Journal of Analytical and Applied Pyrolysis 105 (2014) 5574

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Journal of Analytical and Applied Pyrolysis

journal homepage: www.elsevier.com/locate/jaap

Review of analytical strategies in the production and upgrading of

bio-oils derived from lignocellulosic biomass
Pankaj K. Kanaujia , Y.K. Sharma, M.O. Garg, Deependra Tripathi, Raghuvir Singh
Analytical Sciences Division, Indian Institute of Petroleum, Council of Scientic and Industrial Research, Haridwar Road, Dehradun 248005,
Uttarakhand, India

a r t i c l e i n f o a b s t r a c t

Article history: Lignocellulosic biomass is a promising source of renewable energy and valuable chemicals. It is abun-
Received 19 August 2013 dant in several forms which may be pyrolyzed to give gases, condensates and char. The condensed liquid
Accepted 11 October 2013 obtained through pyrolysis is popularly called bio-oil from which valuable chemicals may be derived
Available online 20 October 2013
in a well dened manner. Chemically, this liquid is a complex mixture of simple organic, inorganic and
macromolecular compounds formed through thermo-chemical breakdown of lignocellulosic biomass.
Keywords:
High oxygen content is responsible for its low gross caloric value which renders it useless for fuel appli-
Bio-oil
cations however; upgrading processes aimed at reducing oxygenates potentially increase its usage at
Biomass
Chromatography
par with fossil fuels. Fast and efcient analytical methods have revealed huge amount of informations
Mass spectrometry from biomass and pyrolysis liquids hence a key component in organizing research in this area. Owing
Nuclear magnetic resonance to the high compositional complexity and diversity in bio-oils originating from different sources, gener-
Fourier transform infrared spectroscopy alized analytical procedures are very difcult to formulate. However, tools of analytical chemistry have
helped in understanding the underlying mechanisms involved in production and upgrading of bio-oils at
molecular levels. With the possibility of commercial large scale production plants coming up in Europe,
concerns with bio-oil quality, stability and upgrading rely strongly on analytical approaches. With this
review we have tried to present outcomes of important research related to chemical analysis of bio-oils.
The discussion is intended to summarize role of prominent analytical techniques in the chemical char-
acterization of bio-oils. On this basis, optimum sample preparation strategies have also been proposed
along with the rationale behind analysis with conclusions.
2013 Elsevier B.V. All rights reserved.

1. Introduction capacity [2,5]. In addition to this, rapid economical and population

Global energy requirements across all major segments rely on
fossil fuels which is responsible for its fast depletion. Improving
lifestyles, growing population and strong economic growth in the
developing world has necessitated search of renewable sources of
energy [1,2]. International Energy Agency has summarized overall
global energy supplies, gross consumption across various sectors
and anticipated demand in the future. It has also estimated the
utilization of renewable and non-renewable fuels for electricity
generation [3]. Worldwide, the transportation sector accounts for
major energy requirement which is met through fossil fuels. Indian
transport system expanding at 10% annually is considered one
of the largest and heavily populated systems with serious fuel
demands [4,5]. Power generation in US is largely achieved through
natural gas whereas coal remains a key feedstock in Indian thermal
power plants which meets nearly 65% of its total installed electrical
Corresponding author. Tel.: +91 135 2525934; fax: +91 135 2660098.
E-mail address: pankajkk@iip.res.in (P.K. Kanaujia).
growth of developing world (China, India, Latin America, Middle
East and Russia) has crucial energy expectation. Limited petroleum
reserves [6] and environmental concerns have necessitated the
exploration of new sources of renewable energy.
Biomass is an abundant renewable source from which fuels
can be obtained in relatively short span, reliable and eco-friendly
manner [7,8]. It provides energy efcient fuels in the form of
bioethanol and bio-diesel which makes up rst generation bio-
fuels. Bioethanol is generally produced from food grade agricultural
biomass and used as additive in gasoline. Its use of up to 25% as
gasoline blend is prevalent in the different parts of globe especially
US and Brazil [9,10]. Bio-diesel on the contrary, is a common bio-
fuel in Europe obtained from vegetable oils and animal fats. It is
also used as diesel additive to reduce levels of particulates and toxic
emissions (e.g. carbon monoxide and hydrocarbons) from vehicular
exhausts [1113].
Bio-oils are obtained through a single step thermo-chemical
conversion of nearly all kinds of wood biomass into a chemically
complex liquid product. An estimated increase of nearly four to
seven folds in energy density is readily achieved during this process

0165-2370/$ see front matter 2013 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.jaap.2013.10.004
56 P.K. Kanaujia et al. / Journal of Analytical and Applied Pyrolysis 105 (2014) 5574
Fig. 1. A schematic pathway of biomass pyrolysis to yield bio-oil.
termed pyrolysis [1416]. During past three decades, researchers
across the globe have emphasized upon evolving bio-oils as a
potential substitute of petroleum [1719]. Bio-oil is a dark col-
ored liquid comprising mixture of organic compounds that serves
as mother liquor to variety of chemicals. Pyrolysis is the destruc-
tive distillation of dried biomass in a reactor at high temperatures
[2022]. Raw and unprocessed bio-oil on the basis of its gross
caloric value may be employed as an alternative to the solid
fuels (wood, coals etc.) used in boilers for power generation. It is a
promising source of numerous value-added chemicals [23]. Consid-
erable efforts are being continuously put in to develop upgrading
strategies to transform it to engine fuel applications. Bio-oils are
often termed clean compared to fossil fuels since they offer several
ecological benets [22].
Pyrolysis causes irreversible breakdown of organic matter at
elevated temperatures in the absence of oxygen that brings tremen-
dous change in the physico-chemical properties of feedstock
leading to the formation of condensates. A schematic sketch of
biomass pyrolysis process to produce bio-oil and its subsequent
value addition is summarized in Fig. 1. The pyrolysis process of lig-
nocellulosic biomass has been review by many researchers [2431].
These reviews are highly informative, fundamental and encom-
pass several aspects of pyrolysis process, reactor designing, process
scale-up and commercialization under global perspectives. Mohan
et al. detailed a comprehensive report on different types of pyrolysis
and highlighted prerequisites for carrying out pyrolytic breakdown
of wood biomass considering their varying chemical compositions
[22]. Similarly, biomass pyrolysis methods have been reviewed and
thoroughly described with mechanisms and kinetics of pyrolytic
reactions and chemometrics [32].
Extensive research in this area reveals a high level of diversity in
selecting biomass comprising agricultural and forest scrap such as
barley straw and hulls [33], rice husk [3436], wheat straw [37,38],
corn (stalks, leaves and husks) [39,40], coconut and peanut shells
[40], fruit bunches and pulps [41,42], waste furniture and wood
(eucalyptus, pine etc.) [38,4346], forage crops [47,48], tea waste
[49], bagasse [40,50], sewage sludge [51], swine manure [52] and
many more. Interestingly, waste plastic and printed circuit boards
from used computers have also been utilized to produce pyrolysis
oil [53,54]. Algae are rich source of energy due to higher photo-
synthetic efciency than trees and therefore considered to possess
high solar energy content. In order to achieve efcient carbon diox-
ide xation, algal biomass has been efciently utilized to produce
bio-oils [5558]. Essential parameters for algal biomass pyrolysis to
extract valuable chemicals, bio-fuels and gases have been reviewed
[59].
Bio-oils are obtained in fair yields (7080% w/w) from almost
all kinds of lignocellulosic biomass and their chemical compositions
vary sharply according to the biomass [60]. Briey, pyrolysis causes
thermal cracking of naturally occurring biomolecules (cellulose,
hemicelluloses, lignin etc.) in the biomass and converts them to
simpler organic molecules. Other constituents of wood such as fats,
mucilage, wax, alkaloids, terpenoids etc. are converted into smaller
compounds and inorganic minerals are reduced to ash during this
process. Bio-oils usually are dark colored, viscous and unstable liq-
uids with considerable water content. Upon storage they undergo
series of chemical reactions triggered by organic acids and inter-
mediates in addition to the re-polymerization of reactive olenic
compounds [61,62]. Ageing also reduces bio-oil quality, increases
its viscosity and ultimately leads to the separation of continuous
 P.K. Kanaujia et al. / Journal of Analytical and Applied Pyrolysis 105 (2014) 5574
and dispersed phases [6365]. Presence of substantial amounts of
organic acids often imparts acidity to the crude bio-oil whose pH
ranges between 2 and 4 [61]. Under these circumstances, storage,
handling and transportation of bio-oils become highly challenging
[60,66,67].
A brief discussion on the analysis of feeds and upgraded bio-
oils has been presented [68]. We also published a short review on
adopting analytical strategies for characterizing pyrolysis oils [69].
This review however, is presented with more detailed discussions
on the update and outcomes of large number of literature reports. It
focuses on all aspects of bio-oil analysis through chromatographic
and spectroscopic methods and techniques. Signicant literature
contributions comprising bio-oil production, upgrading and chem-
ical characterizations have been discussed. Matrices as complex
as bio-oils contain range of chemicals distributed in its aqueous
and organic phases and require efcient pre-treatment steps. The
basis of formulating adequate sample preparation strategy is cru-
cial for any chemical analysis hence it has been discussed in this
review. Prominent research in this area during past two decades has
been summarized with their key features highlighted. In addition to
chromatographic and spectroscopic characterization, hyphenated
platforms due to their unparalleled sensitivity and versatility have
made remarkable access to the molecular level informations. Com-
positional information provided by multiple analytical techniques
has simplied the unambiguous identication of bio-oil compo-
nents with great accuracy. Thermogravimetry has been considered
for discussion since it is widely employed to establish correlation
between biomass composition and bio-oil quality. Bio-oil upgrad-
ing research has gathered signicant momentum in view of rening
potential and process optimization. Tools of analytical chemistry
are essentially involved in the product monitoring at every stage.
The discussions are intended to provide precise informations and
cover as much report as possible.
2. Bio-oil upgrading process
Through bio-oil upgrading, series of processes are implicitly
involved to overcome limitations associated with its use. Obtaining
reliable scale-up and rening processes open up several possibili-
ties to assess its economic viability and ensures usage at par with
conventional fossil fuels. Extraction of value-added products from
bio-oils also requires well organized upgrading approach. The key
objective of this process is removal of free organic acids (formic,
acetic, propionic acid, etc.) through esterication. For this reason,
catalytic esterication is widely studied in addition to solid cat-
alysts [70], acidic ionic liquid catalysts [71], acidic ion-exchange
based zeolites (HZSM-5) [72], H-mordenite H-Y, silicalite [73], alu-
minum silicate catalysts [73,74], propyl sulfonated mesoporous
silica and several others. Pyrolytic lignins often present challenge
in catalytic esterication and extraction of compounds. Lignins get
deposited over the active catalyst surfaces thereby reducing their
efciency. Methanol has been used to enhance the stability and ef-
ciency of modied HZSM-5 zeolite catalyst that minimized lignin
deposition on the catalyst surface maximizing yields of hydro-
carbons [75]. Ion-exchange resins (732- and NKC-9-type) were
used for catalytic esterication of bio-oils monitored through acid
number determination [76,77]. Catalytic hydrotreatment of crude
bio-oil (produced from hydrothermal liquefaction of microalgae)
over palladium on carbon is reported in supercritical water [78].
Catalytic fast pyrolysis in presence of Ga/ZSM-5 catalyst has been
shown to convert furans into aromatic compounds at high temper-
atures [79].
Catalytic upgrading (BEA zeolite and Al-SBA-15) and simul-
taneous analysis through pyrolysis gas chromatography mass
spectrometry (Py-GCMS) is a fast approach reported by Park
57
et al. [80]. Simultaneous hydrotreatment, esterication and crack-
ing of bio-oil in supercritical ethanol using Pd/SO42 /ZrO2 /SBA-15
is described [81]. Bio-oil blending with bio-diesel is considered suit-
able by several of researchers in which adequate bio-oil fractions
were utilized as petroleum additive for transportation and co-
processing at an affordable scale. In this way emulsication of ether
soluble bio-oil fraction with bio-diesel has been attempted after
removing pyrolytic lignins [82,83]. Upgrading of acidic compounds
of bio-oil through ketonic condensation over cerium oxide catalyst
is reported by Deng et al. [84]. Mechanisms of catalytic hydro-
processing of bio-oils have been proposed through three model
compounds representing lignin, cellulose and hemicelluloses (prin-
cipal components of lignocellulosic biomass). These compounds
were processed using palladium or ruthenium catalyst and the
products were then eventually used to suggest reaction pathways
for their formation [85].
Numerous catalyst formulations have been tested over a range
of temperature, pressure and ow rates with chemically differ-
ent bio-oils in a catalytic hydro-processing method optimization
[86]. Effect of reactive aldehydes on the catalytic esterication of
organic acids has been investigated [87]. Other upgrading proce-
dures involved phase separation through addition of ionic salts
followed by methylation of polar oxygenated compounds in ionic
liquids [88]. Recently, several aspects of hydro-deoxygenation and
zeolite cracking of bio-oil to engine fuels have been reviewed by
Mortensen which depicted updates on the catalyst development,
performance evaluation and optimization of efciencies. It also
explained feasibility of reaction mechanisms and kinetics of various
reaction parameters. Additional discussions have been provided to
explore the prospects of process commercialization [89]. Recently,
the upgrading processes for bio-oils have been reviewed [90].
In view of several limitations associated with bio-oil use,
upgrading ensures industrial scalability for its commercial use.
Quantitative information of its chemical composition is abso-
lutely necessary in designing pyrolytic reactors for achieving
optimum efciency. For complete bio-oil characterization, tools
of analytical chemistry facilitate in evolving rening and upgrad-
ing protocols. Very large number of scientic literature present
comprehensive physical and chemical characterization of bio-oils
originating from diverse biomass feedstocks. Majority of reports
have focused largely on qualitative and semi-quantitative anal-
ysis, even so, quantitative analytical measurements have also
been reported. Analysis of bio-oils through gravimetry, volume-
try, electro-analysis and spectroscopy has been widely reported.
Spectroscopic analysis by virtue of its unambiguous results and
conclusions is preferred over others in most of the bio-oil charac-
terization studies. However, Gravimetry is extensively employed
in feedstock characterization and computing the thermodynamic
data for bio-oils. In addition, volumetry is useful in the estima-
tion of ionizable organic acids and bases whereas electro-analytical
approaches have found limited applications in the bio-oil analy-
sis.
3. Sample preparation
Qualitative instrumental analyses may require as many ana-
lytical techniques as possible to reveal the identity of unknowns
present at varying concentrations. In contrast, quantitative analyt-
ical measurement is ensured through large number of variables
governing experimental designing. Majority of the analytical tech-
niques require unavoidable sample pre-treatment steps that ensure
selectivity and specicity in analyte identication. Sample prepa-
ration eliminates matrix interferences by attenuating signals from
the background components. Objective of selection of an adequate
sample preparation method is either enrichment or elimination
58 P.K. Kanaujia et al. / Journal of Analytical and Applied Pyrolysis 105 (2014) 5574

