Pyridinium protic ionic liquids: Effective solvents for delignification of wheat straw

Article abstract of DOI:10.1016/j.molliq.2020.115013

Lignocellulosic biomass covers a vast area of the globe and contains many valuable components that can be envisaged for numerous renewable products. The valorization of these components is hindered by its cumbersome isolation due to the recalcitrant nature of lignocellulosic biomass. Acidic ionic liquids (AILs) have emerged in this field as effective pretreatment solvents. The unique property of disrupting lignin-cellulose-hemicellulose bonds and dissolving lignin makes these ILs significant for the separation of lignin and cellulosic constituents. In this study three protic ionic liquids based on pyridinium cation and hydrogen sulfate anion (HSO₄^?) have been synthesized; [PyH][HSO₄] (IL₁), [PyH][HSO₄. (H₂SO₄)] (IL₂), and [PyH][HSO₄ . (H₂SO₄)₃] (IL₃) and utilized for the isolation of lignin from wheat straw. The synthesized ILs were characterized by NMR and FTIR analyses. The lignin yield was optimized with respect to reaction temperature, time, biomass loading, and type of IL. The lignin removal efficacy of the ionic liquid with highest ratio of acid [PyH][HSO₄.(H₂SO₄)₃] (IL₃) is indicated by high delignification (79%) and lignin recovery (77%) under mild conditions (60 °C, 2 h). The cellulose rich material (CRM) and isolated lignin were characterized by FTIR, SEM, TGA, GPC, and HSQC NMR to assess their structure, molecular weight and stability. The cellulosic part was further hydrolyzed enzymatically to evaluate the capability of the recovered pulp to generate glucose. An appreciable yield of reducing sugars (85%) substantiates high lignin removal.

Full text of DOI:10.1016/j.molliq.2020.115013

Journal of Molecular Liquids 325 (2021) 115013
Contents lists available at ScienceDirect
Journal of Molecular Liquids
journal homepage: www.elsevier.com/locate/molliq
Pyridinium protic ionic liquids: Effective solvents for delignification of
wheat straw
a
Azmat Mehmood Asim a, Maliha Uroos a, , Sadia Naz , Nawshad Muhammad
⁎
b,c,⁎⁎
a
Institute of Chemistry, University of the Punjab, 54000 Lahore, Pakistan
b
Interdisciplinary Research Centre in Biomedical Materials (IRCBM) COMSATS Institute of Information Technology, 54600 Lahore, Pakistan
Department of Dental Materials, Institute of Basic Medical Sciences, Khyber Medical University Peshawar, Pakistan
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Lignocellulosic biomass covers a vast area of the globe and contains many valuable components that can be en-
visaged for numerous renewable products. The valorization of these components is hindered by its cumbersome
isolation due to the recalcitrant nature of lignocellulosic biomass. Acidic ionic liquids (AILs) have emerged in this
field as effective pretreatment solvents. The unique property of disrupting lignin-cellulose-hemicellulose bonds
and dissolving lignin makes these ILs significant for the separation of lignin and cellulosic constituents. In this
study three protic ionic liquids based on pyridinium cation and hydrogen sulfate anion (HSO−4 ) have been syn-
thesized; [PyH][HSO4] (IL1), [PyH][HSO4. (H2SO4)] (IL2), and [PyH][HSO4 . (H2SO4)3] (IL3) and utilized for the iso-
lation of lignin from wheat straw. The synthesized ILs were characterized by NMR and FTIR analyses. The lignin
yield was optimized with respect to reaction temperature, time, biomass loading, and type of IL. The lignin re-
moval efficacy of the ionic liquid with highest ratio of acid [PyH][HSO4.(H2SO4)3] (IL3) is indicated by high
delignification (79%) and lignin recovery (77%) under mild conditions (60 °C, 2 h). The cellulose rich material
(CRM) and isolated lignin were characterized by FTIR, SEM, TGA, GPC, and HSQC NMR to assess their structure,
molecular weight and stability. The cellulosic part was further hydrolyzed enzymatically to evaluate the capabil-
ity of the recovered pulp to generate glucose. An appreciable yield of reducing sugars (85%) substantiates high
lignin removal.
Received 5 November 2020
Received in revised form 4 December 2020
Accepted 7 December 2020
Available online 24 December 2020
Keywords:
Protic ionic liquids
Wheat straw
Lignin
Delignification
Enzymatic hydrolysis
© 2020 Elsevier B.V. All rights reserved.
1. Introduction
specific lignocellulosic biomass source. While cellulose and hemicellu-
lose are mostly considered for their usefulness as a source of energy
Lignocellulosic biomass is a widely investigated material mainly for
the sake of the generation of energy and partially for many useful and
renewable chemicals having numerous applications in industry and
daily life [1]. The remarkable products which can be obtained from eco-
nomical biomass sources are bioethanol, bio-gas, bio‑hydrogen,
hydroxymethylfurfural (HMF), levulinic acid (LA), and lignin-based
products. The fascination of lignocellulosic biomass materials lies in
their ubiquitous and economical availability in nature and huge stocks
from waste materials such as crop residues and municipal waste. Ligno-
cellulosic biomass is largely composed of three components known as
cellulose, lignin, and hemicellulose in varying amounts [2]. The compo-
sition is strictly dependent on the type, region, and season of growth of
and other materials of industrial and daily life importance; lignin is
also very significant. Lignin can be used as a low-value heating fuel,
binder and dispersant [3], also it may become a generous source of
value-added compounds, such as adhesives, polyurethane, polyesters,
biofilms and biologically active polyphenols (aromatics) [4–9]. Lignin
contains a variety of aromatic compounds that are currently obtained
from fossil fuels and it, therefore, may be anticipated as the most com-
petitive candidate for this purpose [4] owing to large deposits of ligno-
cellulosic materials in nature.
