Critical Reviews in Biotechnology, 2010, 1–22, Early Online

© 2010 Informa Healthcare USA, Inc.

ISSN 0738-8551 print/ISSN 1549-7801 online
DOI: 10.3109/07388551.2010.513677

REVIEW ARTICLE

Extraction and Analysis of Polyphenols: Recent trends

C.M. Ajila1, S.K. Brar1, M. Verma2, R.D. Tyagi1, S. Godbout2, and J.R. Valéro1
1
INRS-ETE, Université du Québec, 490 Rue de la Couronne, Québec, Canada, and 2Institut de recherche et de
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développement en agroenvironnement (IRDA), 2700 rue Einstein, Québec, Canada

Abstract
In recent years, there has been an increasing interest in diets rich in fruits and vegetables and this is mostly due
to their presumed role in the prevention of various degenerative diseases, such as cancer and cardiovascular
diseases. This is mainly due to the presence of bioactive compounds, such as polyphenols, carotenoids, among
others. Polyphenols are one of the main classes of secondary metabolites derived from plants offering several health
benefits resulting in their use as functional foods. Prior to the use of these polyphenols in specific applications, such
as food, pharmaceutical, and the cosmetic industries, they need to be extracted from the natural matrices, then
analyzed and characterized. The development of an efficient procedure for the extraction, proper analysis, and
characterization of phenolic compounds from different sources is a challenging task due to the structural diversity of
phenolic compounds, a complex matrix, and their interaction with other cellular components. In this light, this review
discusses different methods of extraction, analysis, and the structural characterization of polyphenolic compounds.
For personal use only.

Keywords:  Polyphenol, extraction, purification, analysis, characterization

Introduction reducing agents, hydrogen donors, and singlet oxygen

Polyphenols are a large family of natural compounds
which are secondary metabolites and are derivatives of
the pentose phosphate, shikimate, and phenylpropanoid
pathways in plants (Ryan et al., 1999). A schematic repre-
sentation of pathways for the formation of polyphenolic
compounds in plants is given in Figure 1. Phenolic com-
pounds play a major role in growth and reproduction,
providing protection against pathogens and predators
besides contributing towards the color and sensory
characteristics of fruits and vegetables (Borochov-Neori
et al., 2009). Phenolic compounds exhibit a wide range of
beneficial properties to health, such as: anti-allergenic,
anti-inflammatory, anti-microbial, anti-oxidant, anti-
thrombotic, cardio protective, and vasodilatory effects
(Chung and Champagne, 2008; Viswanath et  al., 2009;
Singh et al., 2010). Several beneficial effects derived from
phenolic compounds are mainly due to their antioxidant
activity (Viswanath et al., 2009; Tagliazucchi et al., 2010).
Health benefits linked to polyphenols and their applica-
tion have been already well discussed in several earlier
reviews (Terao, 2008; Del Rio et al., 2010).
The antioxidant properties of polyphenolics are mainly
due to their redox properties, which allow them to act as
Address for Correspondence:  Satinder Kaur Brar, Institut national de la recherche scientifique Centre Eau, Terre & Environnement/Centre for
Water, Earth and Environment 490 de la Couronne, Québec, Canada. Tel.: + 418 654 3116; Fax: + 418 654 2600; E-mail: satinder.brar@ete.inrs.ca
(Accepted 02 August 2010)
quenchers (Rice-Evans et  al., 1996). They also act as a
chelator of metal ions, preventing metal catalyzed forma-
tion of free radical species (Salah et al., 1995). Phenolic
antioxidants interfere with the oxidation of lipid and
other molecules by rapid donation of a hydrogen atom to
radicals as shown in the following Equations:
→ ROOH + PP * (1)
ROO * + PPH
RO * + PPH → ROH + PP* (2)

 The phenoxy radical intermediates are relatively stable;
therefore, a new chain reaction is not easily possible.
The phenoxy radical intermediate also acts as a termina-
tor of the propagation route by reacting with other free
radicals (Shahidi and Wanasundara, 1992) and forming
a compound.
ROO * + PP* → ROOPP (3)

RO * + PP* → ROPP (4)

 Structurally, phenolic compounds comprise an aromatic
ring, bearing one or more hydroxyl substituents, and range
from simple phenolic molecules to highly polymerized

1
 2  C.M. Ajila et al.
Abbreviations
Solid–Liquid Extraction (SLE)
Liquid–Liquid extraction (LLE)
Pressurized Liquid Extraction (PLE)
Accelerated Solvent Extraction (ASE)
Super Critical Extraction (SCE)
Super Critical fluid (SCF)
Ultrasonic-Assisted Extraction (UAE)
Microwave Assisted Extraction (MAE)
Hydrochloric acid (HCl)
Ethylenediamine tetraacetic acid (EDTA)
Solid Phase Extraction (SPE)
Peroxidase catalyzed Enzymatic method (PE)
Partial Least Squares (PLS)
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Principal Least Squares (PCA)
Near infrared spectroscopy (NIR spectroscopy)
Fourier transform near infrared reflectance spectroscopy (FT-NIR)
Nuclear Magnetic Resonance (NMR)
High Pressure Liquid Chromatography (HPLC)
Pentose phosphate
pathway
Shikimate
For personal use only.
pathway
Carbohydrate
metabolism
Phenylpropanoid Cinnamic acid
pathways Benzoic acid
Malonyl Co-A
Flavonoids
Figure 1.  Biosynthetic pathways of phenolics formation in plants.
(See colour version of this figure online at www.informahealthcare.
com/bty)
compounds (Bravo, 1998). Most naturally occurring
polyphenolic compounds are present as conjugates
with mono and polysaccharides, linked to one or more
of the phenolic groups, and also may occur as functional
derivatives, such as esters and methyl esters (Shahidi and
Naczk, 1995; Harborne et al., 1999). Phenolic compounds
can be classified into several classes as shown in Table 1.
Natural antioxidants, such as polyphenolics are attract-
ing increased attention due to their potential nutritional
and therapeutic value together with their presumed
safety. The studies on the extraction and characterization
of polyphenolics mainly involve a number of aspects,
such as the nature of samples, type of extraction and
other analytical methods. Due to the vast diversity of phe-
nolic compounds and their sources, no global common
strategy has been developed that will give a general idea
on the analytical studies of polyphenol compounds. The
extraction of polyphenolic compounds mainly depends
 Critical Reviews in Biotechnology
Mass Spectrometry (MS)
Electrospray ionization mass spectrometry (ESI- MS)
Fast atom bombardment mass spectrometry (FAB – MS)
Atmospheric pressure chemical ionization (APCI)
Matrix-assisted laser desorption ionization time-of-flight mass
spectrometry (MALDI-TOF-MS)
Electron impact mass spectrometry (EI MS)
High-speed countercurrent chromatography (HSCCC)
Centrifugal Partition Chromatography (CPC)
Gas chromatography (GC)
Gas Chromatography – Mass Spectrometry (GC-MS)
Laser Diode Thermal Desorption (LDTD)
Capillary electrophoresis (CE)
Capillary zone electrophoresis (CZE)
Micellar electrokinetic chromatography (MEKC)
Background analytes (BGE)
Capillary electrophoresis with electrochemical detection (CE-ED)
Poly Phenol oxidase (PPO), Peroxidase (POD).
Table 1.  Classes of phenolic compounds from natural sources.
Class Structure
Simple phenolics, benzoquinones
Hydroxybenzoic acids C6
Hydroxycinnamic acids, phenylpropanoids C6 –C1
Naphthoquinones C6 –C3
Xanthones C6 –C4
Flavonoids, isoflavanoids C6 –C1 -C6
Lignans, neolignans C6 –C3 -C6
Bioflavonoids (C6 –C3)2
Lignin (C6 –C3 -C6)2
Condensed tannins (C6 –C3)n
(C6 –C3 -C6)n
on the analytical techniques, the nature of the solvents
used which may vary depending on the sources and their
order of magnitudes. Thus, it is necessary to develop an
optimum and appropriate method for the proper extrac-
tion, purification and characterization of polyphenolic
compounds in order to achieve higher accuracy in the
results. There have been many articles published on dif-
ferent aspects of extraction and analytical techniques,
however the studies remain scattered. This review dis-
cusses and summarizes some aspects of different types
of polyphenolic compounds, polyphenol extractions and
related analytical tools for the characterization of these
compounds in a unified manner. In order to identify
the exact methodology to be adopted for extraction and
analysis, it is important to know the different categories
of polyphenolic compounds.
Different types of polyphenolic compounds
Phenolic acid
The name phenolic acid describes phenols that possess
single carboxylic acid functionality. Phenolic acids con-
tain two distinguishing constitutive carbon frame works:

