. Then,
he pH o he black liquo s was lowe ed o 2.5 by slow addi ion o sulphu ic acid (98%
w/w) and le unde s i ing o 30 min. The p ecipi a ed lignins we e il e ed and washed
wice wi h acidi ied wa e (pH 2.5) and d ied a oom empe a u e.
3.3. Lignins Cha ac e iza ion
Chemical composi ion o soda/AQ lignins was examined acco ding o he Labo a-
o y Analy ical P ocedu es o biomass analysis om he Na ional Renewable Ene gies
Labo a o y [
67
], employing he p o ocol NREL/TP-510-42618. A e he acid hyd olysis
o lignin samples, he acid insoluble solid esidue (klason lignin) was eco e ed, whe eas
he liquid ac ion was examined o ca bohyd a es con en by high-pe o mance liquid
ch oma og aphy (1260 HPLC, Agilen , Waldb onn, Ge many, equipped wi h a G1362A
e ac i e index (RI) de ec o and an Agilen Hi-PlexPb column) [
30
]. Mean alues and
s anda d de ia ions we e calcula ed om he iplica es.
The o al phenols con en o soda/AQ lignins was e alua ed acco ding o a sligh ly
modi ied e sion o he Folin-Ciocal eau p o ocol [
30
]. Then, he abso bance o a mix-
u e wi h Folin-Ciocal eau solu ion, Na
2
CO
3
and lignin samples (p e iously dissol ed in
dime hylsul oxide) was measu ed a 760 nm in a UV-Vis spec opho ome e (Lambda 365,
Pe kinElme , Bos on, MA, USA). The o al phenols con en was calcula ed om a calib a-
ion cu e p epa ed om a s anda d solu ion o gallic acid (1–20 mg L
−1
) and exp essed as
g gallic acid equi alen (GAE) mg−1o lignin (on a d y basis). Mean alues and s anda d
de ia ions we e calcula ed om he iplica es.
FTIR spec a o soda/AQ lignins we e acqui ed by a JASCO FT/IR 460 Plus spec om-
e e (Jasco, Japan), wi h an accesso y single e lec ion diamond, wo king a a esolu ion o
1 cm−1, 100 scans, and a spec al ange o 4000–600 cm–1 [30].
Solid-s a e
13
C nuclea magne ic esonance (
13
C NMR) analyses o soda/AQ lignins
we e ca ied ou in a B uke A ance III 400MHz (B uke , Bille ica, MA, USA) a 100.64 MHz
wi h he c oss pola iza ion/magic angle spinning (CP/MAS) echnique a he condi ions
desc ibed by Jiménez-López e al. [30].
13
C–
1
H wo dimensional nuclea magne ic esonance (2D NMR) analyses o soda/AQ
lignins we e eco ded in a B uke AVANCE 500 MHz (B uke , Bille ica, MA, USA) wi h a z-
g adien double esonance p obe. Soda/AQ lignins we e dissol ed in deu e a ed dime hyl-
sul oxide (DMSO-d
6
) and HSQC (he e onuclea single quan um co ela ion) expe imen s
we e eco ded a he condi ions epo ed by Eugenio e al. [
29
] and Ma ín-Samped o
e al. [19]. Residual DMSO (a δC/δH39.6/2.5 ppm) was used as an in e nal e e ence.
Size exclusion ch oma og aphy (SEC) o soda/AQ lignins was conduc ed on a HPLC
(1260 HPLC, Agilen , Waldb onn, Ge many, equipped wi h a G1362A e ac i e index (RI)
de ec o and wo columns PLgel 10
µ
m MIXED B 300
×
7.5 mm). N,N-dime hyl o mamide
(DMF) was pumped as mobile phase a he condi ions desc ibed by Jiménez-López
e al. [
30
]. Columns we e calib a ed wi h polys y ene s anda ds (peak o a e age molecula
weigh s o 570, 8900, 62,500, 554,000, Sigma-Ald ich, San Luis, MO, USA).
3.4. The mal Lignins Cha ac e iza ion
The mog a ime ic analysis (TGA) and di e en ial he mal analysis (DTA) o soda/AQ
lignins we e ca ied ou in ai and ni ogen (SDT Q600, TA Ins umen s, New Cas le, DE,
USA), using a hea ing a e o 10
◦
C min
−1
[
20
]. In addi ion, di e en ial scanning calo ime-
y (DSC) was ca ied ou unde ni ogen (Q2000 calo ime e , TA Ins umen s), using a
Molecules 2021,26, 3819 18 o 21
hea ing a e o
20 ◦C min−1[19]
. In o de o emo e any p e ious he mal his o y om
soda/AQ lignins, he samples we e d ied (60
◦
C o 48 h) and a hea ing and cooling cycle
om 20 o 160 ◦C in he DSC ins umen was pe o med [68].