The utilization of this potential of lignin and ultimately lignocellu-
losic biomass is hindered by the adamant nature of biomass polymeric
structure. Fractionating lignocellulosic biomass into its major fractions
is therefore indispensable for developing an economically viable
biorefinery process. Particularly, an effective and low-cost method for
extraction of lignin from biomass is.
⁎
Corresponding author.
⁎⁎ Corresponding author at: Interdisciplinary Research Centre in Biomedical Materials
(IRCBM) COMSATS Institute of Information Technology, 54600 Lahore, Pakistan.
E-mail addresses: malihauroos.chem@pu.edu.pk (M. Uroos),
essential for facile access to carbohydrate fraction and the genera-
tion of significant products from lignin [4]. Previously the most frequent
commercial method for lignin extraction from biomass has been Kraft
nawshadmuhammad@cuilahore.edu.pk (N. Muhammad).
https://doi.org/10.1016/j.molliq.2020.115013
0167-7322/© 2020 Elsevier B.V. All rights reserved.

A.M. Asim, M. Uroos, S. Naz et al.
Journal of Molecular Liquids 325 (2021) 115013
method (for paper and pulp). However, this process involves high tem-
peratures and environmentally hazardous chemicals that need to be
tackled to develop eco-friendly strategies.
2. Experimental
2.1. Materials
Ionic liquids are green chemicals having amazing features with
solvating ability being the most prominent. The solvent and cata-
lytic abilities of ILs in the field of biomass processing are gaining
growing attention. The isolation of lignin from biomass is accom-
plished using different ionic liquids; the most common among
which are protic ionic liquids (PILs) [10–14]. Protic ILs are the
ionic liquids that possess acidic functionality either in cation or
anion (or both) [15]. Significance of PILs lies in their facile synthe-
sis; relatively mild biomass processing and the valorization of bio-
mass they also afford through efficacious removal of lignin. The
lignin obtained from pretreatment with PILs undergoes very little
change in its structure, thus further processing for bio-refinery
products from lignin is rendered simple and efficient. Achhinivu
et al. [13] utilized protic ILs for the extraction of lignin from corn
stover. In this study Pyrrolidinum acetate ([Pyrr][Ac]) exhibited
good lignin extraction (75%) at 90 °C for a pretreatment time of
24 h. Recently Gschwend and coworkers [16] used ultra-low cost
Brosted acidic ionic liquid for highly efficient lignin removal
(80%). In addition to high lignin removal, the ionic liquid was re-
covered proficiently (>99%) and reused successfully. Hu et al.
[17] succeeded in high lignin removal from Coir and Poplar bio-
mass using [BMIM] [HSO4] in aqueous alcohols. As a result of
pretreatment with this system, CRM (cellulose rich material) con-
taining very low lignin content was obtained (0.95%). The lignin
extraction was characterized with almost the same structure as
that in milled wood from the above two biomass sources. Other ex-
amples where PILs have been employed for pretreatment and
delignification may also be mentioned [18–22].
The analytical grade chemicals i.e. Pyridine (Riedel-de Haën) and
72% sulfuric acid (Sigma) were purchased and used without further pu-
rification. Wheat straw biomass was purchased from the local market in
Lahore Pakistan. All the solvents i.e. ethanol, methanol, acetone, etc.
were distilled before use.
2.2. Synthesis of ILs
Ionic liquids containing clusters of hydrogen sulfate anion and molec-
ular H2SO4 (HSO−4 . (x-1)H2SO4) were synthesized following the method
of synthesis of imidazolium ionic liquids already reported in the litera-
ture [23]. One mole of pyridine was reacted with a different number of
moles (1, 2, and 4 mol) of 72% H2SO4 initially at low temperature
(dropwise addition of the acid at 0–5 °C) and then at room temperature
for one hour. The reaction was monitored by TLC using a solvent mixture
of n-hexane and ethyl acetate (1:1). Very good yields (80–95%) were ob-
tained from all the synthetic reactions using fixed amount of pyridine
and varying number of moles of H2SO4. The reaction showing the synthe-
sis of ILs is given in the following scheme (Scheme 1).
2.2.1. NMR data of PILs
The H NMR data of the synthesized ILs is given below. Notations H1,
H
2, H3 and H4 refer to Fig. 2.
1H NMR (DMSO-D6, 400 MHz): IL1: δ 8.1–8.3 (2H, m, H4), 8.5–8.7
(1H, m, H3), 8.8–8.9 (2H, m, H2), 10.2 (5H, br, H1); IL2: δ 8.01–8.1 (1H,
m, H4); 8.51–8.71 (2H, m, H3); 8.87–8.89 (2H, m, H2); 10.2 (9H, br,
H1) IL3: δ 6.98–7.99 (1H, m, H4); 8.48–8.53; 8.7 (2H, m, H3); 9.48
(20H, br, H1)
Here we describe the use of protic acidic ionic liquids (PAILs)
based on pyridinium cation and clusters on HSO−4 anion (HSO4−
.
(H2SO4)
where X = 1, 2, 4) for lignin extraction from wheat
2.2.2. Pretreatment
x-1
straw. The synthesized ionic liquids, after characterization with
NMR and FT-IR, were used for the pretreatment of wheat straw
to separate the lignin and cellulosic constituents. The study ex-
hibits that anion-clusters based PILs act as promising solvents in
extracting lignin from wheat straw. The lignin extraction has
been optimized regarding temperature, time, and type of IL. To
study the effect of pretreatment, the pulp and extracted lignin
were characterized through compositional analysis (NREL proto-
col), FT-IR, SEM, GPC, HSQC, and TGA. Furthermore, the raw bio-
mass and acquired delignified cellulose pulps were subjected to
saccharification catalyzed by cellulase enzymes (Novozymes). The
release of glucose from the saccharification of biomass samples
was determined using the HPLC method employing the aminex
HPX-87P column.