hydroxycinnamic and hydroxybenzoic structures. The
numbers and position of the hydroxyl groups on the aro-
matic ring creates a variety of structures and compounds.
Hydroxybenzoic acids have common C6–C1 structure
and include gallic, p-hydroxybenzoic, protocatechuic,
vanillic, and syringic acids as presented in Figure 2.
Hydroxycinnamic acids, on the other hand are aromatic
compounds with a three carbon side chain (C6–C3) with
caffeic, ferulic, p-coumaric, and sinapic acids being the
most common (Gallardo et al., 2006). Phenolic acids in
plants have been connected with diverse functions, such
as nutrient uptake, protein synthesis, enzyme activity,
photosynthesis, and structural components. Caffeic acid
is known to selectively block the biosynthesis of leukot-
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Extraction and Analysis of Polyphenols: Recent trends  3
intestinal absorption, caffeic acid was derived from the
hydrolysis of chlorogenic acid in the intestinal tract
(Germano et al., 2006). Recently some studies have dem-
onstrated that intake of rutin and o-coumaric acid can be
beneficial for the suppression of high-fat-diet-induced
dyslipidemia, hepatosteatosis, and oxidative stress in rats
(Hsu et al., 2009).
Flavonoids
Flavonoids constitute the largest group of plant phe-
nolics, accounting for over half of the eight thousand
naturally occurring phenolic compounds (Harborne
et al., 1999). Flavonoids are low molecular weight com-

pounds consisting of fifteen carbon atoms, arranged in

rienes, components involved in immuno-regulation
diseases, asthma, and allergic reactions (Koshihara
et  al., 1984). Caffeic acid and some of its esters might
possess antitumor activity against colon carcinogenesis
(Koshihara et al., 1984) and act as selective inhibitors of
human immunodeficiency virus type I integrase (King
et al., 1999). Chlorogenic acid has been found to inhibit
lipid peroxidation in rat liver induced by carbon tetra-
chloride, a potent liver carcinogen (Huang et al., 1997).
Caffeic and ferulic acid was found to detoxify carcinogen
metabolites of polycyclic aromatic hydrocarbons (Huang
et al., 1996). In fact, the recent interest in phenolic acids
can be attributed to their potential protective role against
For personal use only.
a C6–C3-C6 configuration (Figure 3). Essentially, the
structure consists of two aromatic rings A and B, joined
by a 3-carbon bridge, usually in the form of a heterocyclic
ring. Variations in substitution patterns to ring C result
in the major flavonoids classes, i.e., flavonols, flavones,
flavanones, flavanols (catechins), isoflavones, flavanon-
ols, and anthocyanidins (Hallman and Katan, 1997).
Flavones and flavonols are the most widely occurring
and structurally diverse compounds (Harborne et  al.,
1999). Substitutions on rings A and B give rise to different
compounds of flavonoids and these substitutions may

OH OH
oxidative damage diseases (coronary heart disease,
OH OH
stroke, and cancers). The interrelationship between anti-
oxidative capacity and the vasodilatory activity of deriva- HO O HO O
tives of hydroxybenzoic acid and hydroxycinnamic acid
from wine has been well studied (Mudnic et  al., 2010). OH
OH
The intestinal absorption of phenolic acids has been OH O OH O
demonstrated using Trichilia emetica extracts and during Quercetin Taxifolin
OH
Caffeic acid Cinnamic acid OH OH
O
OH HO O HO O
OH
O OH
OH OH OH
OH O OH O
OH
Kaemferol Myricetin

p-Coumaric acid Ferulic acid HO O HO O

O
O HO
O H3C OH
HO
OH O OH O
OH
OH Baicalein OH Chrysin

Gallic acid HO O
OH HO O
O OH
OH
OH O OH
OH O
Morin
HO OH Galangin
OH
Figure 2.  Structures of phenolic acids. Figure 3.  Structures of flavonoids.

© 2010 Informa Healthcare USA, Inc.

