International Journal of Farming and Allied Sciences

Available online at www.ijfas.com

2013 IJFAS Journal-2013-2-24/1165-1171
ISSN 2322-4134 2013 IJFAS

Oxidative Production of Vanillin from Industrial

Lignin Using Oxygen and Nitrobenzene: A
Comparative Study
Alireza Shakeri1*, Sepideh Maghsodlou Rad2 and Ali Ghasemian3
1. School of Chemistry, University College of Sciences, University of Tehran, P.O. Box 13145-1357,
Tehran, Iran
2. Department of Chemistry, Faculty of Science, Golestan University, Gorgan, Iran
3. Pulp and Paper Department, Gorgan University of Agricultural and Natural Resources Sciences,
Gorgan, Iran
Corresponding author: Alireza Shakeri
ABSTRACT: Vanillin production from lignin oxidation is a biomass-based process that employs a byproduct of the pulp and paper industry. Lignins are phenolic polymers which are found in plant cell walls
with a structure depending strongly on the source of lignin and the process condition, which should be
adjusted for different samples. In this work, lignin was extracted from kraft cooking liquor of Pinus
eldarica. The amount of extracted lignin was 27%, based on oven dry weight of wood. Comparative
oxidation of Pinus eldarica lignin for producing vanillin was separately carried out using oxygen (batch
reactor) and alkaline nitrobenzene. The FT-IR spectrum of vanillin was similar to standard. Finally, the
vanillin concentration in product solution was analyzed by high performance liquid chromatography
(HPLC). The result showed that the amount of vanillin in oxidation of lignin with oxygen was more than
that of with nitrobenzene. The yield obtained from oxidation with oxygen and nitrobenzen was 4.2% and
1.2%, respectively.
Keywords: Vanillin, Lignin, FT-IR, HPLC, Nitrobenzene, Pinus eldarica
INTRODUCTION
Vanillin (4-hydroxy-3-methoxybenzaldehyde), (Fig. 1) is the major flavor constituent of vanilla. It has a wide
range of applications in food industry as a flavor agent and in perfumery as an additive, other applications as
antioxidant additive, antifoaming agent, vulcanization inhibitor and chemical precursor for pharmaceutical and
agrochemicals industries (Borges et al., 2009). The natural source of vanillin is from the seeds of the vanilla plant,
a member of the orchid family. However, the production of natural vanillin from vanilla pods covers only 0.2% of the
market requirement, and its production cost is very high (Mohamad Ibrahim et al., 2009). Vanillin may also be
obtained by chemical synthesis (Tarabanko et al. 1995), but the vanillin obtained in this way may not be called a
natural flavoring. Synthesis of vanillin from renewable sources should result in a greener and more sustainable
process. Vanillin was first synthesized from Eugenol (found in oil of clove) and later synthesized from lignincontaining sulfite liquor, a by-product of wood pulp processing in paper manufacture.

Intl J Farm & Alli Sci. Vol., 2 (24): 1165-1171, 2013

CHO

OMe
OH
Figure 1. Vanillin structure

While some vanillin is still made from lignin wastes, one possible path to produce vanillin based on biomass is
through controlled oxidation of lignin. Lignin is one of the most abundant of organic chemical material resources on
the plant. It is recoverable and available in large quantities from black liquor widely generated by the pulp and
paper industry. Currently, the vast majority of lignin is burned without any industrial utilization. Lignins are phenolic
polymers (Fig. 2) found in plant cell walls, which are responsible, with cellulose, for the stiffness and rigidity of plant
stems.

Figure 2. Lignin structure

The lignin (around 30% of wood composition) is a reticulated polymer composed by phenyl propane units
synthesized by radical coupling of mainly three hydroxypropanoids; coumaryl alcohol, coniferyl alcohol and sinapyl
alcohol (Fig. 3), where the monomers are connected by several ether (non condensed) and covalent carbon
carbon (condensed) bonds (Christiernin 2006). This polymer has two main functions in the tree, binding the fibers
to each other and providing rigidity. Softwood lignin consists almost exclusively of guiacyl type lignin derived from
coniferyl alcohol. Hardwood lignin consists mainly of syringyl units, derived from sinapyl alcohol that has two
methoxy-group, which results in lignin with a larger number of ether bonds. Hardwood lignin also contains on
average 30% guaiacyl units.
CH2 OH