The biomass was crushed and ground and suitable particle size was
obtained using mesh sieves. The subsequent method for pretreatment
and separation of lignin is given in the supplementary information.
2.2.3. Compositional analysis
Compositional analysis of the untreated and pretreated pulp sam-
ples was carried out using the protocol established at NREL [24]. The de-
tailed method is provided in the supplementary information.
2.3. Characterization
The acid to the base ratio in the synthesized ILs was precisely main-
tained using Mettler-Toledo Compact Titrator G20S. The FT-IR analyses
of the ILs, untreated biomass, pretreated samples (CRM), and extracted
Scheme 1. Synthesis of ionic liquids with different mole ratios of H2SO4 and different synthesized ionic liquids with their designations
2

A.M. Asim, M. Uroos, S. Naz et al.
Journal of Molecular Liquids 325 (2021) 115013
lignin were conducted using the Agilent Cary 630 FTIR instrument. The
spectra were recorded within the range 4000–650 cm−. The NMR spec-
tra of the ionic liquids were recorded on Avance AV-400 MHz using
DMSO‑d6 as a solvent. Thermogravimetric analysis (TGA) of the original
biomass (untreated), pretreated biomass (using PILs), and lignin sam-
ples were carried out using Perkin Elmer TGA Pyris Diamond USA. The
change in mass (%) during TGA analysis was observed within the tem-
perature range 65–960 °C with a heating rate of 10 °C/min and the
flow of nitrogen gas at 100 mL/min. HSQC of the lignin samples was
done using Bruker 600 MHz spectrometer (pulse sequence hsqcetgpsi2,
the spectral width of 10 ppm in F2 (1H) having 2048 data points and for
13C 160 ppm in F1 with 256 data points, 16 scans and 1 s interscan
delay). For GPC measurements an Agilent 1260 Infinity instrument
was used which had a Viscotek column set (AGuard, A6000M, and
A3000M). For the detection of the signals, the Agilent 1260 Infinity
RID detector was used. For sample preparation, 20 mg of lignin samples
were dissolved in a mixture of GPC grade DMSO and an aqueous solu-
tion of LiBr (eluent, 1 g/L) and filtered through 0.2 μm syringe filter.
The sample was run at a flow rate of 0.4 mL/min at 60 °C. Ten pullulan
standards (Agilent calibration kit, 180 < Mp < 780,000) were used for
the calibration of the instrument. SEM images were obtained using a
Scanning electron microscope (Model VEGA TESCON) with varying
pressure. Gold sputtering was used and images with different magnifi-
cation (200, 3000 x) were scanned.
Fig. 2. Designation of different protons in the general structure of ionic liquids.
with H2SO4 molecules for the formation of clusters centered on the
anion. The strong hydrogen bond takes the oxygen atom away from sul-
fur and thus wavenumber is reduced. As seen in the spectra of the latter
two ILs (IL2 and IL3), there are peaks in the range 1370–1430 cm−1
which are weak in case of IL1. These peaks are in fact due to hydrogen
bond (O-H) formation between hydrogen atoms of H2SO4 molecules
and oxygen atoms of HSO−4 anion resulting in cluster formation as
shown in Fig. 2. The peaks assigned to different bonds are listed in
Table S1 in the supplementary information.
3. Results and discussion
3.2. NMR analysis of ILS
3.1. FT-IR analysis of ionic liquids
Fig. 3 contains the comparison of different peaks in the 1H NMR
spectra of the ILs used in this study. For IL1 (Fig. 3a), the most downfield
signal (δ=10.22) corresponds to HSO−4 proton and or nitrogen proton.
The spectrum shows that the aromatic protons do not give a well-
defined coupling pattern so these are being termed as multiplets. The
multiplet centered at δ8.3 is assigned to two ring protons denoted as
proton 4 in the pyridinium ring (Fig. 2). The signal for proton at position
3 (centered at 8.60) and 2 (at 8.06) each is a multiplet but the more
downfield among these two is the one which is closer to ring nitrogen
(at position 2 in Fig. 2).
FT-IR spectra of the synthesized ionic liquids are given in Fig. 1.
There are certain peaks in these spectra which correspond to the anionic
part of ILs which is of real interest. Concentrated sulfuric acid bears both
the molecular H2SO4 and anion HSO− therefore, it might contain two
types of frequencies [25]. The single bond S-O frequencies are found
within the range 800–950 cm−1, the double bond S=O stretches appear
from 950 to1300 cm−1 whereas the S-OH bonds fall in the range
1300–1450 cm−1 [25,26]. In IL1, the peak at 862 cm−1 corresponds to
S=O vibration and at 1029 cm−1 belongs to S=O stretching in the cor-
responding HSO−4 . The peaks at about 1150 cm− exhibit various vibra-
tions of SO3 where the most intense vibration peak appears at
1143 cm−1 [25]. In the other two ILs (IL2, IL3) the peaks corresponding
to S=O are featured with weak intensities and are shifted towards
lower wavenumber which is a consequence of strong hydrogen bonds
For IL2 (Fig. 3b), the peak for HSO−4 has shifted slightly upfield (δ=
10.20) because of hydrogen bond formation of an additional mole of
H2SO4 (1:2) with the anion HSO−4 . The signal centered at 8.61 is a mul-
tiplet and it corresponds to the proton at position 4 whereas the peak at
8.078 is again a triplet and is assigned to protons at position 3. In the
same line the NMR spectrum for IL3 (Fig. 4c) exhibits peaks for protons
at positions 2 (multiplet at δ = 8.723), 3 (multiplet at δ = 7.96) and 4
(multiplet δ= 8.50). The most interesting part of the spectrum for IL3
is the peak at 9.48 which is assigned to protons around HSO−4 anion.