 4  C.M. Ajila et al.
include oxygenation, alkylation, glycosylation, acylation,
and sulfation (Hallman and Katan, 1997; Pietta, 2000).
The epidemiological studies point out the possible role
of flavonoids in preventing cardiovascular diseases and
cancer (Chu et al., 2002). Flavonoids are one of the major
antioxidant molecules with vivid functional properties,
such as anti-cancer, anti-atherosclerotic, anti-mutagenic,
anti-viral, anti-neoplastic, anti-allergic, anti-thrombotic,
and vasodilatory activity (Huntley, 2009; Wong et  al.,
2010). In general, the flavonoids exhibit a wide range of
chemotherapeutic, chemopreventive, and antiangio-
genic properties making them a rich class of polyphenols
to combat various diseases.
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Anthocyanins
Anthocyanins are the most important group of water-sol-
uble pigments in the plant kingdom and are responsible
for most of the red, blue, and purple colors of fruits, veg-
etables, flowers, and other plant tissues (Harborne et al.,
1999). Anthocyanins fall under phenolic compounds
in the flavonoids groups as illustrated in Figure 4. They
are generally found in the form of glycosides. The agly-
cones are rarely found in fresh plants. About 250 different
anthocyanins have been isolated from plants (Strack and
Way, 1994).
Anthocyanins help to attract animals, leading to
seed dispersal and pollination (Strack and Wray, 1994).
For personal use only.
Anthocyanins are regarded as an important component
in human nutrition (McDougall et  al., 2007), which is
supported by numerous studies that reported a high
positive correlation of fruit or vegetable pigment con-
tent and antioxidant capacities (Du et al., 2008; Astadi
et al., 2009). Red fruit extracts that are rich in anthocya-
nins are used in folk medicine and have some positive
therapeutic effects as anti-inflammatory agents and in
the treatment of various ailments, including microcir-
culation diseases resulting from capillary fragility and
the prevention of cholesterol induced atherosclerosis
(Wang et  al., 1997). The anthocyanin molecule also
protects the gastrointestinal mucus against oxidative
R1
OH
HO O+
R2
OH
OH
R1 = H; R2 = H; Pelagonidin
R1 = OH; R2 = H; Cyanidin
R1 = OH; R2 = OH; Delphinidin
R1 = OCH3; R2 = OH; Petunidin
R1 = OCH3; R2 = OCH3; Malvidin
Figure 4.  Structure of anthocyanins.
 Critical Reviews in Biotechnology
damage by delaying the onset of stomach, colon, and
rectal cancer (Halliwell et  al., 2001). As a colorant,
anthocyanins are found to be a potential replacement
for banned synthetic food dyes.
Tannins
Tannins are relatively high molecular weight compounds,
which constitute the third important group of phenolics,
and they may be subdivided into hydrolyzable and con-
densed tannins (Porter, 1989). The hydrolyzable tannins
are esters of gallic acid (gallo- and ellagi-tannins), while
the condensed tannins are polymers of polyhydroxyfla-
van-3-ol monomers (Porter, 1989). A third subdivision,
the phlorotannins consist entirely of phloroglucinol and
have been isolated from several genera of brown algae
(Porter, 1989). Tannins are polyphenols, which bind with
protein, basic compounds, such as alkaloids or heavy
metallic ions in a solution and make them insoluble and
induce precipitation (Okuda, 2005).
Lignan
Lignans are some of the minor compounds associated
with dietary fiber and found to produce important physi-
ological effects. Lignans are a group of relatively simple
diphenols. Lignans occur in a wide variety of plant foods,
primarily oil seeds, cereal grains, vegetables, fruits, and
legumes. Flax seed and tea are found to be a rich source
of lignans. Plant lignans generally occur in the form of gly-
cosides. They may occur as monomers as in tea, as a mix-
ture of monomers and oligomers as in the case of broccoli
or predominantly as oligomers as in the case of flax seed.
Plant lignans when ingested are converted by bacteria
in the large intestine into two simple phenols: entero-
lactone and enterodiol. These compounds are called
mammalian lignans and are found to exhibit a number of
significant physiological effects. Enterolactone is a mod-
erate inhibitor of estrogen synthesis and lowers estrogen
levels while enterodiol is a weak inhibitor. Both play a
key role in regulating the level of sex hormone binding
globulin, which is important in controlling the availabil-
ity of androgen and estrogen in the body. Hence, plant
lignans are considered to be one of the several classes of
phytoestrogen and are also effective as antioxidants and
aid in reducing the risk of cancer and cardiovascular dis-
eases (Vanharanta et al., 1999; Arts and Hollman, 2005).
The effects of lignans on cancer have been studied and it
was found that these compounds could provide protec-
tion against ovarian and thyroid cancer in women. Both
enterolactone and enterodiol caused a dose and time
dependent decrease in the number of colon cancer cells
(Qu et al., 2005).
As discussed earlier, the different categories of poly-
phenols constitute varied structures in line with the ben-
eficial properties that they exhibit. In order to quantify
these polyphenols, proper extraction of phenolic com-
pounds from the sample matrix is generally an important
step for further comprehensive analysis and structural
elucidation of the compounds. The major aspects to be

considered for the extraction of phenolic compounds
are the nature of the samples and the analyte type. The
extraction methods should be designed based on the
structural diversity of the phenolics, physico-chemical
behavior of the compounds, such as solubility and par-
titioning behavior of the compounds to be analyzed. In
addition to traditional methods, such as homogeniza-
tion, filtration/centrifugation, distillation, solvent, and
Soxhlet extraction, there are some modern techniques,
such as solid-phase microextraction, pressurized liquid
or fluid extraction, microwave-assisted extraction, and
membrane extraction for the extraction of phenolic
compounds.
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Extraction and Analysis of Polyphenols: Recent trends  5
Solid-liquid extraction (SLE)
Solid-liquid extraction is one of the most commonly used
and easiest methods of extraction for phenolics (Pekić
et al., 1998; Baydar et al., 2004; Lapornik et al., 2005). This
method generally includes initial extraction with differ-
ent solvents, such as methanol, ethanol, acetone or the
aqueous phase of this solvent mixtures, clean up, and fur-
ther fractionation by liquid–liquid extraction (LLE), usu-
ally with ethyl acetate or hexane, and followed by column
chromatography or solid phase extraction. In a general
outline, the solid liquid extraction (SLE) of polyphenols
consists of a direct extraction of the fresh or freeze-dried
plant material with an appropriate solvent using any
extractor, homogenizer or by ultrasonic bath for a par-

ticular time. The polyphenol extraction by solid-liquid

Types of extraction methods
The extraction of phenolic compounds is generally based
on solvents and is time consuming. This can lead to
complex errors in the analytical methods. Even though
solvent extraction produces good recovery of phenolic
compounds, it has some drawbacks, such as the require-
ment of a huge amount of solvents, long extraction time,
limited choice of solvents due to its food quality, and
also the possible degradation of target compounds. The
recent development of advanced extraction techniques,
such as microwave assisted extraction, pressurized liq-
uid extraction, ultrasonic extraction, and supercritical
For personal use only.
extraction is multiphase and involves an unsteady-state
mass transfer operation. The concentration of the solute
and the solid continuously changes during the extraction
(Guerrero et al., 2008). It has been reported that the LLE
step can be eliminated after the solid phase extraction of
polyphenolic compounds from apple peel, when the SLE
is performed using a solvent containing less than 70% of
methanol (Alonso-Salces et  al., 2005). Extraction with
50% solvent or less does not extract enough quantity of
lipids and chlorophyll.
Solubility of the phenolic compounds mainly depends
on the chemical nature and polarity of the compounds.

fluid extraction has aided in surmounting the bottle-
necks of the traditional methods. A global schematic
representation of the traditional and modern extraction
techniques, such as solid-liquid extraction (SLE), pres-
surized liquid extraction (PLE), super critical extrac-
tion (SCE), ultrasonic assisted extraction (UAE), and
microwave assisted extraction (MAE) for polyphenols is
­presented in Figure 5.
Phenolic compounds may form complexes with other
plant components, such as carbohydrates and proteins
and these interactions may affect the solubility of pheno-
lics in the solvents which in turn affect the extraction pro-
cedures. The phenolic extractions by solvents may also
extract non-phenolic compounds, such as chlorophylls,
fats, terpenes, waxes, and others, which are not desired.
This may lead to a requirement for additional procedures

Raw material
Fresh/ dried/ freeze
dried

E SC
E

SL UAE E
PL

MAE

Solvent Extraction at Microwave Supercritical

Acoustic
extraction high pressure energy (300 fluids (super
caviation
(homogenizer/ & MHz- critical CO2)
phenomenon
extractor) temperature 300GHz)

Polyphenolic extract

Figure 5.  Schematic representation of different extraction methods: solid-liquid extraction (SLE), pressurized liquid extraction (PLE),
ultrasonic assisted extraction (UAE), microwave assisted extraction (MAE), super critical extraction (SCE).