OH OH OH

O O
O O O O O
O OH
2,5-dimethylfuran furan-2- 2-methoxyphenol 2-methoxy- 2,6-dimethoxyphenol 2-methoxy-
carbaldehyde 4-methylphenol 4-vinylphenol

O
HO
O O
O
O
3-(4-hydroxy-3-methoxy-
phenyl)acrylaldehyde 1,2,3-trimethoxy-
5-methylbenzene

O O
HO
OH O
O
O
4-(3-hydroxyprop-1-enyl) 1,2,3-trimethoxy-
-2,6-dimethoxyphenol benzene

HO O
O HO
O OH Lignin O
O
2-(4-hydroxy-3-methoxy-
phenyl)acetic acid 4-hydroxy-3,5-di-
methoxybenzaldehyde

HO O

OH O
O

4-(3-hydroxyprop-1-enyl)
-2-methoxyphenol OH

4-hydroxy-2-
methoxybenzaldehyde
O
HO O
O
O
3-(4-hydroxy-3,5-dimethoxy- O
phenyl)acrylaldehyde
OH
4-hydroxy-3-
O methoxybenzaldehyde
HO OH HO OH
HO
O O
O O O O
O
4-allyl-2,6- 1-(4-hydroxy-3-methoxy- 4-allyl-2- 1-(4-hydroxy-3-methoxy- 4-ethyl-2-
dimethoxyphenol phenyl)ethanone methoxyphenol phenyl)propan-1-one methoxyphenol

Fig. 2. Pyrolytic degradation of lignin.

of analytes. Sample pre-treatment is often imperative for analy-
sis and considered extremely labor intensive and time consuming.
Enormous literature is available for adopting a sample prepara-
tion procedure to assess its suitability for variety of samples and
applied analysis. Reviews have been published from time to time
that covered sample preparation for all kinds of analytical tech-
niques applicable to every area of scientic research [9194].
Bio-oil is a complex assemblage of chemicals containing var-
ied functional groups. Wood biomass is a plentiful source of
lignins, cellulose, wax, hemicelluloses, mucilages, terpenoids and
other natural products. Pyrolysis causes thermal cracking of these
biomolecules into simpler organic compounds. Lignins, upon pyrol-
ysis, produce methoxyphenols of which monolignols (guaiacol and
syringol) are prominent. It is one of the major sources of phenol
containing species in bio-oils. Large numbers of such compounds
originating from pyrolysis of lignins are summarized in Fig. 2.
Cellulose upon pyrolysis gives levoglucosan, levoglucosenone, fur-
fural, substituted furans and several small molecules comprising
aldehydes and ketones. Figs. 3 and 4 provide details of several
chemicals formed during pyrolysis of cellulose and hemicellulose
respectively. In this way each class of naturally occurring biomass
based biomolecules degrade pyrolytically to give simpler organic
molecules. This complex mixture of polar and non-polar chemi-
cals gives an oily appearance to the bio-oil along with considerable
amounts of water.
Large numbers of sample preparation methods have been
adopted for pre-treatment of bio-oils. Adopted sample pre-
treatment methods before bio-oil analysis have been summarized
in Table 1. In early literature reports ltration, centrifugation and
phase separation by water addition remained methods of choice
[95]. Chromatography however, has been widely utilized to impart
selectivity and specicity in extraction depending upon the nature
of samples and adopted analysis. Adsorption chromatography in
the form of column chromatography and solid-phase extraction
(SPE) is extensively reported. Presence of large number of chemi-
cals (>400) in bio-oils has restricted widespread utilization of other
 P.K. Kanaujia et al. / Journal of Analytical and Applied Pyrolysis 105 (2014) 5574 59

O
OH
O
O O
HO O OH

1,4:3,6-Dianhydro-alpha- OH
O d-glucopyranose
Levoglucosan
O
OO
Furan-2-carb-
aldehyde
O
Levoglucosenone

OH

O CH2OH
O O
H O
H HO
3-(Hydroxymethyl)furan OH H
-2-carbaldehyde O O
H O
H OH
n 5-hydroxymethyl
-2-furaldehyde
OH Cellulose
HO O

2,3-Dihydroxypropanal
O
O
Glyoxal
O
OH
O
2-Hydroxyacetaldehyde
HO

1-Hydroxypropan-2-one
CO,CO 2,H 2O&Char

Fig. 3. Cellulose pyrolysis products.

highly popular methods like solid-phase micro extraction (SPME)
[96,97], single drop micro extraction (SDME), liquidliquid extrac-
tion (LLE), solid supported liquidliquid extraction (SLE) [98] and
extraction mediated by advanced materials based adsorbent [99].
In the contemporary research, these methods are considered green
and efcient with the requirement of very little sample. These
methods offer promising applications due to the miniaturized for-
mats and associated with high enrichment factors.
Bio-oils usually contain large number of chemicals ranging from
organic, inorganic, coordination complexes to macromolecular lig-
nocellulosic degradation products. The distribution of chemicals
in bio-oils in terms of polarity is quite complex however, enor-
mous informations are available that reveal presence of highly
non-polar to intermediate polarity substituents such as normal and
branched aliphatics (parrafns), alicyclics (cycloparafns), olens
(normal, branched and cyclic) and aromatics (simple to polycyclics
and substituted). Polar and highly polar chemicals comprise mainly
of oxygenates like carboxylates, carbonyls, aryl ethers, phenols, and
alcohols (Figs. 24). Inorganic substances generally get separated
from the mother liquor during pyrolysis and remain into bio-char
though; traces to signicant amounts of inorganic complexes may
be present in the bio-oils. Bio-char on the other hand, is highly

Table 1
Details of bio-oil sample pre-treatment procedures and their underlying mechanisms.