Its integration shows almost four times the number of protons of
HSO−4 anion in IL1. It means that adding four mole equivalents of
H2SO4 increases the number of hydrogen bonds around the anion. How-
ever, the δ value of this proton in IL3 shows it to be far down filed com-
pared to that in IL2 (where it is 10.20) implying that further increase in a
number of protons around HSO−4 results in relatively weaker hydrogen
bonds. This is in accordance the findings in the literature [23,27].
1377
IL3
1380
IL2
1119
1025
980
876
1124
1031
4. Compositional analysis and biomass characteristics
IL1
983
871
The composition of original(untreated) wheat straw and that of
pretreated wheat straw samples was determined from the composi-
tional analysis method following the NREL protocol [24]. It was found
that the wheat straw contains 37.18% glucans, 23.65% xylans. The lignin
is 21.86% as a sum of acid soluble lignin (ASL) and acid insoluble lignin
(AIL). The whole composition of the raw wheat straw as determined
from the compositional analysis is given in the following diagram
(Fig. 4). The amounts of different components are shown in percent of
the total.
1029 1143
862
986
600
750
900
1050
1200
1350
1500
1650
1800
wave number (cm-1)
Fig. 1. Comparison of FT-IR spectra of the three ionic liquids
3

A.M. Asim, M. Uroos, S. Naz et al.
Journal of Molecular Liquids 325 (2021) 115013
Fig. 3. 1H NMR spectra of different pyridinium ionic liquids a) IL1, b) IL2, and c) IL3 (using DMSO‑d6 as a solvent)
The composition of the biomass samples pretreated under opti-
mized conditions is provided in the following Table 1 which shows
the amount of different components as a percentage of the total.
Different characteristics of the pretreated biomass and change in
their amounts are depicted by the graphs in Fig. 5. All the quantities
are expressed in percent w/w of the theoretical maximum (amount in
untreated biomass). Percentage of pulp recovery was calculated
from gravimetrically measured amounts of pulp compared to the
initial amount of biomass. The percent of lignin was determined by
weighing the extracted lignin and comparing it with the amount of lig-
nin in the original biomass. The percentages of hemicellulose removal
and delignification were calculated using the relative amounts of
hemicellulose and lignin in untreated biomass and pretreated samples
from the compositional analysis method. The detailed procedure of
the two methods is described previously.
It is clear from the graph that with the increase in time of pretreat-
ment the pulp recovery is lower as a consequence of delignification
and removal of hemicellulose. The least pulp recovery was obtained in
case of pretreatment with IL3 which is obvious in low lignin content
and hemicellulose component of the pretreated biomass as given in
the above table (Table 2).
The removal of hemicellulose from the biomass as a result of pre-
treatment is more prominent compared to the delignification. In
all the graphs it is obvious that hemicellulose content lowers with
Glucan Xylan Arabinan ASL AIL Ash Extracꢀves
37.18%
19.37%
18.67%
11.56%
3.19%
4.28%
6.73%
Fig. 4. Composition of raw wheat straw used in this study. ASL: acid soluble lignin, AIL: acid insoluble lignin.
4

A.M. Asim, M. Uroos, S. Naz et al.
Journal of Molecular Liquids 325 (2021) 115013
Table 1
Table 2
Composition of pretreated biomass samples determined by compositional analysis; for IL1,
IL2 at 100 °C, and for IL3 at 60 °C for 2 h.
Molecular weight and polydispersity index of isolated lignin using IL1, IL2 at 100 °C and IL
at 60 °C (2 h, 5% loading).
Ionic Liquid Glucan Xylan Arabinan ASL
AIL
(%)
Ash Mass Loss (%)
(%)
Ionic Liquid
Mn (Da)
Mw (Da)
PDI
(%)
(%)
(%)
(%)
IL1
IL2
IL3
1688
1486
1414
8855
7900
7001
5.2
5.3
4.9
IL1
IL2
IL3
55.8
59.6
61.5
8.15
6.39
3.45
1.24
1.06
0.82
1.16 7.84 7.2
1.29 5.87 7.1
0.95 2.84 9.8
18.61
18.31
21.64
Mw: average molecular mass, Mn: number average molecular mass, PDI: polydispersity
index.
increasing time of processing. But the more prominent increase is seen
after 1 h in all the cases. The recovery of hemicellulose is not possible
from the solvent mixture, unlike lignin. Which may be due to the forma-
tion of other products from hemicellulose e.g. furfural [28].
with the constituents of biomass with; IL1 and IL2 at 100 °C for 2 h; IL3
at 60 °C for 2 h
5. Effect of factors on lignin extraction
In previous studies, those ILs are reported for the extraction of lignin
which has higher hydrogen bond basicity [29,30]. This is indicated here
as well when we compare our results with the ILs used in this study.
With more H2SO4 content the ability of the IL to make hydrogen
bonds with the biomass components is also enhanced; as a conse-
quence, more delignified pulp having less hemicellulose content is ob-
tained. The delignification is also more effective as the time of
pretreatment progresses where the peak delignification in the case of
IL1 (59%) and IL2 (69%) is achieved at 100 °C for 2 h and in case of IL3
(79%) at 60 °C for 2 h. Thus ionic liquid IL3 removes the lignin from
wheat straw much more effectively as compared to the other two ILs.