© 2010 Informa Healthcare USA, Inc.

 6  C.M. Ajila et al.
to remove these unwanted impurities from the extracts,
thus increasing the cost of extraction as well as introduc-
ing several analytical artifacts resulting in potential losses
of the analyte.
Pressurized liquid extraction (PLE) and accelerated
solvent extraction (ASE)
Pressurized liquid extraction is a novel extraction tech-
nique which uses organic solvents at high pressures and
temperatures above their normal boiling point. Normally
in PLE, a solid sample is packed in an extraction cell and
extracted with a suitable solvent under elevated temper-
ature (40–200°C) and pressure (500–3000 psi) for short
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periods of time (5–15 min) and the purged sample extract
will be collected into a collection vial by compressed gas.
PLE techniques have been used for the extraction of
polyphenols from many plant materials (Thurbide and
Hughes, 2000; Alonso-Salces et  al., 2001). Accelerated
solvent extraction is a synonymous term for PLE and is a
type of pressurized solvent extraction, which is similar to
SLE. This extraction is performed at higher temperatures
(50–200°C) and pressure (1450–2175 psi) to maintain
the solvents in its liquid state at high temperature. High
temperature accelerates the diffusivity of solvents, which
in turn increases the extraction kinetics (Richter et  al.,
1996; Brachet et al., 2001; Kaufmann and Christen, 2002).
For personal use only.
The high pressure helps the extraction cells to be filled
faster and forces liquid into the solid matrix. Accelerated
solvent extraction is mainly used for the fast extraction
of thermally stable organic compounds. There have
been some reports on the application of accelerated sol-
vent extraction for nutraceuticals and natural products
(Kaufmann and Christein, 2002).
Pressurized liquid extraction/accelerated solvent
extraction possess some advantages over other con-
ventional extraction methods, such as solid liquid
extraction. It consumes less solvent during extraction,
the extraction procedure is less time consuming, and
the handling of the sample is also reduced. Despite the
advantages over conventional methods, this method is
not found to be suitable for thermolabile phenolic com-
pounds as high temperature can have deleterious effects
on their structure and functional activity. However,
pragmatic choice of the solvent, temperature, pressure,
and reaction time can help overcome these problems to
a large extent.
Super critical extraction (SCE)
Supercritical fluid extraction is an alternative efficient
extraction method for polyphenols. The critical point of a
pure substance is defined as the highest temperature and
pressure at which the substance can exist in a vapour-
­liquid equilibrium. At temperatures and pressures above
the critical point, a single homogeneous fluid is formed
known as supercritical fluid (SCF) and is heavy as a liquid
with a penetration power of gas and this makes SCF an
effective and selective solvent (Nahar and Sarker, 2005).
SCF can be produced by heating a gas above the critical
 Critical Reviews in Biotechnology
temperature or by compressing a liquid above the critical
pressure.
The most commonly used SCF is supercritical CO2, a
number of other SCFs, such as ethane, butane, pentane,
nitrous oxide, ammonia, trifluoromethane, and water
are also used. Supercritical CO2 has a low critical tem-
perature (Tc = 31.1°C), is a safe and non-toxic method
and also the absence of light and air may reduce the
risk of degradation reactions. Since CO2 is non-polar, it
is not a good solvent for polar polyphenols. The addition
of organic co-solvents, namely methanol, ethanol, and
acetone enhances the solvating power of CO2 and the
yield of extraction of polyphenols. The critical tempera-
ture of the CO2 mixture is increased, when a co-solvent
is added to the mixture (Gurdial et al., 1993). The addi-
tion of a high concentration of ethanol requires the
extraction to be carried out at subcritical conditions of
40–60°C (Adil et  al., 2007). Supercritical CO2 extraction
with ethanol or methanol as a co-solvent has been used
for the extraction of grape seeds (Palma and Taylor, 1999;
Murga et al., 2000), wine by-products (Louli et al., 2004),
olive leaves (Le Floch et  al., 1998), and pistachio hulls
(Goli et al., 2005). Hydroxycinnamic acid and coumaric
acid derivatives (Murga et al., 2003) are slightly soluble in
supercritical CO2 extraction without co-solvents, where
as quercetin, catechin, epicatechin, and reservatrol are
soluble in supercritical extraction with 5–30% ethanol
(Berna et al., 2001; Chafer et al., 2004) and anthocyanins
are also slightly soluble in subcritical (CO2 + ethanol).
Sermento et al. (2008) developed an extraction method
with supercritical fluid, using ethanol or supercritical
CO2 (scCO2) as a pure solvent and scCO2 with ethanol as a
co-solvent, to obtain polyphenols from cocoa seeds and
further concentrated by polymeric membranes.
SCF is a commonly used method for the extraction of
phenolic compounds. SCF is easily recoverable from the
extract due to its volatility and the non-toxic solvent, in
fact it does not leave any harmful residue in the extract.
Thermally stable compounds and high boiling compo-
nents can be extracted by the SCF method at relatively
low temperatures. Thermally labile compounds can also
be extracted with minimal damage by using low tem-
peratures. Despite the benefits of conserving the struc-
tural integrity of polyphenols, high capital investment is
required for the setting up of this method, equipment,
and elevated pressure is also required for the operation
of the equipment. Further, the compression of the sol-
vent requires intense recycling measures to reduce the
energy cost.
Ultrasonic-assisted extraction (UAE)
Ultrasound-assisted extraction is a well established mild
condition technique for the extraction of phenolic com-
pounds (Choi et al., 1998; Murga et al., 2003; Chafer et al.,
2004; Chemat et al., 2008). Ultrasonic extraction is mainly
based on the acoustic cavitation phenomenon that is
the formation, growth and collapse of micro bubbles
inside a liquid phase submitted to ultrasonic cavitation

