Crop waste, Agricultural waste, Biomass, Rice husk, isolation, extraction, lignin, organosolv, XRD, SEM, elemental analysis, HSQC NMR, ATR, UV-Vis

1. Isolation of lignin by Organosolv process from
different varieties of rice husk: Understanding
their physical and chemical properties
Sandip Kumar Singh (Ph. D. student)
Research guide: Dr. Paresh Laxmikant Dhepe
Catalysis and Inorganic Chemistry Division
CSIR-National Chemical Laboratory, Pune, India
Tel, +91-20-25902024, Fax. +91-20-2502633,
Email: pl.dhepe@ncl.res.in
Websites: http://academic.ncl.res.in/pl.dhepe/group
Keyword: Crop waste, Agricultural waste, Biomass, Rice husk, isolation, extraction,
lignin, organosolv, XRD, SEM, elemental analysis, HSQC NMR, ATR, UV-Vis

2. Highlights & Keywords
1) Organosolv method used to isolate lignin from 3 rice husk (RH I, RH II & RH III)
to understand properties.
2) XRD analysis proves extracted lignin is not contaminated with polysaccharides.
3) ATR and NMR revealed presence of H, G, S & T substructures in varying
concentrations.
Lignocelluloses, Organosolv isolation, Lignin, Physico-chemical properties, NMR

3. Elemental analysis of lignocellulosic rice husk
I, II & III, pulps and ORGLs (oven dry basis)
Sample name[a] I II III
Elemental analysis Rice Husk (RHs)
(%)
C 36.9 37.7 37.2
H 5.6 5.4 5.7
O[f] 47.7 47.1 47.4
C/O ratio 0.77 0.79 0.78
MMF[b] C6.2H11.3O6 C6.4H11.0O6 C6.3H11.5O6
HHV[c] (±0.02 MJ/kg) 11.94 13.74 12.26
Elemental analysis of pulp (%) C 34.2 33.2 32.6
H 4.5 4.4 4.3
O[f] 51.6 50.5 51.1
C/O ratio 0.66 0.66 0.64
MMF[b] C5.3H8.2O6.0 C5.0H8.0O5.7 C5.0H7.9O6.0
HHV[c] (±0.02 MJ/kg) 7.01 6.00 5.92
Elemental analysis ORGLs (%) C 66.9 68.3 66.7
H 7.9 8.1 7.8
O[f] 25.2 23.7 25.5
C/O ratio 2.65 2.88 2.62
MMF[b] C10.1H12.8O3 C11.5H16.2O3 C10.1H12.8O3
HHV[c] (±0.02 MJ/kg) 29.5 30.52 29.21
DBE[d] 4.7 4.4 4.7
pH[e] 6.31 6.46 6.49
Colour light brown light brown light brown
[a]RHs: lignocellulosic rice husks, [b]MMF: monomer molecular formula, [c]HHV: higher heat value, [d]DBE: double
bond equivalence, [e]100 mg sample was suspended in 6 mL millipore water and shaking was done for 5 min. Later
lignin which is insoluble in water was allowed to settle down and then pH was measured. (pH of millipore water
was 6.92 at 25.6 oC) and [f]calculation based on elemental analysis by using (‘O’ wt.%, after ash correction) = 100-
(‘C’ wt.% + ‘H’ wt.%).

4. Higher heat values and double bond
equivalence
 higher heat values (HHVs or also known as calorific value was calculated using
Dulong formula (using equation 1)
𝐻𝐻𝑉 = 0.3383 × 𝐶 + 1.442 × 𝐻 −
𝑂
8
(1)
Where C= weight basis % of the carbon, H= weight basis % of the hydrogen
and O= weight basis % of the oxygen.
 The double bond equivalence (DBE) number (also known as degree of unsaturation)
calculated for extracted lignin based on equation 2 and considering MMF.
𝐷𝐵𝐸 = 𝐶 −
𝐻
2
+
𝑁
2
+ 1 (2)
Where C, H & N = number of carbon, hydrogen and nitrogen atoms obtained
from the monomer molecular formula.

5. UV-Visible spectroscopy of the isolated ORGL
samples from three different rice husk
225 250 275 300 325 350 375 400 425 450 475 500 525 550
Non-conjugated phenolic groups
Pi-Pi interaction of aromatic lignin
316
278
230
204
Absorbance(a.u.)
Wavelength (nm)
(a)
(b)
(c)
290 300 310 320 330 340 350
(c)
(a)
Absorbance(a.u.)
(b)
UV-Visible spectra of ORGL samples (a) RH I, (b) RH II and (c) RH III. 1 mg sample was dissolved
in 10 mL methanol solvent.

6. ATR spectra of isolated ORGL samples
800 1000 1200 1400 1600 1800 2800 3000 3200 3400 3600 3800
915
974
840
1036
1120
11701220
1260
1357
1422
1457
1510
1600
1645
1703
2853
2920
3330
17051710
1740
Transmittance(%)
Wavenumber (cm
-1
)
(c)
(b)
(a)
ATR spectra of ORGL derived from (a) RH I, (b) RH II and (c) RH III.