The efficiency of this IL owes to the clusters of the anion HSO−4 and
H2SO4 molecules which are capable of extensive hydrogen bonding
The pretreatment of the wheat straw samples was carried out under
different conditions of time, temperature, particle size, and type of ionic
liquid. The percentage yields of lignin and cellulose rich material (CRM)
have been calculated by comparing the amounts of isolated lignin and
CRM after pretreatment with the amounts of these components present
in the untreated biomass (theoretical maximum).
The percent yield of extracted lignin was calculated relative to the
amount of lignin in the original biomass (theoretical maximum) using
the following formula.
Ml
Yl ¼
ꢀ 100%
MoꢀMODW
100
where Yl is the percent yield of lignin to be calculated, Ml is the amount
of lignin extracted from the biomass sample, Mo is the percent of lignin
in the original biomass and MODW is the oven dried amount of biomass
sample initially taken.
A discussion of the effect of different pretreatment conditions is
hereby elaborated under the following sub-sections.
IL1
100
80
60
40
20
0
5.1. Effect of temperature
As expected, the extent of pretreatment and the lignin extraction
was enhanced with the increase in temperature. Out of the three
types of ionic liquids, IL1 ([Py] [HSO4]) and IL2 ([Py] [HSO4.H2SO4]
were used for the range 40–100 °C. The third ionic liquid IL3 ([Py]
[HSO4.3H2SO4] was used at 40 °C, 50 °C, and 60 °C. The reason why
this IL was employed at lower temperatures is blackening (charring)
of the biomass at higher temperatures. As shown in Fig. 6 the maximum
lignin recovery was achieved at 60 °C with IL3 (77%) while lignin yield
with IL1 and IL2 came to be 59% and 69% respectively. The observed
amounts of extracted lignin at different temperatures are coherent
with the results in different studies [12,13]. These studies exhibit that
0
30
60
90
120
IL2
100
80
60
40
20
0
0
30
60
90
120
100
80
IL3
100
80
60
40
20
0
60
IL1
IL2
IL3
40
20
0
0
30
60
90
120
40
50
60
80
100
Lignin content
Removal of Hemicellulose
Delignificaꢀon
Pulp recovery
Temperature (°C)
Fig. 5. Main indicators of processing of wheat straw after pretreatment.
Fig. 6. Percentage of lignin extracted from biomass using ionic liquids at.
5

A.M. Asim, M. Uroos, S. Naz et al.
Journal of Molecular Liquids 325 (2021) 115013
100
80
60
40
20
0
the amount of extracted lignin increases with elevating temperature.
This owes to the higher vibrations of the bonds between lignin and cel-
luloses [12]. The ionic liquid IL3 extracted higher amounts of lignin even
at low enough temperature (60 °C) which is due to corresponding
higher acidity (more hydrogen bond basicity); a significant characteris-
tic for appreciable lignin extraction. The following figure exhibits the
amounts (%w/w of total lignin in the initial biomass) of lignin extracted
by employing different ionic liquids Fig. 6 different temperatures
(180–800 μm, 5%, 2 h).
IL1
IL2
IL3
5.2. Effect of time
Pretreatment of the wheat straw was carried out for various time
durations (0.5 h to 2 h). The ionic liquids IL1 and IL2 took relatively
more time to appreciably extract lignin; however, IL3 appeared to be
more effective. Fig. 7 shows the amounts of lignin extracted (% of theo-
retical maximum) using different ILs for varying time durations. In-
creasing the time of pretreatment improves the efficiency of the
process. However, beyond a pretreatment time of 2 h the biomass black-
ening occurred and very low or no yield of lignin resulted. It may also be
noted that the ionic liquid expected to have the highest acidity (IL3) pro-
duces the best result (77%) in coherence with the reported literature
[13,14]. In the previous studies [11,13,28] the pretreatment time for ex-
traction was more extensive (up to 24 h) and the higher lignin yields
were attained at relatively higher temperatures compared to the pres-
ent study temperatures (5% biomass load,60 °C for IL3, and 100 °C for
IL1 and IL2).
5
10
15
Biomass loading (% of IL)
Fig. 8. Effect of biomass loading (%) on pretreatment and lignin extraction (60 °C, 2 h)
5.4. Process optimization
In this study, three ionic liquids, denoted by IL1, IL2, and IL3, were
used for the pretreatment of wheat straw. The ILs were produced by
treating a fixed amount of pyridine (base) with different mole ratios
of 72% sulfuric acid. Many parameters were tested as discussed
above and the most efficient pretreatment was observed in the case
of ionic liquid IL3. A possible justification of the highest efficiency of
this IL is probably its higher acidity (hydrogen bond basicity) which
is expected to be higher compared to the other two ionic liquids
[32]. The graph in Fig. 9 indicates that the maximum amount of lignin
was isolated from pretreatment with IL3. The optimum yield (77%)
was obtained by pretreatment at 60 °C for 2 h. The optimized lignin
yield with IL1 and IL2 was found to be 58% and 69% after 2 h at
100 °C. The previous findings reported in the literature show that ILs
with higher acidities or more ability to make hydrogen bonds can pro-
vide better dissolution and hence effective isolation of lignin
[13,14,33]. Here we see that increasing the number of mole equiva-
lents of acid in ionic liquid improves the delignification of biomass
and hence renders it more viable for further processing. Our
delignification results reveal that pyridinium based PILs containing
clusters (dimers and trimers) on anion are more efficient as compared
to the monomeric ILs, formed by equal mole equivalents of acid and
base, used previously of lignin extraction (Particle size:180-800 μm,
biomass loading 5%, time: 2 h).