(Chemat et al., 2008). These implosive bubbles collapse
lead to extreme conditions of temperature and pressures
(Didenko and Suslick, 2002). In heterogeneous phases,
these hot areas create micro-jets and shock waves that
create strong conditions in the solid phase resulting in
erosion, fragmentation or disaggregation of the sample
to be investigated. The extraction ability of compounds
of interest is generally enhanced and the extraction time
is shortened. In most cases, ultrasonic assistance allowed
simplification of handling and work-up conditions.
Uses of ultrasound energy are considered as a potential
energy assistance which can create significant effects
on the reaction rate of various physical and/or chemi-
cal processes (Chemat et al., 2008). A rapid and relevant
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by Inrs Inst Armand Frappier on 11/12/10
sustainable ultrasound assisted extraction method has
been developed for the extraction of polyphenols from
apple pomace (Virot et al., 2009). An ultrasound assisted
method has also been developed for the isolation of gin-
senosides (saponins) from various types of ginseng using
different solvents and it was found that a sonication-
assisted extraction of ginseng saponins was about three
times faster than the traditional extraction method (Wu
et al., 2001). The ultrasonic extraction was not only more
efficient but also a convenient way for the recovery and
purification of the active ingredients of plant materials.
In addition, the sonication-assisted extraction can be
carried out at lower temperatures which are favorable for
For personal use only.
the thermally unstable compounds. Industrial scale-up
of ultrasonication with different equipment, such as
ultrasonic bath is quite easy and cheaper than other
­conventional techniques (Virot et al., 2009).
Use of an ultrasonic method for extraction is a new
powerful method which is not only a safe and eco-friendly
extraction method but also an efficient and economically
viable method. This method increased the extraction effi-
ciency of the phenolic compounds from different plant
sources, reduced the extraction time, and it can also
be used for the extraction of thermolabile compounds.
Despite the wide utilization of UAE, the method encom-
passes two problems, namely lack of uniformity in the
distribution of ultrasound energy and decline of power
with time (Luque-Garcia and Luque de Castro, 2003).
Nevertheless, the UAE method can be used with the exist-
ing equipment, such as an ultrasonic bath with minimum
alteration in its structure and can be applied for aqueous
extraction where organic solvents can be replaced with
generally recognized as safe (GRAS) solvents, and in turn
can reduce the solvent usage.
Microwave assisted extraction (MAE)
Microwave assisted extraction (MAE) is one of the most
advanced techniques used for the extraction of natural
products from numerous plant materials. It is one of the
faster extraction methods with high performance extrac-
tion ability and imposes less solvent consumption for
thermolabile constituents (Chee et al., 1996). Microwave
energy is a non-ionizing electromagnetic wave of fre-
quency between 300 MHz to 300 GHz and it lies between
© 2010 Informa Healthcare USA, Inc.
Extraction and Analysis of Polyphenols: Recent trends  7
the X-ray and infrared rays in the ­electromagnetic
­spectrum, and produces molecular motion by migration
of ions and rotation of dipoles (Letellier and Budzinski,
1999; Camel, 2001). The principle of MAE is mainly based
on the direct effect of microwaves on molecules by ionic
conduction and dipole rotation. The polar molecules,
such as polyphenols and ionic solutions strongly absorb
microwave energy since they have a permanent dipole
moment and this results in a rapid rise of the tempera-
ture and fast completion of the reaction (Eskilsson and
Bjorklund, 2000; Venkatesh and Raghavan, 2004; Gfrerer
and Lankmayr, 2005). The major physical parameters
important for microwave assisted extraction include
solubility, dielectric constant, and dissipation factors.
Polarity of the solvent is a very important factor since sol-
vents with high dielectric constants (e.g. water) that can
absorb more microwave energy and polar solvents are
usually better solvents than non-polar solvents (Wang
and Weller, 2006). It has been reported that microwave
extraction of phenolics with water is not efficient com-
pared to conventional methods, since water has a high
dielectric constant and a low dissipation factor compared
to other solvents. It is better to use solvents with a high
dielectric constant as well as a high dissipation factor.
Extractability of polyphenol was found to be higher with
acetone than methanol, since methanol has a higher
dielectric constant and higher dissipation factor as com-
pared to acetone. The extractability also depends on the
type of plant material extracted and the solvents used for
the extraction (Proestos and Komaitis, 2008).
In the presence of polar molecules or ionic species,
MAE provides rapid heating leading to collisions with
the surrounding molecules and it can be conducted at
atmospheric pressure. Power and extraction time for
natural products are in the range of 25–750 W and 30s
to 10 min, respectively (Kaufmann and Christen, 2002).
MAE has been used for the extraction of polyphenolics
from different plant sources, such as tea leaves, flax
seeds, radix, vanilla, among others (Pan et al., 2003; Liu
et  al., 2006; Longares-Patron and Canizares-Macias,
2006; Martino et al., 2006). MAE allows the desorption of
compounds of interest from the plant matrix due to the
heating of the free water molecules present in the gland
and vascular systems which leads to localized heating
causing dramatic expansion, with subsequent rupture of
walls, allowing the extracted molecules to flow towards
the organic solvent. The effect of microwave energy is
strongly dependent on the dielectric susceptibility of
both the solvent and solid plant matrix. Most of the time,
the sample is immersed in a single solvent or mixture
of solvents that absorb microwave energy strongly and
the elevated temperature increases penetration of the
solvent into the matrix and the constituents are released