7. ATR band of ORGL derived from RH I, RH II
and RH III samples
Bands (cm-1) Assignment ORGLs Band Location (cm-1)
RH I RH II RH III
3400-3300 O-H stretching 3395 (w) 3320 (w) 3350 (w)
2960-2920 C-H asymmetric stretching in methyl and methylene group 2960 (w),
2925 (w)
2960 (w),
2923(s)
2960 (w),
2923 (m)
2850-2830 C-H symmetric stretching in methyl and methylene group 2850 (w) 2850 (s) 2850 (s)
1740-1680 C=O stretching in unconjugated ketone, carbonyl and ester
groups
1700 (w) 1705 (s) 1740 (s),
1710 (w)
1670-1640 C=O stretching in conjugated p-substituted aryl ketones 1650 (s) 1650 (w) 1650 (s)
1610-1590 Aromatic skeleton vibration plus C=O stretching 1600 (s) 1600 (w) 1605 (m)
1515-1505 Aromatic skeleton vibrations 1510 (s) 1510 (s) 1510 (s)
1470-1450 C-H deformation (asymmetric in -CH3 and –CH2-) 1455 (w) 1455 (m) 1455 (s)
1440-1420 Aromatic skeleton vibrations combined with C-H in plane
deformations
1420 (s) 1425 (w) 1425(m)
1370-1350 Aliphatic C-H stretching in CH3 (not –OCH3) and phenolic -O-H 1355 (w) 1370 (m) 1360 (s)
1270-1260 stretching C-H of G units 1260 (s) 1265 (w) 1265 (m)
1230-1210 C-C plus C-O plus C=O stretching (G condensed > G etherified,
typical of G units)
1215 (s) 1230 (w) 1230 (m)
1170-1160 Typical for H, G, S units of lignin 1165 (m) 1170 (w) 1165 (s)
1120-1115 Aromatic C-H in plane deformation 1120 (s) 1120 (m) 1120 (m)
1035-1030 Aromatic C-H in plane deformation (G>S) plus C-O deformation
in primary alcohols plus C=O stretching (unconjugated)
1035 (s) 1035 (s) 1030 (s)
845-830 p- substituted phenolic 835 (s) 830 (w) 835 (m)
Notes: w: weak, m: medium & s: strong band intensities.

8. 1H & 13C NMR of ORGL samples
1H & 13C NMR spectra of ORGL derived from a, b) RH I; c, d) RH II & e, f) RH III. All the spectra
recorded in the DMSO-d6 solvent.

9. 2D HSQC NMR of ORGL samples
HSQC NMR spectra of ORGL derived from a, b) RH I; c, d) RH II & e, f) RH III and main substructure present in
the lignin.

10. Assignments of 13C/1H HSQC of RH I, RH II
and RH III derived ORGL samples
Assignment δC/δH (ppm) ORGLs
RH I RH II RH III
Cβ-Cβ in phenylcoumaran substructures (Bβ) 53.55/3.47 - -
C-H in methoxyls 56.03/3.75 55.95/3.74 56.04/3.76
Cγ-Hγ in cinnamyl alcohol end group (Iγ) 60.07/4.03 59.88/4.026 60.07/4.04
- 60.26/3.6 - -
Cγ-Hγ in β-O-4’ substructures (Aγ) 60.32/3.42 59.97/3.58 59.88/3.49
- - - 62.18/4.12 and 4.25
- 63.86/3.33 - 63.14/3.34
Cγ-Cγ in β-5’ phenylcoumaran substructures (Bγ) 63.04/3.68 - -
- - - 65.06/3.98
- - - 69.09/5.20
Cα-Hα in β-5’ phenylcoumaran substructure (Bα) 87.60/5.46 - -
C8-H8 in tricin (T8) 94.57/6.57 - 94.70/6.57
C6-H6 in tricin (T6) 99.29/6.22 - 99.32/6.22
C2,6-H2,6 in (S) 103.95/6.99 - 103.93/7.00 and
104.44/6.70
- - - 104.60/7.48
- - - 104.60/7.48
C’2,6-H’2,6 in tricin (T’2,6) 104.68/7.34 - 103.93/7.00
C2-H2 in (G) 110.71/6.93 110.36/6.91 110.86/6.92
C3,5-H3,5 in p-hydroxybenzoate (PB3,5) - 115.7/6.62 115.6/6.53
C3,5-H3,5 in (H) 115.7/6.9 115.6/6.92 115.76/6.93
C5-H5 in (G) 115.85/6.77 115.6/6.76 115.76/6.78
C6-H6 in (G) 119.11/6.78 118.92/6.76 119.29/6.78
C2,6-H2,6 in (H) - 128.2/7.22 128.4/7.23
C2,6-H2,6 in p-hydroxybenzoate (PB2,6) - 130.9/7.49 130.5/7.47
Note: (-) not assigned.

11. Conclusions
 In summary, the organosolv lignins (ORGLs), with 12±3% yield and 93±5% mass
balance were isolated from diverse rice husk (RH) substrates using organosolv
procedure (water:ethanol, H2SO4) carried out at 180˚C for 1 h.
 In RH I and RH III derived ORGL samples, presence of T units and higher
concentration of G units are evident compared with RH II derived ORGL sample.
 In particular, RH III derived ORGL sample has properties and functional groups
similar to both RH I and RH II derived ORGL samples.

12. [1] S.K. Singh, P.L. Dhepe, Isolation of lignin by organosolv process from
different varieties of rice husk: Understanding their physical and chemical
properties, Bioresour. Technol., 221 (2016) 310-317.
[2] http://www.sciencedirect.com/science/article/pii/S0960852416312998
[3] http://academic.ncl.res.in/pl.dhepe/publications
Further reading..!