CH2 OH

MeO
OH
P-coumaryl
alcohol

CH2 OH

OMe

OMe
OH
sinapyl
alcohol

OH
coniferyl
alcohol

Figure 3. Monomers of the Lignin

1166

Intl J Farm & Alli Sci. Vol., 2 (24): 1165-1171, 2013

The widely used chemical process to transform wood into pulp (kraft pulping or cooking) utilizes alkaline
pulping liquor (NaOH and Na2S). The main purpose of these chemicals is to dissolve the lignin in order to promote
cellulosic fibres separation from the wood matrix (Baptista et al., 2006).
In this project lignin was extracted from kraft black liquor of Pinus eldarica and analyzed by FTIR. Then Lignin
was oxidized with oxygen and also with nitrobenzene. In order to de-polymerization lignin to monomer the batch
experiments of lignin oxidation have been performed in a jacketed reactor with injection of oxygen. The vanillin
concentration in product solution with 2 ways were used an internal standard and analyzed with HPLC.
MATERIALS AND METHODS
All reagents and solvents for synthesis and analysis were commercially available and used as received without
further purifications.
Preparation of pulp samples
The kraft cookings were performed in a batch reactor, taken directly from digester. The Pinus chips were
cooked using the following constant conditions: active alkali charge, 25% (as NaOH); Sulfidity, 30%; liquor to wood
ratio, 6:1; cooking temperature, 170C (Ola and Ann-Soffi, 2006).
Preparation of lignin samples
The liquors with pH=14, were acidified with 2N H2SO4 until pH=1.5. The resulting precipitates were centrifuged,
washed by distilled water, and finally dried at 40C under vacuum over P 2O5 (Baptista et al., 2008).
FT-IR spectroscopy
Infrared spectra were recorded using KBr disks on a FT-IR PerkinElmer spectrophotometer (System 2000,
Perkin Elmer, Wellesly, MD, U.S.A.) to determine the functional groups present in the isolated lignin and compared
this lignin with a commercial lignin sample. The lignin samples used for FTIR were in powder form and the analysis
were performed in replicates (n = 5). The obtained spectra of the samples (on KBr pellets) were in the frequency
-1
range of 4000400 cm (Rajeev Bhat, 2009).
Preparation of Vanillin under oxygen condition
The first step to start the batch reactor experiments is to prepare the liquid mixture: The 500 ml of aqueous
solution with 60 g/l of lignin and 80 g/l of sodium hydroxide was prepared. The thermostatic bath was already
turned on with a 101C set point, Nitrogen was admitted to the reactor and another valve was opened to remove air
(oxygen) from the gas phase. After 10 minutes, the feed of nitrogen was stopped. At this time, the final pressure of
the system was increased to 6.5 bar with the injection of oxygen, then both valves are closed and the system is
ready to start the oxidation reaction. The heating step normally took 120 minutes to be completed, and in that time
digester was hand shaken every 10 minutes. Finally the vanillin concentration in product solution was analyzed
with HPLC (Araujo et al., 2010).
Preparation of Vanillin with Nitrobenzene
-1
Approximately 0.2 g (oven dried) of wood sample with NaOH aqueous solution (7 ml; 2 mol L ) and
nitrobenzene (0.5 ml) was loaded in to a 500 ml flask and heated up to 170C and reflaxed for 2.5 hours, with
-1
analyses repeated twice. The oxidized material was added (2.5 ml; 4 mol L ) to the aqueous phase. The combined
organic phases were evaporated. The sample was transferred to a 50 ml volumetric flask and the volume
completed with methanol/water solution (1:1 v/v). Following, the solution was filtered in a regenerate cellulose
membrane, pore size 0.45 m. The lignin oxidation products were then analyzed using high performance liquid
chromatography (HPLC) (Claudio et al., 2008).
High-Performance Liquid Chromatography (HPLC)
The HPLC system consists of a Chem- Station for LC system (KNAUER, Germany) with a liquid
chromatograph, a UV-visible detector, a quaternary pump, a vacuum degasser and a manual sampler.
Chromatographic separation was performed on a Lichrospher 100 RP-18 column (250*4mm i.d., 5m particle size)
-1
at room temperature; the mobile phase, at a flow rate of 1 mL min , comprised a mixture of two solvents: 50%
water, and 50% methanol. Elute was continuously monitored by UV detector at 280 nm (Yong-Hong et al., 2004).