S. No. Sample preparation Mechanism References

1. Mechanical phase separation

1.1 Filtration, centrifugation, phase Difference in solubility and particle size [90,111]
separation by water
1.2 Liquidliquid extraction (LLE) Difference in solubility in two immiscible liquids [33,62,84,97,103,104,105,106,107]

2. Chromatography
2.1 Column (normal and reversed phase) Difference in rate of movement of solutes through [38,49,95,106,108]
stationary phase in column
2.2 Solid-phase extraction (SPE) Adsorption of solute over adsorbent through normal or [37,97]
reversed phase mode
2.3 Ion-exchange Interaction of solute with ion-exchange resins [99,110]
2.4 Size exclusion Difference in molecular size [99,100,101,102]
2.5 High performance thin layer (HPTLC) Difference in rate of movement of solutes through [109]
stationary phase in planar support
2.6 Solid-phase microextraction (SPME) Adsorption of analyte over adsorbent ber followed by [45,96,97]
thermal desorption

3. Electrophoresis
3.1 Capillary electrophoresis (CE) Difference in migration of charged species in electric [224,235]
eld
60 P.K. Kanaujia et al. / Journal of Analytical and Applied Pyrolysis 105 (2014) 5574
Fig. 4. Major pyrolytic components from hemicelluloses.
oxygenated heavier lignocellulosic remainder along with residual
carbonaceous mass which is obtained during biomass pyrolysis.
Keeping the chemical composition of bio-oil into account,
no single sample preparation approach is sufcient enough to
isolate compound(s) from complex background interferences how-
ever; the chemical composition of bio-oil has been revealed to a
large extent. Porous layer open tubular silica gel columns have
been used for group separation of polar oxygenated compounds
[100]. Volatiles in the bio-char and oil have been extracted to
assess their toxicities through Headspace-SPME (HS-SPME). For
this, eucalyptus bio-oil was vacuum distilled to obtain residual
tar which was subjected to HS-SPME followed by analysis with
two dimensional gas chromatography time of ight mass spec-
trometry (GC GCTOFMS) [45]. Low molecular weight (MW)
aldehydes in bio-oils were extracted through HS-SPME after deriva-
tization in solution as well as on-ber derivatization. The aldehyde
derivatives were then thermally desorbed and analyzed with gas
chromatography mass spectrometry (GCMS) [101]. Polycyclic aro-
matic hydrocarbons (PAHs) were extracted and enriched from
bio-oil through LLE, SPE and SPME. With solvent extraction, PAHs
were extracted into acetonitrile and hexane whereas normal phase
SPE was employed to trap the analytes over silica placed after pyrol-
ysis chamber. The compounds were also adsorbed over SPME ber
and eventually analyzed [102].
Due to the versatility, efciency, and simplicity, SPE is the most
extensively used sample pre-treatment tool utilized in almost all
the areas of basic and applied science [103]. It is a method of
choice for sample preparation for complex matrices prior to analy-
sis with highly sensitive spectroscopic techniques with an objective
of eliminating matrix interferences and enrichment of target ana-
lyte(s). Two SPE formats are popular and are utilized in the majority
of applications. In the rst approach, target analytes are retained
over adsorbent while matrix interferences are washed away. Sec-
ond approach involves retention of matrix interferences over the
solid-phase while target analytes pass out in the efuents. Variables
that directly affect analyte recoveries (such as nature and amount
of adsorbent, rinsing and eluting solvents) need optimization for
operational ease and efcacy. Literature reports provide sufcient
details related to bio-oil pre-treatment through SPE involving nor-
mal phase, reversed phase and ion-exchange layouts.
Reversed phase (C18 ) SPE is reported for extraction of several
oxygenated compounds in bio-oil [37]. Bio-oil fractionation has
been demonstrated through ion-exchange and size exclusion chro-
matography to segregate components on the basis of their polarity
and pKa [104]. Gel permeation chromatography (GPC) was used
for the extraction of phenols from high MW lignins in bio-oil sam-
ples with tetrahydrofuran as an optimum solvent [105]. Through
GPC, Scholze et al. separated and characterized pyrolytic lignin to
explain reaction mechanisms during pyrolysis and aging processes
[106,107]. Retrospective investigation of bio-oil through GPC led
to the conclusion that higher MW compounds present in pyrolysis
oils actually originated from lignins and derived compounds.
LLE has been efciently used to fractionate bio-oils obtained
from sawdust, rice husk, lignin and cellulose. In addition to ioniza-
tion suppression through pH manipulation and soxhlet extraction,
several solvents were optimized to maximize enrichment of target
compounds in LLE extracts [108,109]. In a separate studies involv-
ing bio-oil fractionation, extracts were simply obtained as water
soluble and insoluble (i.e. dichloromethane or hexane soluble)
[33,61]. In general, fractionation typically involved extraction with
solvents with varying polarities such as benzene, toluene, hexane,
dichloromethane, chloroform, carbon tetrachloride, ethyl acetate,
 P.K. Kanaujia et al. / Journal of Analytical and Applied Pyrolysis 105 (2014) 5574
Fig. 5. Scheme of liquidliquid extraction strategy to fractionate bio-oil components.
diethyl ether, acetonitrile, methanol and water. Partitioning of ana-
lytes is governed by several factors like polarity of solvents and
analytes, functional groups within the compounds, pKa and types
of physical and chemical interactions [88,110,111]. Characteriza-
tion of bio-oil on the basis of fractions obtained as benzene soluble
and insoluble is reported on non-polar and polar GC columns [112].
Fig. 5 demonstrates plausible extraction design through which bio-
oil components can be segregated into different chemical classes on
the basis of LLE.
Column chromatographic separations of bio-oil components
have been reported in which fractions were obtained with suit-
able solvents. In a study, pyrolysis oil was fractionated into two
distinct fractions i.e. n-pentane soluble and insoluble. The pentane
soluble extract was further separated chromatographically over
activated silica gel column and eluted successively with pentane,
toluene and methanol to produce aliphatic, aromatic and polar frac-
tions respectively [49,113]. In a similar set of experiment, bio-oil
fractionation through successive elution with pentane, benzene,
dichloromethane, ethyl acetate and methanol is reported. Impor-
tant correlation were made on the basis of compounds obtained
in each fraction, authors have concluded that polar, aliphatic and
aromatic fractions were obtained in maximum concentrations
at lower, moderate, and higher pyrolysis temperatures respec-
tively [111]. Naik et al. performed extraction of bio-oil with 1:1
mixture of chloroform and diethyl ether, loaded this extract on
silica gel column followed by elution with pentane, carbon tetra-
chloride, benzene, diethyl ether, chloroform and methanol [38].
Application of silica gel column for group type separation of oxy-
genated compounds of bio-oils similar to petrochemical group
separation into aliphatic and polyaromatic compounds is reported
[100].
High performance thin layer chromatography (HPTLC) has been
demonstrated for separation of anhydrosugar i.e. levoglucosan and
cellobiosan along with glucose, arabinose, xylose and cellobiose
from fast pyrolysis oil. The study envisaged the use of disposable
HPTLC plates for which pre-treatment and derivatization of sam-
ples were not necessary [114]. Ion-exchange chromatography nds
immense applications in the removal of bio-oil acidity components.
Elimination of acetic and formic acids from bio-oils is successfully
attempted by many researchers. In a study, neutralization of free
61
acids with calcium oxide was proposed followed by its removal
through ltration. It also focused upon use of an anion-exchange
adsorbent (Dowex-22) to trap acids by manipulating pH of the
sample [115]. In a separate method, several fractions of bio-oil
were obtained through use of specic coolants operating at certain
prescribed temperatures that were circulated along condensers
leading to selective condensation of several classes of molecules
according to their dew points in the pyrolysis vapor stream. In this
way, vapors and aerosols of oligomer rich heavy ends, middle cut of
monomeric phenols and furans along with aqueous phase contain-
ing light oxygenates were separated [116]. Fig. 6 highlights the role
of chromatographic methods in sample preparation and outlines
some common procedures for handling complex matrices such as
bio-oils.
Inorganic analysis comprising metal content estimation is gen-
erally carried out more reliably and conveniently with atomic
absorption spectroscopy (AAS) and inductively coupled plasma
atomic emission spectroscopy (ICP-AES). Sample preparation pro-
cedures usually involve acid digestion and alkali fusion followed by
dissolution [95].
An essential aspect of sample preparation is derivatization
which is often imperative before analysis especially through chro-
matography. The primary objective of derivatization involves
imparting thermal stability to the labile compounds and enhances
their chromatographic properties. Derivatization is at times crucial
in improving the sensitivity of certain detection techniques. Despite
several advantages, derivatization is occasionally tedious, time con-
suming and introduces error in quantitative measurements besides
generating unwanted artifacts in some analysis [117]. In gas liq-
uid chromatography, derivatization of polar organic compounds is
always obligatory. In mass spectrometry, derivatization is equally
valuable as soft ionization techniques and helps in elucidating
structural identity of so many classes of chemicals that do not pro-
duce molecular ion [96]. In Nuclear magnetic resonance (NMR),
formation of derivatives containing phosphorus is highly useful in
deriving conclusive informations. Overall, derivatization research
has led to the development of large number of analytical proto-
cols, reagents and reaction conditions for large number of analytes
[118,119]. Bio-oil analysis also involves several derivatization reac-
tions that have been employed before analysis.
62 P.K. Kanaujia et al. / Journal of Analytical and Applied Pyrolysis 105 (2014) 5574
Fig. 6. Schematic presentation of bio-oil fractionation through chromatography; the analytes are written in order of their elution from chromatographic column sequentially
eluted with gradual change in solvents polarity.
Literature reports on bio-oil preparation, optimization of
pyrolysis conditions and upgrading have involved huge amount
of analytical methods and techniques. As and when required,
researchers have adopted suitable sample preparation methods to
ngerprint bio-oil compositions. However, majority of the inves-
tigations are devoid of rigorous sample preparation steps. The
reason probably is due to enormous variations in complexity of
bio-oils which limit the generalization of extensive sample pre-
treatment protocols. In our opinion, solid-phase extraction with
several available adsorbents has well known potential in separat-
ing bio-oil components. Afnity chromatography manifested by
molecularly imprinted polymers (MIPs) may offer selective isola-
tion of compound classes. Some work in these areas is underway at
our laboratory. In addition, nanostructures with unique properties
such as carbon nanotubes and graphenes have enormous surface
area that may be utilized in various approaches of chromatogra-
phy. SLE is inexpensive and an efcient approach for eliminating
interferences from water soluble compounds.
4. Physical properties of bio-oil
Biomass pyrolysis oils differ signicantly in physical and chem-
ical properties from petroleum based fuels. American Society for
Testing and Materials (ASTM) has given few guidelines and cer-
tain specications of pyrolysis liquid produced from biomass. For
estimation of some physico-chemical properties, ASTM gives nor-
mative references of the standards developed for characterization
of petroleum and lubricants [120]. Oasmaa et al. reported norms
and standards for pyrolysis oil along with properties and quality
[121]. Physical properties of bio-oils are extensively studied and
very well documented in the scientic literature [16,17,122139];
hence only limited discussion is presented here. The caloric val-
ues reported for various bio-oils and are signicantly lower than
conventional hydrocarbon fuels and accounted due to high oxy-
gen content and presence of water. Lower heating value of bio-oils
has been estimated to be around 60% of that of hydrocarbon fuels
[17]. Heating value of bio-oil is approximately 18 MJ/kg which is
signicantly lower than those of hydrocarbon fuels.
Presence of high amounts of oxygenated species can be
adjudged from its ready miscibility with polar organic solvents
(ethanol, methanol etc.). Proximate analysis of bio-oils derived
from vegetation biomass reveals 4550% oxygen (w/w) as com-
pared to 42% in wood biomass [22]. Water accounts for majority of
oxygen content in bio-oils with small amounts of gases which are
formed during pyrolysis (CO2 and CO). Oxygen has been reported
to be present in more than 300 compounds broadly identied as
hydroxyaldehydes, hydroxyketones, sugars, carboxylates and phe-
nolates [140]. VTT, Finland pioneered systematic research in this
area and presented detailed physical properties of pyrolysis oils and
methods to determine them [141,142]. These documents focus on
nature of feedstocks and developments in the analytical test meth-
ods in order to elaborate as many properties of bio-oils as possible.
They also highlight outcome of several round robin exercises in
biomass pyrolysis oil analysis and emphasize on health and safety
concerns.
Studies have been carried out to investigate role of additives
in lowering viscosity and imparting storage stability to bio-oils.
For this purpose ethyl acetate, methyl isobutyl ketone, methanol
and several others were screened. Accelerated aging tests were
conducted at 90 C to measure the effect of these additives on
bio-oil stability and viscosity [143]. Norms, standards, analysis
and outcomes of round robin conducted by International Energy
Agency-European Union for comparing accuracy of chemical and
physical characterization methods for fast pyrolysis liquids have
been summarized and presented [144]. Determination of critical
physical properties such as water content, viscosity, solid con-
tent, pH, stability, elemental analysis and pyrolytic lignins was
the objective of test. On the basis of performance in round robin
test, important conclusions were given to ensure accuracy, reli-
ability, repeatability and reproducibility of results. Based on these
round robins important conclusions were inferred which included
importance of liquid sample handling and good estimation of
water and density. Determination of oxygen by difference was
variable while its direct determination was poor. Additionally,
high variations were obtained for nitrogen, viscosity, pH, and
solids. The results also emphasized upon improvement in the
 P.K. Kanaujia et al. / Journal of Analytical and Applied Pyrolysis 105 (2014) 5574 63

