Characterization of extracted lignin was performed by Fouriertransform
infrared (FT-IR) spectroscopy, enzymatic assay for
hemicellulose contamination, Thermogravimetric analysis (TGA)
and molecular weight distribution using gel permeation chromatography
(GPC). Functional groups of lignin (lignin:KBr ratio of 1:99
by weight) were analyzed by FT-IR spectroscopy (Spectrum 2000,
PERKIN ELMER, USA) at 4000e400 cm1 wave number at 4 cm1
resolution and 128 numbers of scan. The FT-IR spectra were averaged
from triplicate measurement. Hemicellulose contamination
was determined by hemicellulase hydrolysis (2 mL of black liquor:
10 mL of sodium acetate buffer pH 4.8: 3 mL of Accellerase 1500
enzyme) at 50 C for 48 h. The reducing sugar released was
measured by DNS assay (Miller, 1959) reported as glucose equivalence.
Gel permeation chromatography for the lignin molecular
weight distribution determination was performed in LiCl/DMF
system (Sjöholm, Gustafsson, & Colmsjö, 1999). The black liquor
(2 mL) was subjected to GPC on a 1.5 100 cm Sephadex LH 20
column in DMF containing 0.8% (w/v) LiCl in order to exclude the
hydrophobic ability of lignin molecules. Fractions (1.5 mL) were
collected and the lignin quantity was assayed by UVeVIs spectroscopy
at 280 nm in terms of absorbance. Thermogravimetric
analyzer (Pyris Diamond TG/DTA, PerkinElmer, USA) was used to
determine thermal stability of lignin samples with a heating rate of
10 C/min in nitrogen atmosphere and the temperature ranging
between 30 and 500 C.