1167

Intl J Farm & Alli Sci. Vol., 2 (24): 1165-1171, 2013

RESULTS AND DISCUSSION

In this project the concentration of lignin was determined using ASTME 1721-95 standard method. The amount
of extracted lignin was 27% based on oven dry weight of wood.
FT-IR analysis
Enhancing the industrial application of lignin will require not only improving the lignin extraction processes, but
also determining its precise structure and the functional groups, to develop new applications (Boeriu et al., 2004).
Gosselink et al. reported that the most important chemical functional groups in lignin include the hydroxyl,
methoxyl, carbonyl, and carboxyl groups in various numbers and proportions, depending on origin and extraction
processes. Fig. 4 shows the infrared spectrum of lignin isolated from black liquor was similar to standard (Biljana et
1
al., 2001). The FTIR spectra of the lignin samples showed that bands in the range of 34143435 cm could be
assigned to the OH group of the phenolic compounds present in the samples. The medium intensity band at 2933
-1
cm was assigned to the vibration of the methoxy (OCH3) group, and bands in the strong intensity range of 1463,
1
1507, and 1603 cm were assigned to the aromatic skeleton or the aromatic ring/aromatic methyl group vibration.
1
The band at 1420 cm was due to the CH group (i.e., the bending vibration of the aromatic group). The bands at
1
12171220 and 1137 cm might have been due to the presence of syringyl ring breathing with CO stretching and
1
aromatic CH plane deformation. The other weak intensity bands in the range of 1044, 852, and 743 cm could
have been due to the presence of a guaiacyl group, CH out-of-plane deformation (CH2=CH2), and CH
1
deformation and ring vibration. The other weak intensity bands at 616619 and 533 cm could have be due to CS
stretching and to the CC=O band, respectively. These FTIR results are on par with some previous reports about
lignin obtained from other plant sources [sun et al. 1999, Hattalli et al. 2002 and Tejadoet al. 2007). In general, the
FTIR results revealed high resemblance between commercial lignin and the kraft lignin isolated from Pinus Eldarica
black liquor waste. This similarity indicates the purity of the sample and thus provides a strong basis for its
commercial exploitation in the food industry.

Figure 4. FT-IR spectra of kraft lignin extracted from Pinus eldarica

Amount of Vanillin
For the lignin that was oxidated under oxygen pressure, as it showed in Fig. 5, the spectra of vanillin standard
was in 7-8 min and after injection of sample it appeared in 7.67 min (Fig. 6), and also for the lignin oxidized with
nitrobenzene, the spectra of sample appeared in 7.43 min (Fig.7). The quantification of existence vanillin was
performed using an internal standard. The yield of vanillin obtained from oxidation was around 4.2% (wt vanillin / wt

1168

Intl J Farm & Alli Sci. Vol., 2 (24): 1165-1171, 2013

lignin) and oxidized with nitrobenzene was 1.2% (wt vanillin / wt lignin). These data were in good agreement
with Gogotov et al..s study in which they reported that nitrobenzene oxidation produced approximately equal
amounts of vanillin and syringic aldehyde and only traces of p-hydroxy-benzaldehyde and Araujo Jose and et
al. which reported that batch oxidation produced 3.5% to 7.6% vanillin yields with respect to the lignin.
Interestingly, the concentration of vanillin in the oxygen condition (batch) oxidation products was much higher
than that in nitrobenzene oxidation product.
It is assumed that the oxygen degradation breaks ether linkages between cinnamic acid and lignin
(Fidalgo et al., 1993) and that oxygen is a milder oxidant than nitrobenzene for conversion ferulic acid
into vanillin.