Table 2
Standard methods applicable for the determination of physico-chemical properties of bio-oils as per ASTM.

S. No. Physical property ASTM standard Method of determination

1. Ash content D482 Thermogravimetry method

2. Pour point D97 Specied cooling of a heated sample and monitoring of sample movement
3. Volatiles in bio-char D3175 Weight loss through gravimetric analysis under rigidly controlled conditions
4. Fixed carbon/ash content of bio-char D3174 Gravimetry under controlled conditions
5. Density D1298 Hydrometer method
D4052 Digital density meter
D369 (withdrawn) Specic gravity by pycnometer method

6. Water content D1744 (withdrawn) KarlFisher method volumetric method

D95 Distillation method
E203 KarlFisher reagent volumetric method

7. Carbon residue D189 Destructive distillation method of carbon residue determination

D4530 Micro-method (gravimetric analysis at 500 C under inert nitrogen
atmosphere)

8. Viscosity D88 Saybolt viscosity by viscometer method

D445 Capillary method through viscometer
D2170 Flow of liquid through calibrated glass capillary method

9. Flash point D93 Flash point by specied rate of heating

D3828 Small scale cup test in which sample is placed and ignition spark is created

10. Heating value D3286 (withdrawn) Gross caloric value of coal and coke by Isoperibol Bomb Calorimeter
D240 Heat of combustion by Bomb Calorimeter method
D4809 By Bomb Calorimeter method

11. Total acid value. D974 Color-indicator titration

D664 Potentiometric titration method
D3339 Semi-micro color-indicator titration

12. Elemental analysis D5373 Estimation of CO2 , H2 O and NOx (oxidization of sample containing C, H and N)
D5291 Determination of gases obtained after conversion from their respective
elements

13. Sulfur analysis D4239 Infrared determination of SOx after combustion of sample
D4294 Energy dispersive X-ray uorescence spectrometric method
D2622 Wavelength dispersive X-ray uorescence spectrometric method

14. Surface tension D971 Measurement of interfacial tension by movement of a platinum ring from
surface of uid with higher surface tension
15. Solid content D2276 Particulate contaminant in by line sampling (ltration through membrane)
16. Lubricity D2783 Load under one ball is measured against three xed balls covered by sample
17. Boiling range fractionation D2887 Boiling range distribution by gas chromatography