into the surrounding hot solvent (Lay-Keow and Michel,
2003; Santana et al., 2009).
MAE is a relatively new method, which has received
increasing attention as an alternative extraction method
with many advantages over other extraction methods
 8  C.M. Ajila et al.
such as ultrasonication, comprising shorter time,
reduced solvent usage, higher extraction rate, improved
extraction yield, and the possibility of agitation during
extraction that improves the mass transfer phenom-
enon and even extracts minute traces of constituents.
The main disadvantages of MAE are its high capital cost
and possible filtration of the sample, if fine particles are
used for the extraction of compounds.
Ultrafiltration (UF)
Recently, membrane separation methods, such as
ultrafiltration (UF) have been used for the separation
and purification of polyphenols (Hossain, 2005; Nawaz
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by Inrs Inst Armand Frappier on 11/12/10
et al., 2006). Ultrafiltration is generally used to separate
desirable components from a mixture of compounds.
Ultrafiltration is commonly used as a clarification method
in the juice processing industries and is more effective in
terms of energy, environmental, and processing benefits
compared to the conventional filtration technologies
(Maier et  al., 1994). Ultrafiltration mainly depends on
the particle size of the compounds present in the mixture
and the driving source for transport across the membrane
is the differential pressure between the membranes
and it usually operates at 2–10 bar (Nawaz et al., 2006).
Generally, ultrafiltration membranes can separate mole-
cules with molecular weight ranges of more than 3000 Da
For personal use only.
going upto 100 kDa and higher and thus the system can
easily separate polyphenol compounds (Garcia et  al.,
1999). Ultrafiltration membrane techniques have been
also used for the separation and concentration of poly-
phenolic compounds from the juice of Echinacea herb
using some commercially available membranes, such
as polyethersulphone and regenerated cellulose; from
grape seeds using ethanol with higher extraction rates,
high extraction selectivity, short extraction time, and sig-
nificant labor savings. It has also been used for almond
skins (Hossain, 2005; Nawaz et al., 2006; Prodanov et al.,
2008).
Ultrafiltration with semi-permeable membranes was
found to be an easy and rapid method for the separation
and concentration of low molecular weight polyphe-
nolic compounds from a mixture of compounds. The
membrane separation method has more advantages
compared with conventional solid-liquid separation
techniques in terms of longer life, higher flux, resistance
to temperature, pH and organic solvents, and better
defined c­ leaning protocols (Borneman et al., 2001).
Enzyme assisted extraction
Recently, several types of enzymes, especially carbohy-
drate-hydrolyzing enzymes, such as pectinase, cellulase,
hemicellulase, and glucanase have been used for the
extraction of polyphenolics to break the cell wall com-
plex polyphenols (Meyer et al., 1998; Landbo and Meyer,
2001; Sørensen et al., 2005). These enzymes may disinte-
grate the plant cell wall matrix and facilitate polyphenol
extraction (Renard et al., 2001; Pinelo et al., 2006; Stalikas,
2007; Le Bourvellec et  al., 2009). Pectinase has been
 Critical Reviews in Biotechnology
used for the extraction enhancement of ­polyphenols
from apple pomace (Zheng et al., 2008). It has been also
reported that alpha-amylase and cellulase have been uti-
lized to release phenolic acids in barley (Yu et al., 2001)
and use of a combination of commercial enzymes, such
as Viscozyme, Termamyl and Lallzyme in spent barley
grain (Bartolomé and Gómez-Cordovés, 1999). Non-
starch polysaccharide hydrolyzing enzymes also play a
role in the release of bound phenolics located in the cel-
lular wall of vegetables and fruits. Polyphenol extraction
can be improved by the use of carbohydrate-hydrolyzing
enzymes, such as pectinase, cellulose, hemicellulase,
glucansae, and xylanase, among others (Landbo and
Meyer, 2001). The cell wall complex polyphenol can be
released by using these enzymes and play an important
role in disintegrating the plant cell wall matrix and thus
enhancing the polyphenol extraction (Le Bourvellec
et al., 2009).
Enzyme assisted extractions for polyphenolics have
many advantages over conventional methods such as
ultrafiltration. This method was found to be an easy and
rapid method for the separation and concentration of
polyphenolic compounds. It enhances the disintegra-
tion of the plant cell wall which in turn improves the
­polyphenolics extraction.
Variable factors affecting extraction
Type of solvents
A variety of solvents have been used for the extraction of
antioxidants especially for the polyphenolic compounds
and the activities of these compounds are directly
related to the extraction solvents (Chavan et  al., 2001).
Extraction yield of the compounds mainly depends on
the solvent and the method of extraction (Goli et  al.,
2004). Commonly used solvents for the extraction of
polyphenols from plant material are water, aqueous
methanol, and aqueous mixtures of ethanol, metha-
nol, and acetone, among others (Sun and Ho, 2005).
Sometimes, the polyphenolics bind with other plant
components, such as carbohydrates and proteins and
these interactions may lead to the development of some
complexes that may be difficult to solubilize in organic
solvents. Thus it is difficult to develop a general proto-
col for the phenolic extraction from plant materials and
needs close screening strategies to establish a viable
analytical method.
Polyphenols extracted with ethyl acetate showed
improved antioxidant activity from natural antioxi-
dants (Tsuda et  al., 1994; Marinova and Yanishlieva,
1997). It has been reported that many cereals were
found to be soluble in polar solvents and the hydroxyl
group in the phenolics determines the choice of sol-
vents. The solvents mainly used for the extraction of
cereal polyphenolics are methanol, ethanol, propanol,
acetone, ethyl acetate, dimethylformamide, and their
combinations (Naczk and Shahidi, 2006). In some
cases, 1% HCl is used with the solvents for extraction