5.3. Effect of biomass loading
Previous studies, regarding pretreatment of biomass and lignin ex-
traction, show that low biomass loading is more effective than higher
biomass loading. This is because the contact of IL ions and the biomass
particles increases when small weight is loaded [11,31]. In this case,
too, the PILs gave the results accordingly in favor of low biomass load-
ing. But in our study, the maximum yield is obtained using IL3 at pretty
mild conditions (60 °C, 2 h). This is attributed to the higher hydrogen
bond-forming content of this IL. As exhibited by the graph in Fig. 8,
the removal of lignin is more efficiently achieved in case of low loading
of biomass. Here biomass loading of 5% (77%), gave the highest amount
of lignin followed by 10% (64%), and the least lignin extraction was real-
ized with 15% (53%) biomass loading. The low biomass loading allows
the ions of IL and the biomass particles to interact more freely because
of small sized particles [31]. Another probable reason might be the in-
creased density of the reaction mixture in case of heavier biomass
loading.
100
80
100
77%
80
69%
60
IL1
60
40
20
0
58%
100
IL1
lL2
IL3
40
IL2
IL3
20
IL3
IL2
0
IL1
40
30
60
90
120
50
60
Temperature (°C)
80
Time (minutes)
Fig. 7. Percentage of lignin extracted from biomass using ionic liquids at different.
Fig. 9. Comparison of different ionic liquids used in this study for their efficiency.
6

A.M. Asim, M. Uroos, S. Naz et al.
Journal of Molecular Liquids 325 (2021) 115013
1650
120
100
80
60
40
20
0
1513
1733
a
b
1430
b
a
d
c
c
d
0
200
400
600
800
Temperature (°C)
Fig. 11. Thermogravimetric analysis (TGA) of different biomass samples a) untreated,
b) IL1 treated (100 °C, 2 h), c) IL2 treated (100 °C, 2 h) and d) IL3 treated (60 °C, 2 h, 5%)
600
900
1200
1500
1800
Wave number (cm-1)
biomass samples implies the effectiveness of pretreatment. Thermo-
graph shows reduced stabilities of the biomass samples after treatment
with different ILs. The biomass sample treated with IL3 (d) is the least
stable; this is an outcome of the most reduced crystallinity, which
shows the efficacy of this IL in pretreatment and delignification of
wheat straw. The overall sequence of stability of treated and untreated
biomass samples is: untreated > IL1 > IL2 > IL3.
Fig. 10. FT-IR spectra of the biomass samples; a) untreated, b) IL1 treated (100 °C, 2 h),
c) IL2.
6. Analysis of biomass
This is based on the shape and the onset temperatures of TGA curves.
The onset temperature of the untreated biomass sample is 332 °C
whereas, for the biomass samples treated by IL1, IL2, and IL3, it is
310 °C, 220 °C, and 205 °C respectively. The given results can be justified
based on the change in morphology of the biomass after pretreatment.
The untreated biomass is more recalcitrant owing to the highly crystal-
line network of cellulose, hemicellulose, and lignin. After pretreatment,
the appreciable removal of lignin makes the biomass more amorphous
compared with the original (untreated). The removal of lignin occurs
as different types of bonds; lignin-cellulose and lignin-hemicellulose
(responsible for highly crystalline nature), are broken. Along with the
removal of lignin, some amount of cellulose and hemicellulose is also
lost resulting in a further reduction instability.
6.1. FT-IR analysis
The following diagram (Fig. 10) exhibits a comparative overlap of
the FT-IR spectra of untreated biomass and IL-treated biomass samples.
The peaks at 1430, 1371, 1318, 1162, 1119, 1025, and 897 cm−1 are as-
sociated with cellulose. The signal at 897 cm−1 is assigned to the glyco-
sidic linkage (at C1) and the peak at 1025 cm− corresponds to the C-O-C
stretching in glucopyranose ring [34]. This peak is obvious in all the
samples but in the case of biomass sample pretreated using IL3 (mole
ratio 4:1) it is relatively weaker. This is ascribed to some loss of carbohy-
drates during pretreatment which may be possibly due to higher acidity
of the PIL. The peak at about 1160 cm− is attributed to C-O anti-
symmetric stretching. The CH2 symmetric bending is represented by
the peak at 1430 cm− [35]. The signal intensity of this band has reduced
which is an indication of less crystalline cellulose in the IL-pretreated
samples of biomass [35]. The peak at 1319 cm− is due to CH bending
in carbohydrates and 1365 cm− is for CH bending in cellulose. The
peak at 1513 cm− is assigned to the aromatic ring conjugated bonds
which is a characteristic of lignin. This peak (1513 cm−) has signifi-
cantly reduced intensity verifying appreciable removal of lignin from
the biomass after pretreatment. The peak at 1650 cm− is also character-
istic of phenolic OH bonds in lignin. This peak has also been rendered
weak in the pretreated biomass. Out of all the ionic liquids used, the
IL3-pretreated sample exhibits the most pronounced reduction in peak
intensity. This is in coherence with the yields of lignin as stated above
treated (100 °C, 2 h) and d) IL3 treated (60 °C, 2 h, 5%)
6.3. Scanning Electron Microscopy (SEM)
The original biomass and regenerated CRM were subjected to scan-
ning electron microscopy (SEM) to verify the effect of pretreatment
on the morphology of the polymeric network of biomass. Previous stud-
ies on the utilization of protic ILs in biomass valorization demonstrate
that an increase in acidity disrupts the morphology [13]. This was ex-
pected in the case of our study also and it is clear from the SEM images
(Fig. 12) that the PILs used in this study have affected the morphological
arrangement of biomass. The SEM image of untreated biomass shows a
flat structure that undergoes disruption when treated with the PILs. This
accords with the results obtained in the previous studies [13,35]. The
flakiest structure is obtained in the case of ionic liquid IL3. This is in
agreement with the percentage of extracted lignin (highest in case of
IL3) and FT-IR spectra of the biomass samples (100 °C, 2 h), c) IL2 treated
(100 °C, 2 h) and d) IL3 treated (60 °C, 2 h, 5%)
6.2. Thermo-gravimetric Analysis (TGA)
Thermogravimetric analysis was done to assess the stability varia-
tion caused by pretreatment. Thermographs of untreated and
pretreated biomass is furnished in Fig. 11. It is evident that there are
two plateaus for each curve; the initial plateau indicates a phase of
moisture removal loosely bound on the surface after which a steep fall
occurs as a result of a loss of mass from biomass due to degradation
[36]. Finally, the curve straightens and goes horizontal up to the end;
and in this phase, the graph corresponds to the charring of carbohy-
drates [37]. The difference in the stability of the untreated and treated
7. Saccharification
To evaluate the effect of delignification and potential of recovered
pulp from untreated and treated biomass enzymatic saccharification
was performed at 50 °C and 250 rpm for 7 days. The amount of sugars
was determined using the HPLC technique equipped with an aminex
HPX-87P column. The graph (Fig. 13) exhibits the variation in quantity
7

A.M. Asim, M. Uroos, S. Naz et al.