determination of pyrolytic lignin and measuring stability of the
liquid [144].
A very brief detail of ASTM methods that have been used for
determining physico-chemical properties of bio-oils is given in
Table 2.
5. Chemical characterization of bio-oil
During early 90s, complete chemical characterization of bio-oil
remained a challenge for analytical scientists for quite some time.
Macromolecules produced from lignins and sugars cause difculty
in the analysis due to their high MW. Bio-oils contain highly reactive
chemical species that self-react upon storage and/or handling. Pop-
ular analytical techniques which have been widely employed for
bio-oil analysis are gas chromatography (GC), high performance liq-
uid chromatography (HPLC), GPC, GCMS, liquid chromatography
electrospray ionization mass spectrometry (LC-ESI-MS), Fourier
transform infrared spectroscopy (FTIR), NMR and thermo gravimet-
ric analysis (TGA). In bio-oil analysis, results from many analytical
techniques complement each other leading to unequivocal identi-
cation of its chemical components. Important prerequisite of GC
based detection is the volatility of analytes due to which gases,
volatile non-polar and chemical derivatives of polar organic com-
pounds in bio-oils are largely amenable. HPLC is usually associated
with the analysis of polar and thermo-labile organic compounds
while MW distribution of compounds within a sample is reliably
estimated with GPC. Mass spectrometer is a powerful detector to
chromatographic systems as well as an independent technique for
studying behavior of molecules and advanced gaseous phase reac-
tions [145]. GCMS and LCMS have immensely contributed in the
identication of bio-oil components. FTIR analysis reveals pres-
ence of functional groups in compounds and helps in establishing
molecular structure. NMR is essential in determining the nature
and types of covalently bonded nuclei (hydrogen or carbon) within
a molecule. It gives an estimated number of such nuclei under
observation through peak area integrations.
Physico-chemical characterization of bio-oil largely relies on
bulk property measurements and expressed as functions of respec-
tive chemical properties. On the contrary, spectroscopic analysis
gives valuable structural insights of such complex matrix. Upgrad-
ing of bio-oils for specic purposes requires monitoring the
progress and efcacy of chemical reactions that helps in developing
molecular level understanding of bio-oils and converted products.
The ensuing discussion intends to summarize the role of different
analytical techniques in deconvoluting compositional complexity
of biomass pyrolysis oils. Sincere efforts have been made to cover
as many reports as possible and to avoid monotonous discussions,
only prominent contributions in the analysis of bio-oils are pre-
sented.
5.1. Thermogravimetric analysis
Thermoanalytical techniques are commonly represented by
thermogravimetry and derivative thermogravimetry (DTG). They
have been widely utilized to study the thermal behavior of fossil
64 P.K. Kanaujia et al. / Journal of Analytical and Applied Pyrolysis 105 (2014) 5574
fuels [146151] and bio-fuels [152,153]. Thermogravimetric data
reveal plausible thermal behavior of the biomass and derived prod-
ucts. TGA involves monitoring the change of sample mass against
time or temperature at a specied rate of heating. Kinetics of ther-
mal events may be determined by the application of Arrhenius
equation corresponding to separate slopes of constant mass degra-
dation and serves as a basis of generating valid thermodynamic
gures. TGA is a promising technique to study bio-oil properties
as a fuel, since the measured mass losses depend on volatility and
molar masses of fractions under investigation. It has been used to
compare evaporation and cracking behavior of different bio-oils
and their fractions [153,154].
Bio-oil vapors comprise of assemblage of true vapors and minute
droplets (aerosols) along with number of non-volatile compounds
formed during vapor condensation in irreversible manner. TGA
plays signicant role in characterization of evaporation, ther-
mal decomposition and combustion properties of bio-oils. Since a
lighter product is normally better for combustion, TGA of biomass
and bio-oil often produces data that may be used to improve bio-
oil quality and energy content. It has been observed generally that
lower the parent biomass lignin content, better is the quality of
obtained bio-oil.
Feedstock biomass characterization through TGA is highly use-
ful that predicts thermal behavior of biomass and suitable pyrolysis
conditions. TGA coupled to FTIR was utilized for the analysis of
bio-oil obtained from pyrolysis of wheat straw in the presence of
potassium chloride [155]. In one of the studies involving proximate
analysis of bagasse through TGA, 6870% (w/w) volatiles, 2729%
(w/w) xed carbon and 3% (w/w) ash content were estimated [50].
TGA analysis of softwood bark and hardwood biomass [111,112],
empty fruit bunches [41], wheat-hemlock [38] and tea waste [49]
has been reported that revealed presence of cellulose, hemicellu-
lose and lignin. In some cases, ash content of feedstock was found
to signicantly inuence the yield of organics so that higher the ash
concentration lower is the yield of pyrolysis liquid [41]. Character-
ization of various biomass samples and derived bio-oils with TGA
and DTG shows three steps of weight losses i.e. (i) at around 100 C
due to water evaporation, (ii) at 250350 C due to lignin pyrolysis
and (iii) at 350500 C due to the combustion process. This esti-
mation gives valuable information about the sample reactivity so
that wood biomass having lower lignin showed higher reactivity.
Tar composition from dissimilar biomass also varied signicantly
[156].
Rice hulls were pyrolyzed inside a thermogravimetric analyzer
under helium atmosphere to determine kinetics of devolatilization
reactions. The pyrolysis experiments were conducted by heating
rice hulls from room temperature to 1173 K at constant heating
rates [157]. Evaluation of effect of high temperature aqueous pre-
treatment of wheat straw to modify bio-oil composition from fast
pyrolysis is reported [158]. In a separate study, volatile compounds
from TG-FTIR experiment were swept immediately into gas cells
to minimize secondary reactions [40]. Ash content of macroalgae
was estimated at 550 C together with the volatile matter and xed
carbon by means of TGA [159].
Apart from feedstock analysis, thermal stability of bio-oil was
also monitored systematically by Adjaye et al. in 1992. Fresh bio-
oil sample was distilled under vacuum at different temperatures
and its density, viscosity, distillation characteristics and composi-
tion were measured. The study concluded that yield of oxygenated
compounds like acids, ethers, alcohols and volatile aliphatic hydro-
carbons having moderate thermal stability decreased with the
increase in temperature above 200 C. In contrast, concentration
of aromatic hydrocarbons, naphthenes, aldehydes and ketones
showed maximum stability at around 175 C [63]. TGA and DTG
studies of blend of ether soluble bio-oil fraction with bio-diesel
have been reported. The study was undertaken to estimate ageing
and thermal stability of this mixture [83]. Optimization of reaction
temperature for cellulose pyrolysis is reported [160]. Fast pyrolysis
bio-oil produced in a cyclone reactor was evaluated thermally and
leftover solids were weighted at the end. The fractions of char and
unreacted biomass were measured by methods based on TGA. In
this way, temperatures of maximum mass losses were estimated
to lie at 137 and 287 C and it was assumed that bio-oil contained
very few components originating from holocellulose [123].
TGA data were used to obtain vital clues of crude and upgraded
bio-oil compositions [161]. In separate study, bio-oils obtained
from vacuum pyrolysis of softwood bark and hardwoods were frac-
tionated by solvent extraction and characterized with GCMS, TGA
and GPC. The TGA data showed very close proximity with GCMS
based on which components of bio-oils were classied into dis-
tinct chemical classes [110]. Qiang et al. compared TGA and DTG
curves for bio-oil which was analyzed in nitrogen and air. The
thermal behaviour exhibited similarities in nitrogen and air before
400 C while at temperatures above 400 C, burning of char residues
occurred in air. DTG curves of the bio-oil heated in nitrogen and
air showed two and three distinct peaks respectively. It was con-
cluded that rst two peaks corresponded to the loss of volatiles in
both atmospheres while third peak in the latter was due to burn-
ing of char residues [35]. TGA experiments have been reported for
calcium enriched bio-oil obtained from rice husk and important
thermo-chemical parameters such as degradation kinetics, acti-
vation energy, and pre-exponential factor were evaluated with
integral methods [162]. Separately, fuel properties of bio-oil + bio-
diesel mixture have been characterized [163]. The TGA and DTG
analysis of bio-oil obtained from pine wood and its blend with
bio-diesel is also reported. On the basis of overall description of
bio-oil boiling point distribution and cracking temperatures, DTG
curves were resolved into six major families of compounds. It was
proposed that DTG curves were good indicators of boiling point
distribution and overall composition of bio-oils. Furthermore, it
was assumed that families do not interact during evaporation and
cracking [127]. Very interesting application of TGA exists for cata-
lyst characterization in which it was used to determine the amount
of deposits in Pt/ZrO2 catalysts. The catalyst was used to obtain
hydrogen from bio-oil by steam reforming of acetic acid as model
oxygenated compound [164].
5.2. FTIR analysis
In FTIR analysis, mid-infrared region is extensively utilized to
reveal the presence of various functional groups in molecules. FTIR
analysis is of vital signicance to study the fundamental vibrations
and associated rotationalvibrational structure within a molecule.
Scientic literature contains huge informations on updates and
upcoming trends in modern infrared spectroscopy [165169]. FTIR
is essential in qualitative and quantitative component analysis,
thermal deterioration, ageing studies, quality control and quality
assurance applicable in almost all areas of contemporary chem-
ical and biological research [170176]. Enormous applications
of FTIR analysis for bio-oils exist accompanied by simple and
straightforward interpretation of the spectra. It generally com-
prises peaks above 3050 cm1 corresponding to CH stretching
vibration which indicates presence of aliphatic hydrocarbons. Peak
above 3100 cm1 corresponds to olenic CH stretch, between
3300 and 3400 cm1 corresponding to OH stretching vibrations
caused by carboxylic acids and/or alcohols. Peaks between 1450
and 1600 cm1 indicate a C C stretching vibration caused by
aliphatic or aromatic structure. The C O peak can be seen between
1600 and 1800 cm1 indicating either carboxylic acid/ester or alde-
hydes/ketone groups. CO stretching peak (in acids, esters, ethers
and alcohols) is seen between 1000 and 1100 cm1 . Aromatic rings
 P.K. Kanaujia et al. / Journal of Analytical and Applied Pyrolysis 105 (2014) 5574
can also be determined by the presence of CH out of plane bands
between 700 and 850 cm1 .
TG-FTIR investigation on the inuence of KCl over wheat straw
pyrolysis was reported in 1998 by Jensen et al. Comparison was
made between the pyrolysis yield from unwashed and washed
wheat straw in the presence of KCl with TG-FTIR data [155]. TG-FTIR
has been reported for monitoring the pyrolysis products from sev-
eral other agricultural residues (coconut shell, sugarcane bagasse,
corn stalks, peanut shell etc.) [40]. Characterization of the water
insoluble fraction of bio-oil suggested that oxygen content sig-
nicantly effects intensity of carbonyl absorption bands [177].
FTIR analysis of pentane, toluene and methanol extracts of bio-
oil showed aliphatic, aromatic and polar compounds respectively
[113,178]. Bio-oils from subcritical and supercritical water lique-
faction of wood biomass comprised largely of heavy oils, FTIR
analysis of which showed the presence of hydrocarbons, aldehydes,
ketones, hydroxybenzenes and esters [179].
In a well planned pyrolysis and upgrading study, bio-oil with
low oxygen content, high H/C ratios and high heating value was
produced from soybean stalk and deoxy-liqueed in a closed
reactor. It proposed dissociation of OH and recombination of
CH bonds into hydrocarbons which was accompanied by the
release of CO and CO2 . The bio-oil was characterized by FTIR
and usual oxygenates were observed. Further upgrading of this
bio-oil yielded nal product which was distilled and decolorized
over activated clay to obtain high quality bio-fuel [180]. Func-
tional group analysis of bio-oil obtained from rice husk suggested
nitrogenous compounds (NO2 stretching and NH bending) with
signals between 1550 and 1490 cm1 , in addition to other usual
informations [35]. FTIR analysis of bio-oil from lignin pyrolysis in
attenuated total reectance (ATR) mode showed predominance of
hydrocarbon structures along with smaller amounts of phenols and
carboxyl groups [181]. FTIR characterization of bio-oils obtained
from rapeseed cake [122], apricot and peach pulp [42,113], pre-
treated pine wood [46] and linseed [182] revealed presence of
usual functional groups. A ber optic near infrared system based
on reectionabsorption phenomenon has been proposed for pre-
dicting water content in bio-oils. Spectral informations in near
infrared region were obtained with varied levels of water con-
tent after applying multivariate analysis [125]. Bio-oil produced
from hydrothermal liquefaction of macroalgae was found to be
a complex mixture with more than 180 compounds including
ketones, aldehydes, phenols, alkenes, fatty acids, esters, aromatic
compounds and few nitrogen containing heterocyclic compounds
[159]. IR analysis of wheat-hemlock biomass derived bio-oil along
with its supercritical CO2 fraction is reported [38].
FTIR analysis of bio-oil and bio-char from tea waste indicated
importance of CH bending vibration at 1378 cm1 that provided
evidence of methyl groups [49]. Storage and thermal stability of
ether soluble fraction of bio-oil and bio-diesel blends were inves-
tigated and changes in chemical compositions were monitored by
FTIR with ATR method [83]. Reactivity of various carbonyl function-
alities in bio-oils have been predicted on the basis of FTIR analysis
and considered important in upgrading process. Carbonyl distri-
bution in bio-oil has been correlated with hemicellulose, cellulose,