of polyphenols as it leads to acid hydrolysis of cell
walls. This acid ­hydrolysis is mainly employed for the
extraction of bound ­phenolics (Ramachandra et  al.,
1977; Sripriya et  al., 1996). It is also reported that the
extraction of polyphenols from plant material may be
influenced by the ratio of solvent to sample volume
(Naczk and Shahidi, 2006). The extraction of polyphe-
nols is also affected by the time of extraction. Longer
extraction time may enhance the chances of oxidation
of phenolics, unless reducing agents are added to the
solvent system (Naczk and Shahidi, 2006).
pH
The pH is an important factor for the extraction of phe-
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by Inrs Inst Armand Frappier on 11/12/10
nolics. Usually, low pH value of the extraction solution
can prevent the oxidation of phenolics. It has been
reported that millet polyphenolics are stable at acidic
conditions and feeble or labile at alkaline conditions.
Millet polyphenolics may lose their identity by oxida-
tion or could be removed by chelating with metal ions
(Chethan and Malleshi, 2007). Alkaline hydrolysis has
also been reported for the extraction of bound phenolic
acids. Krygier et al (1982) reported that alkaline hydro-
lysis may lead to some degradation of hydroxycinnamic
acid derivatives and a later report stated that this can
be prevented by the addition of 1% ascorbic acid and
10 mM ethylenediamine tetraacetic acid, EDTA (Nardini
For personal use only.
et al., 2002).
The determination of optimum pH and solvent is an
important factor for the extraction of polyphenols. The
pH of the extraction medium depends on the nature
of phenolic compounds to be extracted and the source
of plant material. The solvent to be used for the extrac-
tion of compounds varies with the source and also the
type of phenolic compounds to be extracted. In fact, an
integrated combination of pH and solvent can lead to
enhanced extraction of polyphenols as pH determines
the solubility of the polyphenols and the solvents address
the efficient contact.
Purification and fractionation of phenolics
The extracts of phenolic compounds from plants may
contain different type of contaminants and interfer-
ing substances. Thus, it becomes crucial to remove the
impurities prior to the identification and quantification
of phenolic compounds. Extracts of phenolic com-
pounds may be concentrated for further studies under
vacuum, and the lipids and other unwanted compounds
can be extracted with petroleum ether, ethyl actetate or
diethyl ether (Naczk and Shahidi, 2004). In some phe-
nolic compounds, such as anthocyanins, it should be
noted that high concentration may cause loss of labile
acyl and sugar residues, in order to prevent this loss, it
is better to use neutral organic solvents, boiling water or
weak organic acids (Antolovich et  al., 2000). The puri-
fication and fractionation of the phenolic compounds
can be carried out mainly by solid phase or liquid-liquid
procedures.
© 2010 Informa Healthcare USA, Inc.
Extraction and Analysis of Polyphenols: Recent trends  9
Solid phase purification
Solid phase purification is one of the commonly
used procedures for the isolation, purification and
­pre-concentration of phytochemicals, especially phe-
nolics (Glowniak et  al., 1996; Klejdus and Kuba, 2000).
Alkylated silica gels (mainly C8, C18) are commonly
used as sorbents for SPE and also the combination of two
cartridges with different sorbents (C18 and quaternary
amine) are used for improved separation selectivity.
However, polymeric solvents, such as RP 105 and OASIS™
HLB were also found to be the most suitable materi-
als for extraction of phenolic compounds (Žiaková and
Branšteterová, 2002). Solid phase purification is faster
and more reproducible and produces cleaner extracts
compared to classical liquid-liquid extraction procedure.
Generally, C18-bonded silica reversed-phase sorbents,
styrene-divinylbenzene copolymers or graphitized car-
bon black (Hoffsommer et al., 1980; Gawdzik et al., 1990)
were used for the purification of phenolics. In some cases,
combined SPE procedures with C18 and anion exchange
columns were used for the isolation of plant materials
(Buiarelli et al., 1995; Glowniak et al., 1996). Sometimes,
the co-polymers have some limitations during the condi-
tioning step due to the hydrophobicity of their surfaces,
which in turn leads to drying of the conditioned sorbent
(Klejdus et al., 1999). It has been reported that solid phase
purification using some new polymeric sorbents with
chemically modified polar organic compounds (e.g. solid
phase columns with RP 105, OASIS HLB polymeric sor-
bents) showed higher recovery and improved extraction
efficiency (Klejdus and Kuba, 2000). These compounds
increase the wettability of the polymeric material and
the sorbents retain the polar phenolic compounds much
more easily.
Solid-phase microextraction (SPME) is a relatively
new sample extraction and purification technique which
brings unique capabilities to the chromatographic analy-
sis of samples. It has potential to reduce the solvent con-
sumption and also any issues related to the disposal of
solvents used for the sample preparation. Solid-phase
micro-extraction (SPME) is a solvent free, rapid and inex-
pensive method for the extraction of organic compounds,
such as polyphenolics from aqueous and gaseous samples
(Potter and Pawliszyn., 1994). In this extraction method,
sorbent coated silica fibers are used to extract analytes
from the sample mixture. For sample extraction from
aqueous media, the fiber is usually immersed to extract
the analytes and the fibers are then directly transferred
for further chromatographic analysis, where the samples
are finally separated and analyzed. Polydimethylsiloxane
and polyacrylate are two major coating fibers used for the
analysis of phenolic compounds by solid phase microex-
traction methods (Potter and Pawliszyn., 1994). A solid-
phase micro-extraction technique has been developed
using a polyacrylate coated fiber for the extraction and
determination of phenolic compounds in contaminated
wastewater (Moder et  al., 1997). Recently, an environ-
mentally friendly sample pre-treatment system based on
 10  C.M. Ajila et al.
solid-phase microextraction (SPME) has been developed
for the analysis of polyphenols from wine and grapes
using gas chromatography-mass spectrometry (Vinas
et al., 2009)
Solid phase extraction is commonly used due to its
advantages over other techniques, such as liquid-liquid
extraction methods. The solid extraction methods are easier
to automate with other systems, such as on-line coupling
of analytical columns. Cleaner extracts can be obtained
by the SPE method compared to other methods. The solid
phase extraction automated method can reduce the time
required for the purification and extraction of compounds.
It can handle small volumes of sample and requires only a
small volume of solvents compared to liquid-liquid phase
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by Inrs Inst Armand Frappier on 11/12/10
purification. The major disadvantage of the method is
the restriction of the sample size, so that large volume of
samples cannot be processed by this method.
Liquid-liquid phase purification
Liquid-liquid extraction is a commonly used procedure
for the purification of phenolic compounds from plant
extracts (Conway and Petrovski, 1995). Generally in this
method, the plant extracts were dried under reduced
pressure and temperature, based on the nature of phe-
nolic compounds. The dried residue was soaked in boil-
ing water and cooled for 12–24 h and then filtered. The
For personal use only.
resinous residue precipitate was washed with water and
the filtrate was defatted by petroleum ether. The aqueous
solution was extracted with diethyl ether and the phe-
nolic acids in the diethyl ether layer were further treated
with 5% Na2CO3 to transform the phenolic acid into
water soluble sodium salts. Later, the bicarbonated layer
was acidified with HCl and extracted with diethyl ether.
The resultant ether extract was washed thoroughly with
water to remove the acid and water in the extract was
finally removed by adding anhydrous sodium carbonate.
Liquid-liquid phase separation of phenolics was mainly
based on their partitioning between two immiscible
liquids (Conway and Petrovski, 1995) and these mobile
phases usually can use a miscible auxiliary solvent which
can aid in the partitioning of the analytes between the
two immiscible solvents (Degenhart et al., 2000).
Liquid-liquid phase purification has many disadvan-
tages over SPE, such as consumption of large volume of
solvents, intensive requirements of time and labor, may
need evaporation steps to remove excess solvent, deter-
mination of proper solvent for each sample was difficult
and emulsified sample may cause a problem in LLE and
there is also a chance of contamination during the pro-
cess. The major advantage of this method was its ability
to work with a large quantity of samples compared to
other techniques.
Quantification and identification of polyphenolics
Quantification and identification of phenolic compounds
mainly depends on different parameters, such as the
chemical nature of the compounds, extraction method
used, storage time and conditions, particle size, selection
 Critical Reviews in Biotechnology
of standards, interfering substances and impurities, and
assay methods. There are large numbers of spectromet-
ric methods that have been used for the quantification
of phenolic compounds from plant material. With the
advent and advancement of analytical science, there
has been an array of modern tools, such as HPLC, GC,
LC-MS, GC-MS, FT-IR, NMR, among others.
UV-Vis spectroscopy
The spectroscopic methods for analysis of phenolic com-
pounds are mainly based on different principles and the
various structural groups present in the phenolic com-
pounds. A number of spectrophotometric methods have
been developed for the quantification of phenolic com-
pounds. The Folin-Ciocalteu method is the commonly
used spectrophotometric method for the estimation of
total phenolic compounds which was developed by Swain
and Hills (1959). This method is an electron transfer based
assay and is a modified method of the Folin-Denis assay
(AOAC, 1980). This is mainly based on the reduction of
the phosphomolybdic-phosphotungstic acid reagent to a
colored complex in an alkaline condition in the presence
of phenolic compounds. However, this method is not very
specific and detects all phenolic groups and extractable
proteins and also there is a problem with interference by
reducing agents, such as ­ascorbic acids.
A precise, reproducible and repeatable Prussian blue
based method has been developed by Pueyo and Calvoa
(2009) based on the reduction of Fe(III) to Fe(II) and
the subsequent detection of Fe(II) by formation of the
hexacyanoferrate(II) chelate (Prussian Blue). Recently, a
new spectrophotometric-enzymatic method, peroxidase
catalyzed enzymatic method (PE) has been developed
for the determination of total phenolics from tea and
wine (Stevanato et  al., 2009). The possible mechanisms
involved are presented in Figure 6. As: (a) an oxidation
4-aminophenazone (4-AP)
Horseradish peroxidase H2O2
Oxidation of amino group
Phenoxyl radical (electrophile)
Formation of qunione imine dye
Absorbance at 500nm
Figure 6.  Possible mechanism of the reaction of 4-amino
phenazone (4-AP) with phenol in the spectrophotometric-
enzymatic method.