3.5. An ioxidan Ac i i y o Lignins
The an ioxidan ac i i y o soda/AQ lignins was pe o med using T olox equi -
alen an ioxidan capaci y me hods acco ding o Re e al. [
69
], wi h sligh modi ica-
ions [
52
]. ABTS
+•
was p oduced by he eac ion be ween a s ock solu ion o 2,2
0
-azino-
bis(3-e hylbenz hiazoline-6-sulphonic acid) diammonium sal (ABTS) and po assium pe -
sul a e. P io o use, he ABTS
+•
s ock solu ion was dilu ed wi h phospha e bu e saline o
ge an abso bance o 0.7
±
0.02 a 734 nm. Then, 1 mL o he ABTS
+•
s ock solu ion was
mixed wi h 10
µ
L sample (1 mg mL
−1
) o con ol (bu e ). The abso bance o he eac ion
mix u e was measu ed a 734 nm du ing 6 min in a UV–Vis spec opho ome e Lambda
365 (Pe kinElme , Bos on, MA, USA). T olox was employed as a s anda d and esul s we e
showed in mg o T olox equi alen (TE) g
−1
lignin. Mean alues and s anda d de ia ions
we e calcula ed om he iplica es.
4. Conclusions
In o de o inc ease he compe i i e and sus ainable p oduc ion o ene gy and high
added- alue p oduc s om ca bohyd a es con ained in lignocellulosic ma e ials, including
ag o o es y esidues, he alo iza ion o lignin-en iched esidues gene a ed du ing hese
ans o ma ion p ocesses is c ucial. In addi ion, he use o hese lignin-en iched esidues
will also con ibu e o he es ablishmen o he ci cula bioeconomy, which seeks o maxi-
mize he usage and alue o all aw ma e ials, p oduc s and esidues. Ne e heless, dep h
knowledge o lignin is necessa y o de ine i s alo iza ion ou e. In his s udy, esidual
lignins solubilized du ing soda/AQ pulping o o ange and oli e ee p uning esidues,
wo o he mos abundan ag o o es y esidues gene a ed in Spain, we e isola ed and
chemical, he mal and an ioxidan p ope ies analyzed. Bo h lignins showed ex ensi e
β
-O-4’ linkages deg ada ion, as e ealed
13
C–
1
H wo dimensional nuclea magne ic es-
onance analyses. Consequen ly, a high OH phenolic con en , in e ed by solid s a e
13
C
NMR, and low molecula weigh and polydispe si y alues, showed by size exclusion
ch oma og aphy, we e obse ed o bo h lignins. In addi ion, lignins displayed a high
p opo ion o sy ingyl uni s, con aining di e en na i e as well as soda/AQ lignin de i ed
uni s. Based on hese chemical p ope ies, o ange and oli e lignins exhibi ed ela i ely high
he mal s abili y and good an ioxidan p ope ies. This an ioxidan capaci y could be used
in blends wi h o he syn he ic polyme s in o de o educe hei oxida i e deg ada ion.
Mo eo e , he ela i ely high he mal s abili y desc ibed o bo h lignins makes hem
po en ial addi i es o imp o e he he mo-oxida ion s abili y o syn he ic polyme s.
Au ho Con ibu ions:
Concep ualiza ion, M.E.E. and D.I.; me hodology, M.E.E., R.M.-S., J.I.S., B.W.
and D.I.; so wa e, M.E.E., R.M.-S., J.I.S., B.W. and D.I.; alida ion, M.E.E., R.M.-S., J.I.S., B.W. and
D.I.; o mal analysis, M.E.E., R.M.-S., J.I.S., B.W. and D.I.; in es iga ion, M.E.E., R.M.-S., J.I.S., B.W.
and D.I.; esou ces, M.E.E., R.M.-S., J.I.S., B.W. and D.I.; da a cu a ion, M.E.E., R.M.-S., J.I.S., B.W. and
D.I.; w i ing—o iginal d a p epa a ion, D.I.; w i ing— e iew and edi ing, M.E.E., R.M.-S., J.I.S.,
B.W. and D.I.; isualiza ion, M.E.E., R.M.-S., J.I.S., B.W. and D.I.; supe ision, M.E.E., and D.I.; p ojec
adminis a ion, M.E.E., B.W. and D.I.; unding acquisi ion, M.E.E., B.W. and D.I. All au ho s ha e
ead and ag eed o he published e sion o he manusc ip .
Funding:
This esea ch was unded by Comunidad de Mad id and MCIU/AEI/FEDER, EU ia
P ojec s SUSTEC-CM S2018/EMT-4348 and RTI2018-096080-B-C22, espec i ely. B.W. acknowledges
inancial suppo om MINECO (Spain) and FEDER (EU) (p ojec MAT2015-71117-R) and om
MICINN (Spain) o a JIN con ac (PID2019-107022RJ-I00).
Ins i u ional Re iew Boa d S a emen : No applicable.
In o med Consen S a emen : No applicable.
Da a A ailabili y S a emen : No applicable.
Molecules 2021,26, 3819 19 o 21
Acknowledgmen s:
Uni e sidad de Jaén and Uni e sidad de Có doba a e acknowledging o oli e
and o ange ee p uning esidues, espec i ely. The con ibu ion o COST Ac ion LignoCOST
(CA17128), suppo ed by COST (Eu opean Coope a ion in Science and Technology), in p omo ing
in e ac ion, exchange o knowledge and collabo a ions in he ield o lignin alo iza ion is g a e ully
acknowledged.
Con lic s o In e es : The au ho s decla e no con lic o in e es .
Sample A ailabili y: No applicable.
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