lignin contents in feedstocks. The observation that carbonyl com-
pounds in light and heavy bio-oil fractions reacted differently with
methanol over amberlyst solid acid catalyst was also studied upon.
Since carbonyl stretch was observed as a broad band due to the
presence of various carbonyl containing functional groups, there-
fore it was deconvoluted to unveil detailed information [183].
As discussed, bio-oils contain substantial amounts of reactive
organic species that tend to repolymerize, rearrange and fragment
to form stable products. The scenario often causes difculty in
bio-oil storage, handling, transportation and upgrading. Series of
deterioration reactions during bio-oil storage have been studied
65
that resulted in the formation of several new functional groups.
The study was focused on evaluating bio-oil quality during ther-
mal treatments or long term storage at ambient temperatures.
FTIR analysis showed signal at 880 cm1 was assigned to CH out
of plane deformation vibration of terminal olens indicating start
of thermal deterioration process [184]. Accelerated ageing studies
from several minutes to 24 h were carried out to determine stabil-
ity of bio-oils and underline ageing process. FTIR spectra recorded
before and after the accelerated ageing steps showed increase in
relative concentration of CO stretch (phenols, carboxylic acids,
esters and ethers) and C O (carbonyl) functional groups [185].
Upgraded bio-oil after emulsication with bio-diesel was mon-
itored for stability through FTIR [163,186]. In a study, structural
and physico-chemical changes in bio-oils obtained from liquefac-
tion of swine manure with crude glycerol were monitored by FTIR
which indicated reduction in aliphatic structures and increase in
more oxidized and condensed polycyclic aromatic components
resulting from addition of crude glycerol to swine manure [52].
Bio-char obtained after pyrolysis of pine sawdust in a selective con-
densation method of pyrolysis was characterized by FTIR which
indicated N H, C H, C C and C O C bonds [187]. Bio-oil and char
from microalgae has also been characterized [188]. FTIR founds
immense application in bio-oil analysis and upgrading which is
evident through large number of reports. The analysis is simple,
straightforward and useful in generating bio-oil reference database
and ngerprinting.
5.3. Gas chromatographic analysis
GC is a versatile separation technique employed for volatile
and thermally stable organic compounds. Widespread utilization
of a variety of detectors has exponentially expanded the dimen-
sions of GC among which mass selective detection is one of
the most popular. Comprehensive two-dimensional GC (GC GC),
static headspace extraction-GC [189], fast GC [190] are prominent
techniques that have evolved over very short period and reviewed
periodically. Research in GC stationary phases has remained a large
attraction for analytical scientists with ionic liquids having signi-
cant applications as stationary phase and reported in the analysis of
bio-diesel blends [191]. Developments in GC methods, technology
and applications have been reviewed regularly [192,193]. Flame
ionization detection (FID) is an attractive GC detector owing to its
wide linear dynamic range, sensitivity, accuracy and precision. It is
a universal detector with broad response range for varied organic
compounds [194,195]. Ease of operation, maintenance, cost and
reliability makes FID a favorable detection system. Gases are often
conveniently analyzed with GC equipped with thermal conductiv-
ity detector (TCD). Petroleomics involve immense applications of
these simple detectors [196198]. Bio-oils contain chemicals with
varying volatility and polarity so that polar organic compounds are
difcult to analyze with capillary GC columns due to their low
solubilities in widely employed organic solvents. This challenge
is met through chemical derivatization as discussed previously
[96,117119]. Bio-oils always contain macromolecules arising from
thermal degradation of lignins, hemicelluloses and celluloses that
damage GC columns.
In 1986, Elliott employed GC-FID for estimating concentrations
of the tar components from biomass pyrolysis gasication conden-
sates [199]. GC-FID has been used quite extensively for the analysis
of bio-oil components. It was utilized with fused silica capillary
column [63,104], quantication of PAHs [200] and determination
of bio-oil components from chromated copper arsenate treated
wood [201]. In a round robin test conducted by International Energy
Agency and European Union, GC analysis was included as a primary
technique for bio-oil characterization. The recommended station-
ary phase was bonded, cross linked mid polar capillary column
66 P.K. Kanaujia et al. / Journal of Analytical and Applied Pyrolysis 105 (2014) 5574
containing 14% cyanopropyl-phenyl. In the same test, participating
laboratories reported GC-FID analysis of organic acids, aldehydes,
ketones, alcohols, sugars, phenols and PAHs [144]. GC-FID analyses
of acetic acid [116], low molecular weight chemically derivatized
aldehydes [101] and sewage sludge pyrolysis liquid [202] have been
reported. In addition, components in bio-oil fractions obtained with
supercritical CO2 have also been determined [38].
Group type analysis of oxygenated organic compounds in bio-
oils with the use of silica gel column and GC GC-FID is reported
[100]. Extraction of bio-oils with LLE, SPE and column chro-
matography has been performed and analyzed with GC-FID using
intermediate polarity capillary column [113,178]. Separately, gases
evolved during cellulose pyrolysis were collected in a large bag
and analyzed with GC [160]. In addition, total masses of perma-
nent gases obtained during biomass pyrolysis have been calculated
from GC-FID/TCD [123]. In large number of similar applications,
characterization of uncondensed gases such as CO, CO2 , CH4 , H2 ,
C2 H4 , C2 H6 , C3 H6 , C3 H8 , C4 H8 , and C4 H10 has been preferred by
GC-FID/TCD [33,39,41,43,48,108,126,188,198,203206].
Since a decade, GC GC has rapidly progressed and emerged as
a powerful analytical tool for the analysis of organic compounds.
Briey, GC GC offers discrete separations where the components
of sample are separated rstly in one dimension and then sub-
sequently separated in the second dimension. Excellent results
are obtained if the two or more separation mechanisms are inde-
pendent of each other. For convenience, the technique is termed
multidimensional GC. GC GC plays a vital role in separation of
large number of compounds with very close boiling points such as
in bio-oils. Due to the promising results, this technique has found
immense applications and has been reviewed [207,208]. Relation-
ships between molecular structures and retention times in GC GC
analysis have been studied [209]. Application of GC GC-FID has
been demonstrated for identication and quantication of compo-
nents in complex mixtures of tobacco oils [210].
Pyrolytic products from cellulose, lignin and sewage sludge
were analyzed by multidimensional GC coupled to high resolution
mass spectrometer (HRMS) [211]. Identication of fast pyrol-
ysis oil components and upgraded products with GC GC-FID
and GC GC-TOFMS is also reported [161,212]. In another study,
pyrolytic products from a pyrolysis rig were analyzed through
GC GC-FID (gaseous) and GC GC-TOFMS [204]. Volatile organic
compounds released from bio-pitch have been characterized with
GC GC [45]. Qualitative and quantitative characterization of bio-
oils obtained from three different sources is reported by Sfetsas
et al. with GC-FID and GC GC-TOFMS [213].
5.4. Mass spectrometric analysis
Mass spectrometry (MS) provides crucial elemental and
structural composition of organic molecules. Key technological
advancements in sample introduction, ionization and analyzers
in the past two decades have revolutionized the research which
has largely focused upon biological perspectives. Reviews have
appeared that highlighted signicant technological advances and
diverse application of MS [214,215]. Development in ionization
techniques has evolved matrix assisted laser desorption ioniza-
tion (MALDI) as a powerful ionization method for thermolabile
molecules especially biomolecules. Desorption electrospray ioniza-
tion (DESI) is manifested by direct analysis in real time (DART) mass
spectrometers. High resolution mass spectrometers (HRMS) are
nowadays quite common and signify exponential progress in mass
analyzer research. Petroleum analysis has been highly facilitated
with HRMS especially time of ight (TOF) and Fourier transform ion
cyclotron resonance (FT-ICR). Excellent reviews have been written
in this area detailing fundamentals and basic to advanced applica-
tions of HRMS in various R&D sectors [216,217].
Accurate mass measurements have deconvoluted informations
from complex matrix like petroleum where as much as 250 chemi-
cals have been known to have same nominal masses [218]. Bio-oils
have comparable degree of compositional complexity; hence suf-
cient scope exists in the investigations utilizing higher mass
resolving power of MS to extract accurate molecular level informa-
tions. Researchers in of the area of bio-oil analysis have extensively
involved quadrupole analyzer based mass spectrometers. This ana-
lytical approach is quite suitable for characterization of small
molecular weight organic compounds from aliquots obtained after
sample preparation and offers labor and cost effectiveness. The
analysis may involve GC to introduce sample to achieve chromato-
graphic separations. The obtained results are conclusive in electron
ionization (EI) mode since mass spectral database exists. Recently,
widespread utilization of TOF-MS coupled to multidimensional GC
has also been witnessed.
Tars resulting from wood carbonization were examined that
provided simple guidelines to biomass pyrolysis oil followed by
GCMS analysis under electron ionization (EI) mode utilizing
quadrupole analyzer. Prior to the analysis, samples were extracted
with dichloromethane which extracted non-polar plus moderately
polar compounds. Highly polar components however constituted
bulk of sample and were found difcult for analysis [199]. To
overcome this, ion-exchange and size exclusion liquid chromato-
graphic conditions were developed followed by GCMS methods
of analysis [104]. GCMS characterization and stability analysis
of wood derived bio-oil have been performed in which more
than 85 individual compounds including oxygenated ones and
C6 C11 hydrocarbons were identied. Oxygenated compounds
mostly included acids, cyclic alcohols, aliphatic alcohols, aldehydes,
cyclic ketones, substituted furans, ethers and alkyl substituted and
methoxy phenols. Hydrocarbons consisted mainly of long chain
parafns, olens, aromatics, cycloparafns (non-condensed and
condensed) and polycyclic aromatic hydrocarbons [63]. Fast pyrol-
ysis oil derived from polystyrene has been analyzed for PAHs
through GCMS equipped with ion trap analyzer and the mass spec-
tra of compounds were interpreted for compositional clues along
with comparison with relative retention indices obtained with FID
[200].
Phenolic compounds from bio-oil were separated from high
MW lignins and then extracted into toluene for GCMS charac-
terization [105]. Compositions of eight pyrolytic lignins obtained
from different feedstocks were investigated with GCMS equipped
with analytical pyrolyzer (Py-GCMS) [177]. Wide range of organic
compounds in bio-oils has been identied with GCMS that
consisted mainly of ketones, phenols, aldehydes, ethanol, acids, etc.
[108,160]. In a similar study, pentane soluble fraction of bio-oil was
fractionated over column chromatography and sub-fractions con-
taining aliphatic, aromatic and moderately polar compounds were
analyzed with GCMS [113,178]. Identication of bio-oil obtained
in a cyclone reactor was extensively carried out with GCMS
and the identied compounds were categorized into (i) holocel-
lulose derived compounds viz. propanol, acetic acid, propanone
and furfural; (ii) lignin derived compounds viz. phenol, cresol, gua-
iacol, xylenol, methyl guaiacol, ethyl guaiacol, syringol, vanillin
and eugenol and (iii) PAHs such as pyrene, uorene, naphthalene,
phenanthrene, byphenyl and anthracene [123].
Complex nature of bio-oils is apparent from studies that involve
GCMS analysis of bio-oil from rice husks [35] in which more
than 100 peaks were displayed in chromatogram with only few
could be evaluated with semi-quantitative approach [36]. Com-
parison of bio-oil components with GC GC-FID and GCMS has
been shown [161]. On the basis of solvent extraction followed by
GCMS characterization, bio-oil components were classied into
distinct groups such as volatile non-polars, volatile polars, mono-
lignols, polar with moderate volatility, sugars, extractive-derived
 P.K. Kanaujia et al. / Journal of Analytical and Applied Pyrolysis 105 (2014) 5574
compounds, heavy non-polars and heavy polars [110,111]. GCMS
analysis of bio-oils components obtained through sub- and super-
critical water liquefaction of woody biomass revealed presence of
heavy oils which comprised of hydrocarbons, aldehydes, ketones,
hydroxybenzenes and esters [179]. GCMS analysis of bio-oil from
fruit pulp [42], soya bean stalk [180], sewage sludge [202], switch-
grass/alfalfa stems [47], wheat straw [37] and pine sawdust [219]
has also been reported.
Electrospray ionization (ESI), a soft ionization technique is typ-
ically used in liquid chromatography mass spectrometry (LCMS)
systems in order to obtain molecular weight information of polar,
thermolabile molecules. Molecular mass distribution and chem-
icals present in bio-oil obtained from lignins are reported with
LCMS [181]. GCMS was also utilized for analyzing polar com-
pounds after chemical derivatization (silylation) [181]. Bio-oil
analysis through positive and negative ESI-MS showed 320 peaks
with mass distribution ranging between 100 and 1100 mass units
which were substantiated through size exclusion chromatographic
data [220]. LC-ESI-MS and chemometrics however, have been
reported for the ngerprinting of components present in oil and
petroleum products [221]. TOF-MS in conjunction with GC GC is
reported to identify and classify components (group type analy-
sis) in ash pyrolysis oil and hydro-deoxygenated oils [212]. Bio-oil
produced in an Auger reactor from pyrolysis of pine and oak wood
was analyzed through GCMS [124]. One of the GCMS analy-
sis of bio-oil obtained from wood suggested that one-fourth of
all the identied compounds were furfural or furfural derivatives
formed by dehydration of monosaccharides present in cellulose or
