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

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

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.%).

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.

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

Absorb

ance (

a.u

.)

Wavelength (nm)

(a)

(b)

(c)

290 300 310 320 330 340 350

(c)

(a)

Ab

so

rba

nce

(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.

ATR spectra of isolated ORGL samples

800 1000 1200 1400 1600 1800 2800 3000 3200 3400 3600 3800

91

59

74

84

0

10

36

11

20

11

70

12

20

12

60

13

57

14

22

14

57

15

10

16

00

16

45 17

03

28

53

29

20

33

3017

05

17

10

17

40

Tra

nsm

itta

nce

(%

)

Wavenumber (cm-1

)

(c)

(b)

(a)

ATR spectra of ORGL derived from (a) RH I, (b) RH II and (c) RH III.

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.

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.

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.

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.

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.

[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..!