Journal of Molecular Liquids 325 (2021) 115013
Fig. 12. SEM images of different biomass samples; a) untreated, b) IL1 treated.
glucose from the untreated wheat straw and optimized pretreated sam-
ples after different time durations. The raw biomass sample was run
without any ionic liquid-treatment and it yielded only 21% glucose of
the theoretical maximum. It was found that the extent of yield was en-
hanced with increasing time of pretreatment at respective tempera-
tures. It is apparent that the sample obtained after pretreatment with
IL1 gave the lowest yield of reducing sugars at all time-durations. The
amount of TRS obtained in this study is appreciably comparable and
even better than the yields in previous studies on PILs found in the
literature [35,38]. The biomass sample treated by IL2 provides the
highest TRS value (84%) after pretreatment at 100 °C for 2 h. While
under the same conditions, the yield of glucose from the IL1 treated
sample was 67%. This is in accordance with the previous studies
where the increase in saccharification yield with increasing extent of
delignification is reported [28,35,38]. However, for IL3 highest
delignification does not produce the highest amount of TRS. This may
be attributed to the deformation of the cellulosic structure of pulp due
to carbonization which in turn may be resulted from higher acidic con-
tent of the IL3 from enzymatic saccharification after 7 days (50 °C,
250 rpm).
100
80
60
40
20
0
8. Analysis of extracted lignin
8.1. HSQC NMR
Fig. 14 depicts structures of different fractions having relative posi-
tions marked as α, β, and γ. The wheat straw lignins obtained through
different methods have slightly different ratios of various fractions.
But a general inference is that it has a higher Guiacyl component than
Syringyl fraction [39]. This is also true about the lignin extracted using
IL3 under optimum conditions in this study.
Fig. 15 shows the prominent peaks in HSQC NMR within the range
ẟ1- ẟ9 (H NMR) and ẟ50 to ẟ130 (C NMR). The peaks of major compo-
nents are labeled respectively. The Guiacyl (G) unit is the most abun-
dant followed by Syringyl (S), Coniferyl (C), and other units, Tricin
Untreated
IL1
IL2
IL3
Biomass samples
Fig. 13. Yield of Glucose of untreated and pretreated wheat straw samples.
8

A.M. Asim, M. Uroos, S. Naz et al.
Journal of Molecular Liquids 325 (2021) 115013
Fig. 14. Significant bonds and subunits of lignin.
(T) and other less common units. The β-O-4 linkage (Aβ) has the highest
percentage of all the ether linkages which is an indication that there is
not much structural disruption in the structure of lignin recovered
from wheat straw under mild conditions (60 °C, 2 h). The aromatic por-
tion of the HSQC NMR spectrum (ẟC/ẟH: 90–150/6.0–7.7) is given in
Fig. 16. The peaks assigned to different positions of Guiacyl fraction of
lignin are shown. It is reported in the previous literature that peak for
G2 can be taken as the reference peak for other signals (G2cond., G5
and G6) [40]. This is because it mostly does not undergo any modifica-
tion or condensation during the extraction. However, G2 may be shifted
to G2 cond. (condensed form) due to modification in the G5 and G6 po-
sitions. From the areas of contours in the spectrum, it was found that the
intensity of G5 and G6 is lower than G2 and there is a prominent peak of
G2 cond. (H NMR 7.0–7.9; and 110–114C NMR). Thus it shows a moder-
ate condensation of lignin during the process. Some previous studies in-
dicate more severe degradation and condensation of the lignin
extracted using protic ILs [21,28]. In this case, we also found condensa-
tion of polymer which is obvious from the appearance of G2 cond. The
peak in the spectrum, but the extent of condensation is lesser
comparatively.
8.2. Gel Permeation Chromatography (GPC)
The Gel Permeation Chromatography was conducted for the lig-
nin extracted from wheat straw by pretreatment using the three
ILs. The GPC determined values of number average molecular weight
(Mn), average molecular weight (Mw) and polydispersity of different
lignin samples are given in Table.2. It is known previously that the
organosolve lignin mostly bears higher molecular mass compared
to ionosolve lignin [41,42]. Previous studies suggest that disruption
of lignin side chain linkages is caused by IL anions specifically protic
ILs [43]. In the present study, the molecular mass of the lignin de-
creases with the increasing ability of IL to make hydrogen bonds
and thus disrupt the lignin linkages. We can see that the lignin
from ionic liquid IL1 has the highest molecular mass and that from
IL3 has the lowest while lignin from IL2 being intermediate. The poly-
dispersity index (PDI) of lignin also differs by varying the ionic liq-
uid. IL1 and IL2 extracted lignin shows higher PDI compared to the
lignin isolated using IL3; thus showing more uniform structural fea-
tures of lignin extracted with the first two ionic liquids [44]. Thus the
above observations indicate that clustered ionic liquids extract
Fig. 15. HSQC NMR spectrum of the lignin extracted from wheat straw using IL3 at 60 °C for 2 h.