hemicellulose. Other compounds originating from wood carbohy-
drates were acetic acid, hydroxyl acetaldehyde, hydroxyacetone,
ethyl acetate, 1-hydroxy-2-butanone and 5-hydroxymethyl fur-
fural [154].
Removal of organic acids (acetic and formic) from bio-oil has
been reported to be monitored with GCMS [125]. Qualitative
interpretation of the mass spectral data of bio-oil components
at different electron beam energies has been shown to provide
molecular weight distribution in bio-oil. In this study, analysis was
performed with TOF-MS at a resolution of 8000 and samples were
introduced into MS through direct insertion probe. Thereafter, EI
mass spectra were acquired at varying electron beam energies
i.e. 12, 15, 30, and 70 eV which indicated a decrease on peaks
and m/z range with increasing electron beam energies [34]. Rela-
tionship between feedstock chemical pre-treatments and bio-oil
properties has been established on the basis of GCMS analysis
of bio-oil which showed that chemical concentrations of compo-
nents arising from degradation of hemicelluloses, cellulose, and
lignin were lower in treated than untreated pine wood [46]. Char-
acterization of slow pyrolysis oil obtained from linseed is reported
with GCMS [182]. Separately, pyrolysis products obtained from
technical lignins have been classied into three groups such as
coke, liquid and gas. Liquid fraction was analyzed with GCMS
and GC-FID along with GC GC-TOFMS and GC GC-FID. Gases
were characterized and semi-quantitative studies were carried out
by headspace GCMS/FID analysis [204]. Py-GCMS studies have
also been carried out to evaluate high temperature aqueous pre-
treatment method to modify or upgrade bio-oil from fast pyrolysis
of wheat straw [158]. GCMS characterization of bio-oil obtained
from pyrolysis of whole tree biomass [126], corn cobs/stover
[39], waste furniture sawdust [43], barley biomass/byproduct,
rice husk [184] and softwood/hardwood [222] has been reported
[33] along with hydrothermal liquefaction of algal biomass [159].
Semi-quantitative identication of 104 volatile organic compounds
released from distillation residues is reported with GC GC-TOFMS
[45]. Bio-oil stability has been monitored through accelerated age-
ing processes with GCMS [185]. GCMS has also been used for the
analysis of volatile compounds in bio-oil samples GCMS [183].
67
Low molecular weight aldehydes have been derivatized and ana-
lyzed with GCMS [101]. Bio-oil obtained through stage fractions
from pyrolysis vapor streams was characterized [116].
PAHs evolved from pyrolysis reactions has been investigated
in which alkylated benzofurans, indenes and naphthalenes dom-
inated above other PAHs and their presence were conrmed
through retention indices values. Around 63 PAHs have been iden-
tied and some of them were also quantied [102]. Analysis of
wheat-hemlock derived bio-oil, fractionated from supercritical CO2
is reported. Common elements in the biomass ash were determined
by inductively coupled plasma mass spectrometry (ICP-MS) [38].
GCMS analysis of pentane soluble (aliphatic) sub-fraction of bio-
oil obtained from tea waste was carried out which showed presence
of n-alkanes, alkenes and branched hydrocarbons while the major-
ity of linear hydrocarbons were distributed in the range of C11 C29 .
A semi-quantitative estimation of hydrocarbon distribution was
presented by considering mean of the percentage chromatographic
peak areas that revealed 72.87% n-alkanes, 16.44% n-alkenes and
1.84% branched hydrocarbons [49]. GCMS and PIONA (Parafns,
Isoparafns, Olens, Naphthenes, and Aromatics) analysis of oxy-
genated compounds in hydrotreated biomass fast pyrolysis oil
distillate fractions is reported [128].
Py-GCMS was used to compare the water insoluble solids
isolated from bio-oils which revealed their origin from lignin
[106,107]. Chen et al. utilized several inorganic salts to induce
phase separation of bio-oils; the upper hydrophilic layer contained
water soluble polar compounds (organic as well as inorganic) while
the lower layer contained non-polar lignins. Both the layers were
analyzed by GCMS after applying various upgrading processes
[88]. GCMS was used to characterize the volatile components
from vacuum batch reactor and the oils were extracted using
benzene followed by methanol and GCMS analysis to study the
ageing prole [112]. Bio-oil produced from swine manure through
co-liquefaction with crude glycerol upon GCMS characterization
indicated that crude glycerol addition facilitated the esterication
reaction [52]. In another study, mixture of food waste oil fractions
and BEA zeolite/Al-SBA-15 catalysts were introduced directly to
Py-GCMS and the resultant vapor phase products were simulta-
neously analyzed [80]. A Comparison between water content of
bio-oil was made between KarlFisher titrations and azeotropic
distillation using toluene. GCMS correction was applied to
azeotropic distillation which closely agreed with KarlFischer titra-
tion results [223].
For the rst time, HRMS was employed to evaluate bio-oil
composition using laser desorption ionization (LDI) and linear
ion trap-orbitrap analyzer. Bio-oil obtained from pyrolysis of pine
wood was analyzed with orbitrap and the data were compared
with FT-ICR-MS data to establish the minimum resolution required
for unambiguous detection of analytes. Orbitrap analyzer has been
reported to provide sufcient resolution with which authors
could determine chemical compositions for over 100 molecular
compounds [224]. The same group of researchers has recently
compared high resolution orbitrap, quadrupole-TOF (QTOF) and
FTICRMS mass spectra of bio-oils in negative ESI modes. With this
study, it seems the authors have clearly differentiated polyhydroxy
cyclic compounds from phenolics. Heteroatom class distribution
and double bond equivalence have also been performed with
FTICRMS to obtain clearly distinguished phenolics (lignin derived)
[225]. Bio-oils obtained from mallee and pine wood were subjected
to GCMS analysis on the basis of which, researchers concluded
that bio-oil from pine lacked syringols since pine is softwood
whereas syringyl units are only found in hardwood lignins [127].
GC GC-TOFMS analysis of different bio-oil samples diluted in
organic solvent led to the identication of nearly 300 compounds,
whose spectra were eventually deconvoluted for group type
classication [213]. MALDI-TOF-MS, LDI-TOF-MS and temperature
68 P.K. Kanaujia et al. / Journal of Analytical and Applied Pyrolysis 105 (2014) 5574
resolved analytical pyrolysis eld ionization mass spectrometry
(Py-FIMS) have been reported for pyrolytic lignins precipitated
from different aged bio-oil [226].
Gaseous and liquid pyrolytic products from pine, oak, and sweet
gum sawdust were analyzed with GCMS more than 100 chemi-
cals were identied and several quantied [227]. Functional group
distribution in bio-oil from chemical pre-treatment of wood feed-
stock has been elucidated with GCMS based method [201]. Change
in pyrolysis conditions and selection of different feedstocks has
been reported to alter bio-oil stability [228]. Pyrolysis oil obtained
from wheat and oat straw has been analyzed with GCMS [229].
Compositional characterization of hydrotreated fast pyrolysis oil
[230], bio-oils derived from pine sawdust [187], seaweed [231] and
sugarcane straw [232] were achieved through GCMS. Py-GCMS
and bench top GCMS were employed to simultaneously moni-
tor the components of bio-oil with nearly 75% identication and
quantication of GC peaks [233]. Thermo-chemical liquefaction
and pyrolysis of algal biomass have been compared and product
characterization involved ICP-MS and GCMS [188].
Mass spectrometry has emerged as a powerful tool in the com-
positional characterization of bio-oils derived from lignocellulosic
biomass. Ongoing rigorous use of the technique for bio-oil analysis
is apparent by the presence of enormous literature informations.
There is a strong need for inter-laboratory prociency testing in
the form of round robin tests so that uniform analytical guidelines
and results evaluation criteria evolve in due course of time. Since
mass spectrometric results provide conclusive evidence of com-
pounds in fresh and aged bio-oil samples, therefore storage and
stability issues associated with it must be addressed. Outcome of
round robin tests also emphasized that GCMS analysis of bio-oil
must be included in future tests so that the validity of laboratory
developed methods is evaluated [144].
5.5. Nuclear magnetic resonance analysis
Nuclear magnetic resonance (NMR) is one of the important
spectroscopic techniques employed in analytical and bioanalyt-
ical chemistry. NMR spectrometers are sophisticated and largely
present in research and academic institutions across the world.
The operating frequencies of modern NMR spectrometers have
extraordinarily increased to nearly 1 GHz. The technique is nowa-
days essentially employed in range of experiments on solid, liquid,
and gaseous samples, over a wide temperature span. Inherent
limitation associated with NMR is its relatively low sensitivity
as compared to other spectroscopic techniques. Hence, trace and
ultra-trace level quantitation are always represented by poor lim-
its of detection that is several orders of magnitude poorer than mass
spectrometry or emission/absorption/uorescence based tech-
niques. NMR is exceptionally used in the structural analysis of
petroleum fractions. It is an essential tool for carrying out group
type analysis and directly measures aromatic and aliphatic carbon
(13 C) and hydrogen distributions (1 H). It also provides important
information on the carbon and hydrogen structural groupings in a
molecule. 13 C NMR studies of coal and asphaltenes are the pioneer
NMR applications in petroleum analysis [234,235].
In bio-oil analysis, major advantages of NMR are that the whole
bio-oil can be dissolved in a suitable solvent (such as deuterated
dimethylsulfoxide) and a qualitative assessment of the oxygen con-
taining functionalities can be determined simply from integration
of appropriate regions of the 13 Cand 1 H NMR spectra. Bio-oils have
been largely studied with NMR and the 13 C NMR spectra are par-
ticularly instructive because of their large chemical shift regions.
13 C and 1 H NMR offer a reasonable trade-off between functional
group identication and analytical measurement effort. Accuracy
however depends upon selection of chemical shift regions, baseline
compensation, and correction for incomplete longitudinal relax-
ation effects.
Elliott in 1986 reported NMR to provide additional conrma-
tion of the chemical functional groups in bio-oil. The only concern
at that time was the solubility of components in deuterated acetone
or chloroform. In addition, the 13 C NMR spectra were of poor quality
due to low signal-to-noise ratio and imperfect baseline attenua-
tion. The results were attributed to the presence of high molecular
weight compounds and insufcient solubility of the components
[199]. Pyrolytic lignin recovered from crude pyrolysis oil was char-
acterized by 13 C NMR to conclude that high degree of thermal
splitting occurred during pyrolysis [106]. NMR analysis was also
carried out in 1987 for pyrolysis oil, oil fractions, and pyrolytic
lignins [236,237]. In the studies that evaluated effect of pyrolysis
conditions like sweeping gas and steam on product formation were
investigated through 1 H NMR in which pentane soluble fraction
of bio-oil was investigated. In addition, percentage of aromatics,
parafns and olens in pyrolytic oils is also reported which helped
to optimize crucial pyrolytic parameters and manipulate the yields
[42]. Chemical structures present in bio-oils derived from pyroly-
sis of lignins have been characterized through NMR with 1 H, 13 C
and 31 P to show complete degradation of the lignin. Hydroxyl and
carboxyl groups were quantied using 31 P NMR after derivatiza-
tion with 2-chlorophosphalone [181]. This study is an outstanding
demonstration of NMR application to quantitatively establish pres-
ence (or absence) of any species in uncomplicated manner. 31 P NMR
spectra are devoid of too many peaks from other nuclei and can be
studied in a wide chemical shift scale.
Determination of bio-oil functional groups, hydrogen distri-
bution and carbon aromaticity is reported in from pyrolysis of
wood and bark [124], linseed [182] and macroalgae (Enteromor-
pha prolifera) [159]. 1 H, 13 C and Distortionless Enhancement by
Polarization Transfer (DEPT) NMR were used to characterize fast
pyrolysis oils from switchgrass, alfalfa stems, corn stover, guayule
plant and chicken litter in order to quantitatively classify carbon
and hydrogen. DEPT was used to quantify protons and number of
carbon atoms in off-resonance decoupled 13 C spectra to extract
further information on the types of molecules that are found in
bio-oil [238]. In a similar study, several biomass fast pyrolysis oils
were characterized by 13 C and DEPT NMR to determine functional
group compositions related to their energy content. The spectra
were compared with gasoline and diesel and divided into distinct
regions according to the chemical shift ranges appropriate for var-
ious functional groups. The spectral intensities of each region were
quantied and carbon proton substitution numbers were deter-
mined. In addition, chemometrics was used to extract more useful
information from spectra [239]. 13 C NMR suffers from low sensi-
tivity due to its low natural abundance and long relaxation times.
Joseph and coworkers proposed chemical shift assignments and
correction factors based on 13 C and 1 H NMR measurements of large
number of compounds. Semi-quantitative assessment of molecular
composition of bio-oils on 8 h time scale was provided for pro-
cess development [240]. 1 H NMR study carried out for determining
hydrogen distributions in crude bio-oil and its fraction with super-
critical CO2 indicated nearly 50% water. However, the supercritical
CO2 fraction was found to contain aromatics, phenolics, olenics,
ring attached methylenes, aromatic exocyclics CH3 , CH2 and CH
protons [38]. Similar estimations were also obtained for bio-oil
obtained from tea waste [49].
Thermal stability of bio-oil and bio-diesel blends has been
monitored with 1 H NMR [163,186]. Similarly, thermal stability
of ether soluble fraction of bio-oil and its blend with bio-diesel
was also studied. Structural changes in the blends have also been
measured along with proton and carbon assignments with 1 H and
13 C NMR experiments respectively [83]. Hydrotreated and distilled
bio-oils with different oxygen contents were used to produce light
 P.K. Kanaujia et al. / Journal of Analytical and Applied Pyrolysis 105 (2014) 5574 69