Figure 5. HPLC spectra of standard Vanillin

Figure 6. HPLC spectra of sample oxidized under pressure of oxygen

1169

Intl J Farm & Alli Sci. Vol., 2 (24): 1165-1171, 2013

Figure 7. HPLC spectra of sample oxidized with Nitrobenzene

CONCLUSION
In this study, we isolated lignin from black liquor a waste product of Pinus eldarica - extraction and then the
controlled oxidation of lignin to produce vanillin in a batch reactor and also with nitrobenzene was studied. The
reaction system involved a step where vanillin was formed from lignin and because of the similarity of coniferyl
alcohol to the vanillin structure; this monomer was oxidized to vanillin. Other monomers were also oxidized but in
this project, recognition of the vanillin was important. As explained, the yield of vanillin obtained from oxidation with
oxygen (batch reactor) was more than that of with nitrobenzene.
REFERENCES
Araujo Jose DP, Grande Carlos A, Rodrigues Alirio E. 2010. Vanillin production from lignin oxidation in a batch reactor, Chemical Engineering
Research and design 88:1024-1032.
Baptista C, Robert A, Duarte AP. 2006. Effect of pulping conditions on lignin structure from maritime pine kraft pulps. Chemical Engineering
Journal 121:153158.
Baptista C, Robert D & Duarte AP. 2008. Relationship between lignin structure and delignification degree in Pinus pinaster kraft pulps,
Bioresource Technology 99:23492356.
Boeriu CG, Bravo D, Gosselink RJA, &Van Dam JEG. 2004. Characterization of structure dependent functional properties of lignin with infrared
spectroscopy. Ind. Crops Prod. 20:205218.
Biljana B, Sally A, Ralph Richard S, Reiner Rajai, H Atalla. 2007. Lignin modification in the initial phase of softwood kraft pulp delignification with
polyoxometalates (POMs), Holzforschung 61:731-738.
Borges Da Silva EA, Zabkova M, Araojo JD, Cateto CA, Barreiro MF, Belgacem MN, Rodrigues AE. 2009. An integrated process to produce
vanillin and lignin-based polyurethanes from Kraft lignin, Chemical Engineering Research and Design 87:12761292.
Christiernin M. 2006. Lignin composition in cambial tissues of poplar. Plant Physiology and Biochemistry 44:700706.
Fidalgo ML, Terron MC, Martinez AT, Gonzalez AE, Gonzalez-Vila ET, Galletti GC. 1993. Comparative study of fractions from alkaline
extraction of wheat straw through chemical egradation, analytical pyrolysis, and spectroscopic techniques, J. Agric. Food Chem.
41:1621- 1626.
Gogotov AF, Rybal'chenko NA, Makovskaya TI & Babkin VA. 1996 Catalytic nitrobenzene oxidation of lignins, Russian Chemical Bulletin,
45(12):2854-2857.
Gosselink RJA, Snijder MHB, Kranenbarg A, Keijsers ERP, Jong E & Stigsson LL. 2004. Characterisation and application of Nova Fiber lignin,
Ind. Crops Prod. 20:191203.
Hattalli S, Benaboura A, Ham-Pichavant F, Nourmamode A, Castellan A. 2002. Adding value to Alfa grass (Stipa tenacissima L.) soda lignin as
phenolic resins, Lignin characterization. Polym. Deg. Stab. 75:259264.
Li YH, Sun ZH, Zheng P. 2004. Determination of Vanillin, Eugenol and Isoeugenol by RP-HPLC. Chromatographia 709713.
Lima CF, Luiz CA, Barbosa A, Marcelo CR, Silverio FO, Colodette JL. 2008. Comparison between analytical pyrolysis and nitrobenzene
oxidation for determination of syringyl/guaiacyl ratio in Eucalyptus spp.lignin. Bioresources 3(3):701-712.
Mohamad Ibrahim MN, Sipaut CS, Mohamad Yusof NN. 2009. Purification of vanillin by a molecular imprinting polymer technique. Separation
and Purification Technology 66:450456.

1170

Intl J Farm & Alli Sci. Vol., 2 (24): 1165-1171, 2013

Ola W, Ann-Soffi J. 2006. Separation of lignin in kraft cooking liquor from a continuous digester by ultrafiltration at temperatures above 100 C,
Desalination 195:187-200.
Rajeev Bhat HP, Khalil SA, Karim AA, 2009. Exploring the antioxidant potential of lignin isolated from black liquor of oil palm waste, C. R.
Biologies 332: 827831.
Sun R, Tomkinson J, Bolton J. 1999. Effects of precipitation pH on the physico-chemical properties of the lignins isolated from the black liquor of
oil palm empty fruit bunch fibre pulping. Polym. Deg. Stab. 63:195200.
Shamsuri A, Abdullah DK. 2012. A preliminary study of oxidation of lignin from rubber wood to vanillin in ionic liquid medium. Oxidation
Communications 35(3):767775.
Tarabanko VE, Fomova NA, Kuznetsov BN, Lavanchenko NM, Kudryashev AV. 1995. On the mechanism of vanillin formation in the catalytic
oxidation of lignin with oxygen. Reaction kinetic and catalyze letter 55(1):161-170.
Tejado A, Peaa C, Labidia J, Echeverria JM & Mondragon I. 2007. Physico-chemical characterization of lignins from different sources for use
in phenol-formaldehyde resin synthesis, Biores. Technol 98:16551663.

1171