9

A.M. Asim, M. Uroos, S. Naz et al.
Journal of Molecular Liquids 325 (2021) 115013
Fig. 16. The relative intensities of different peaks of Guaiacyl unit in HSQC NMR of the lignin
lignin having fairly high molecular weight with better uniformity of
structure.
robust evidence for the presence of lignin. The absorption band from
1400 to 1500 cm−1 characterizes aromatic skeletal vibrations combined
with C-H symmetric stretching. The peak at about 1370 cm−1 pertains
to phenolic OH and that at about 1330 cm−1 exhibits C=O stretching
of syringyl unit. Absorption band at 1220 cm− characterizes C-C
stretching combined with C-O and C=O stretching. The peaks at
1140 cm−1 and 1116 cm−1 are the frequencies absorbed by C-H in
plane vibrations and ether linkage (C-O-) respectively. The absorption
bands within 800–850 cm−1 correspond to C-H out of plane vibrations
of Guaicyl units. A minor degradation effect can be observed in the case
of IL3 in which some peaks have reduced intensities. This owes to the
high acidity of this IL as pointed out by previous studies on lignin extrac-
tion using PILs [14,43].
8.3. FT-IR analysis of lignin
FT-IR spectra of lignin fractions isolated after pretreatment with
ILs are given in the following diagram (Fig. 17). The peaks in the
FT-IR spectrum which are generally assigned to lignin vary depend-
ing upon the type of lignin (e.g. hardwood lignin, softwood lignin).
But there are some common peaks which may be generalized to de-
scribe the spectra. Some important peaks are being considered here
for our purpose, which has been assigned the wavenumber values
from previous literature [3,45].
The band at 1730 cm−1 is for unconjugated C=O stretching and that
at about 1660 cm−1 for conjugated C=O stretching. The peaks between
1500 and 1600 cm−1 correspond to aromatic skeletal vibrations and are
8.4. Thermogravimetric analysis of extracted lignin
Thermograms of the extracted lignins after pretreatment with ILs
are furnished in Fig. 18. The change in mass (%) was observed within
the temperature range 60–960 °C. Different types of lignins have been
analyzed and reported using thermogravimetric analysis in literature
[46,47]. According to these studies, the major degradation of organosolv
1513
1220
1375
c
1112
817
b
100
90
a
80
70
c
60
50
40
30
b
a
500
1000
1500
2000
2500
0
200
400
600
800
1000 1200
Wave number (cm-1)
Temperature (°C)
Fig. 17. FT-IR spectra of lignin extracted from wheat straw using a) IL1, b) IL2, c) IL3.
Fig. 18. TGA curves of the three samples of lignin from a) IL1, b) IL2 and c) IL3.
10

A.M. Asim, M. Uroos, S. Naz et al.
Journal of Molecular Liquids 325 (2021) 115013
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As shown in Fig. 15, there are two plateaus for each curve. The initial
plateau indicates a phase of water removal after which a steep fall oc-
curs as a result of the loss of mass from biomass. Finally, the curve
straightens and goes horizontal up to the end. The two lignin samples
(IL1 and IL2) show a comparative degradational pattern but the lignin
extracted from IL3 shows much resistance under the same conditions.
This may, perhaps, be due to carbonization (charring) or ash content.
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9. Conclusion
Three ionic liquids have been synthesized from pyridine and H2SO4
and employed for pretreatment of biomass and consequent extraction
of lignin. The clustered protic ionic liquids are based on pyridine and
sulfuric acid in varying mole ratios. Results depict that the ILs are effi-
cient enough to extract lignin from wheat straw under mild conditions.
FT-IR of biomass samples supports the lignin removal which is further
strengthened by good TRS values and reduced stability evidenced by
TGA. The optimum TRS value (85%) could be furnished by the biomass
samples after pretreatment at 100 °C for 2 h with 5% biomass loading.
The optimum amount of extracted lignin (77%) was achieved by pre-
treatment with IL3, the ionic liquid containing 4 mol ratio of sulfuric
acid. The optimum conditions for lignin removal are 60 °C, 2 h, with
5% biomass loading. The characterization of the isolated lignin was
done using HSQC NMR, Gel Permeation Chromatography, FT-IR, and
TGA. The HSQC analysis showed some condensation of lignin during
the extraction process. The GPC results showed high molecular weights
and polydispersity indicating the less degraded structure with fair uni-
formity. Thermogravimetric analysis (TGA) further substantiated the
presence of stable lignin samples.
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Author statement
Azmat Mehmood Asim did experimental work as well as paper write
up, Sadia Naz also helps in paper writing and did some characterization
of the samples. Maliha Uroos and Nawshad Muhammad conceive the
idea and supervised the project.
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Declaration of Competing Interest
The authors have no conflict of interest (financial or otherwise).
Acknowledgements
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This work was financially supported by Higher Education Commis-
sion (HEC) Pakistan, project numbers: 8639/Punjab/NRPU/R&D/HEC/
2017 and TDF-03/294. Institute of Chemistry, University of the Punjab
is acknowledged for its support towards this project. Some analyses
were made at Chemical Engineering Department Imperial College
London; we highly acknowledge the facilitation and generous support
there.
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Appendix A. Supplementary data
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