naphtha, jet, diesel, and gas oil boiling range fractions followed The study concluded that phenols present in the bio-oil showed
by 13 C NMR analysis for oxygen containing species. Interestingly, chemical instability by giving time dependent uorescence inten-
it was proposed that presence of very broad peak in the spectra sities [244]. Bio-oil analysis with AAS, ICP-AES, X-ray diffraction
resulted due to the presence of polymeric material with slow (XRD) and optical microscopy has been carried out. In addition,
molecular motions [107]. 1 H NMR analysis for bio-oil water elemental analysis (C, H, N and S) and KarlFisher titration for
determination was compared with conventional methods like moisture is also reported [95]. Brix method to accurately esti-
KarlFisher titration and azeotropic distillation [223]. In an mate water content in bio-oils has been reported which estimated
attractive research investigation, 31 P was used for determining that small carbohydrates obtained after the pyrolysis lower bio-oil
three principal forms of phenolic hydroxyls present in lignins (p- quality and stability due to their reactivity. Brix is a commercial
hydroxyphenyl, guaiacyl and syringyl structures). Other functional method for quantifying sugar content of grapes and wine how-
groups present in lignins such as primary hydroxyls, carboxylic ever, demonstrated to possess applicability for pyrolysis oil [245].
acids and two diastereomeric forms of arylglycerol-beta-aryl In another study, bio-char was characterized for its surface mor-
ether units were quantied from 31 P NMR experiment after phology through scanning electron microscopy (SEM) [187]. SEM
phosphitylation [201]. and XRD have been extensively used in characterizing catalysts that
1 H NMR analysis of bio-oils obtained from wheat and oat straw are commonly employed in the upgrading of bio-oils and have not
is reported which concluded that oils were highly heterogeneous been discussed in this review.
chemically and contained phenols, carbonyl groups, carboxylic
acids, aromatic compounds, monoaromatic compounds, PAHs,
6. Future perspective
esters, n-alkanes, alkenes, and nitrogenous compounds [229]. Com-
position of hydrotreated bio-oil is also reported to be determined
Rapid development of advanced analytical methods in bio-oil
with 1 H, 13 C and 31 P NMR experiments [230]. Separately, from
13 C NMR analysis, it was concluded that carbohydrate fraction analysis has enhanced our understanding about its composition
and correlation of several variables responsible for quality, upgrad-
decreases and aromatic fraction increases when pyrolysis temper-
ing and stability. Nature of biomass feedstocks is an important
atures in a uidized-bed reactor were increased [219]. Quantitative
13 C and 31 P NMR were used to characterize the pyrolysis oil from variable that governs nal bio-oil properties in addition to the
adopted pyrolysis process, reactor design, pressure and tem-
Kraft lignin and the data indicated that aliphatic hydroxyl, carboxyl,
perature conditions. Gravimetry together with spectroscopy and
and methoxyl groups were eliminated during pyrolysis [241]. In
chromatography has extracted important informations from com-
continuation, analysis of pyrolysis products from softwood kraft
plex environmental, biological and other matrices. There exists a
lignin, cellulose, and loblolly pine wood by heteronuclear single
huge potential of value addition to bio-oils as they are mother liquor
quantum correlation NMR (HSQC-NMR) provided chemical shift
to large number of valuable chemicals as well as prospective fuel
assignment for 27 different types of C H bonds present in bio-oils
applications. Great deal of research in this area had major focus
and indicated presence of two different types of methoxyl groups in
on setting up rening and upgrading processes for fast pyrolysis
samples. Levoglucosan, furfurals and phenols were the major com-
oils. Chemical groups that are formed during pyrolysis of lignocel-
ponents in all samples; however, most aliphatic and aromatic C H
lulosic biomass are very well known today. Also it is quite simple
bonds were produced from lignin component [242].
to ascertain amounts of macromolecular constituents in the feed-
stocks. Improvements in pyrolysis processes have been fascinating
5.6. Other techniques
the researchers to maximize yields and quality of bio-oils and it
remains as one of the favorite area of contemporary research. Ana-
Applications of other instrumental analytical techniques besides
lytical insights have simplied overall bio-oil understanding that
those discussed above are very limited due to the complex nature
ranges from compositional revelations to modelling and chemo-
of bio-oil as a matrix. Presence of very large number of compounds
metric approaches in process optimizations. Despite vast analytical
with varied functional groups in bio-oils has necessitated devel-
informations which we have about bio-oil composition and moni-
opment of universal extraction and analytical protocols. Very few
toring of upgrading processes, reliable rening scale is still far away.
studies have been undertaken for bio-oil characterization with such
Chromatographic approaches of analysis offer several formats
techniques which have proven capabilities in other areas of mod-
of extraction with increasing types of adsorbents, optimization of
ern research. Characterization of phenolic fraction of bio-oil with
which leads to efcient segregation of different chemical classes of
micellar electrokinetic chromatography is reported. The technique
bio-oils. Nanomaterials exhibit extraordinary physical, chemical,
is chromatographic that closely resembles capillary electrophore-
mechanical and electrical properties and therefore offer tremen-
sis (CE). Components of a sample are separated by differential
dous applications in sample pre-treatment, catalysts formulation
partitioning between micelles (pseudo-stationary phase) and a
in upgrading and specic extractions. In sample preparation,
surrounding aqueous buffer solution (mobile phase) [243]. Char-
their enormous adsorption potential may be very well utilized
acterization of hydrotreated fast pyrolysis liquids is also reported
as such or with covalent/non-covalent modication of surfaces.
using CE [230]. HPLC analysis equipped with retention index detec-
Magnetic nanoparticles with or without functionalization offer
tor is reported for polar bio-oil components [47]. Another method
large variety in selective extractions as well as desired sample
of HPLC separation facilitated by ultraviolet detection has been
pre-treatments. Principles of immunoafnity chromatography in
reported for low molecular weight aldehydes present in bio-oils
the form of molecularly imprinted polymers need to be explored
after derivatization with 2,4-dinitrophenylhydrazine [101]. Multi-
for specic rening applications. LCMS and CE in conjunction
dimensional liquid chromatographic methods were optimized for
with suitable sample pre-treatment methods promise wide range
estimation of phenols from bio-oil [105].
of applications in the identication of unknown chemicals with
Bio-oil from pyrolytic lignins has been ngerprinted with size
very wide range of polarity.
exclusion chromatography coupled to ultraviolet and retention
index detector to obtain average molecular weight, dispersity,
and molar mass at the maximum peak height [181,226]. Laser 7. Conclusion
induced uorescence was used to simulate and monitor the effect
of ultraviolet exposure on the chemical stability of bio-oil pro- Biomass derived fast pyrolysis oils are potential source of
duced from pine lumber, pine bark, oak bark, and oak wood. future energy requirements and value-added chemicals for diverse
70 P.K. Kanaujia et al. / Journal of Analytical and Applied Pyrolysis 105 (2014) 5574
industrial applications. A concerted effort towards value addition
of bio-oils is absolutely essential to circumvent increasing burden
on limited petroleum resources. Owing to the current state of
research and thrust being put in this area, it is very well known
by now that bio-oils comprise of numerous chemical species and
differ signicantly with each other in several physical and chemical
properties. Presence of signicant amounts of oxygenated com-
pounds has necessitated understanding of such complex matrices
at molecular levels so that pilot or industrial scale upgrading
becomes practical. Analytical chemistry offers unequivocal identi-
cation and quantitation of target analytes as well as the matrix as
a whole. Spectroscopic analysis has always dominated over other
tools of analysis due to obvious reasons, however large number of
physico-chemical property determinations heavily relies on clas-
sical methods of analysis. The discussions highlighted important
research in this area and enumerated various approaches and tech-
niques adopted by to deconvolute vital compositional informations
that varied from one sample to the other. In this process, we might
have missed some signicant ndings which are inadvertent. Some
discussion on upgrading and TGA has been included in addition
to the spectroscopic techniques just to emphasize the importance
of correlation between feedstock characterization and obtained
bio-oil quality. Unstable chemical species in bio-oils revealed by
TGA necessitates development of efcient extraction protocols that
must be adopted immediately after bio-oil production for explicit
rening and value addition applications. Other spectroscopic tech-
niques like mass spectrometry, NMR, FTIR and chromatography
have simplied the bio-oil analysis with high validity, precision and
accuracy.
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