of the amine group of 4-aminophenazone (4-AP) by
­hydrogen peroxide in the presence of horseradish per-
oxidase; (b) an electrophilic attack of the oxidized 4-AP
by a phenoxyl radical; and (c) the formation of a quino-
ne-imine colored adduct (Fiamegos et  al., 2000). The
interference of sulfite, citrate and ascorbate were found
to be insensitive to the PE method, however, the enzymes
required are often in the pure phase and are costly.
The vanillin method has been used for the analysis
of condensed tannins and proanthocyanindins using
catechin as a standard (Goldstein and Swain, 1963).
This method is more sensitive to polymeric proantho-
cyanidins than monomeric flavan-3-ols and seems to be
sensitive, simple and specific. The 4-(dimethylamino)-
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by Inrs Inst Armand Frappier on 11/12/10
cinnamaldehyde (DMCA) assay is another method
used for the determination of proanthocyanidins and
reacts only with indoles and terpenes (McMurrough
and McDowell, 1978). DMCA does not react with a wide
range of flavonoids, including dihydrochalcones, fla-
vanones, and flavononols as well as phenolic acids. The
rhodanine and sodium nitrite method has been used
for the estimation of gallotanins (Inoue and Hagerman,
1988) and ellagic acid (Wilson and Hagerman, 1990) in
plant extracts. Al(III) based spectrophotometric methods
has been employed for the estimation of total caffeic acid
(Lamaison and Carnet, 1990), total flavonoids (Zhishen
et al., 1999), and total tannins (Zaporozhets et al., 2004).
For personal use only.
A pH differential method has been employed for the
quantification of anthocyanins based on their behavior
in acidic media at different pH (Sondheimer and Kertesz,
1948).
Recently, chemometric techniques to analyze spectra,
such as partial least squares or principal least squares
have been employed to adjust the over estimation of
polyphenol content of crude extracts due to the overlap-
ping of spectral responses (Van der Voort, 1992; Kramer,
1998). A mathematical relation has been developed
between the information collected from chemometric
techniques (such as, spectrum) and chemical indices
(such as, concentration of a component) which helps
determine the concentration of polyphenolics.
NIR spectroscopy
Advancement in both chemometrics and instrumentation
resulted in development of many rapid methods related to
multivariate spectroscopic techniques for predicting the
quantification and structural data of compounds. Near
infrared (NIR) spectroscopy is a recent powerful, rapid,
non-destructive analytical tool used in the agricultural,
petrochemical, textile, nutritional, and pharmaceutical
Table 2.  Application of NIR spectroscopy for the identification of plant polyphenolics.
Source Analyte
Red wine fermentation Phenolic compounds
Radix Salvia Miltrorrhiza Phenolic acids
Camellia sinensis Catechins
Green tea Total polyphenols
Vaccinium corymbosum L Total polyphenols, flavonoides, anthocyanins, ascorbic acids
© 2010 Informa Healthcare USA, Inc.
Extraction and Analysis of Polyphenols: Recent trends  11
industries which requires minimal processing prior to
analysis (Haarhaus et  al., 1995; Rodriguez-Saona et  al.,
2001; Falla et al., 2006; Sinelli et al., 2008). The NIR region
of the electromagnetic spectrum lies between the visible
and infrared regions with a wavelength between 750 and
2500 nm. The NIR region gives information concerning
the relative proportions of C---H, N---H and O---H bonds
which are the primary structural components of organic
molecules (Osborne et al., 1993). Quantitative measure-
ments of NIR spectroscopy have been carried out, mainly
based on the correlation between sample composition
and the absorption of light at different wavelengths in the
near infrared region measured either by reflectance or
transmission spectroscopy.
NIR spectroscopy has been used to analyze the flavin
and moisture content in black tea (Hall et al., 1988) and
also for the quantitative analysis of alkaloids and pheno-
lic substances in green tea leaves (Schulz et al., 1999). The
antioxidant activity of green tea leaves has been carried
out by NIR spectroscopy (Zhang et  al., 2004). Recently,
Chen et al. (2008) reported simultaneous analysis of free
amino acids, caffeine, total polyphenols, and amylase in
green tea by NIR spectroscopy. The analysis of catechin
in tea has also been performed by Fourier transform
near infrared reflectance (FT-NIR) spectroscopy (Schulz
et  al., 1999). There have been some attempts to deter-
mine the concentration of phenolic compounds, such as
malvidin-3-glucoside, pigmented polymers, and tannins
in fermenting and finished red wine by NIR spectroscopy
(Cozzolino et  al., 2004). Several reports discussed the
analysis of nutraceutical compounds, such as total phe-
nols, flavonoids, anthocyanins, and ascorbic acid con-
tents by FT-IR/NIR spectroscopy as presented in Table 2.
NIR spectroscopy has emerged in the past 30 years as a
precise method for analysis and prediction of the quality
of food and other agricultural products. Recently, it has
been used for the identification and analysis of bioactive
compounds from different plant sources. It has many
advantages over the conventional chromatographic
techniques due to its speed of analysis and minimum
sample preparation (Chen et al., 2008). The major advan-
tages of NIR are that it can record the response of the
molecular bonds of its chemical constituents to the NIR
spectrum and can build a characteristic spectrum which
can act as a finger print of the samples. The application of
multivariate statistical techniques, such as principle least
squares or discriminate analysis gives an idea of how to
understand the spectral properties of the sample. The
major limitations of this technology are calibration of the
sample analysis and the cost of the instrument.
Detection References
UV/NIR Cozzolino et al., 2004
FT-NIR Li and Qu, 2010
FT-NIR Chen et al., 2008
FT-NIR Chen et al., 2008
Near and mid IR Sinelli et al., 2008
 12  C.M. Ajila et al.
NMR spectroscopy
Nuclear magnetic resonance (NMR) is a powerful
complementary spectroscopic technique used for the
structural elucidation of complex phenolic compounds
isolated from different plant sources. NMR spectra of
phenols are generally complex as a reference compound
is needed and the technique requires relatively large
amounts of the compounds. However, 2D-NMR spec-
trometry can be used for structural analysis without a
reference standard. The NMR analysis of compounds
mainly includes 1H NMR and 13C NMR, two-dimensional
homonuclear (2D1H–1H) correlated NMR spectroscopy
(COSY), heteronuclear chemical shift correlation NMR
(C–H HECTOR), totally correlated NMR spectroscopy
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by Inrs Inst Armand Frappier on 11/12/10
(TOCSY), Nuclear Overhauser effect in the laboratory
frame (NOESY) and rotating frame of reference (ROESY)
as provided in Table 3.
Even though NMR spectroscopy is an expensive, recent
and rarely used method for phenolic compounds, it has
many advantages over other techniques. NMR analysis is
often more accurate and precise than the standard HPLC
methods. Expensive standards are not required for the
NMR analysis of compounds. It also gives information
on the impurities and the isomeric structures of the com-
pounds. NMR can be less time consuming (no equilibra-
tion time), easy to perform and more specific leading to a
high reproducibility.
For personal use only.
Chromatographic methods
Different types of chromatographic techniques have
been used for the separation, preparative isolation, puri-
fication, and identification of phenolic compounds. Even
earlier techniques such as thin layer chromatography and
column chromatography has been widely used for the
identification and purification of phenolic compounds.
Nowadays, HPLC techniques are the most widely used for
the separation and quantification of numerous phenolic
compounds. Different types of mobile phases and sup-
ports are used for the analysis of phenolic compounds,
such as flavonones, flavonols, procyanidins, and antho-
cyanins. Development of reverse phase chromatography
has led to a great impact on the purification of phenolic
compounds. The phenolics are generally detected by dif-
ferent analytical methods, such as UV-Vis/fluorescence
(Tasioula and Okogeri, 2001; Alonso et  al., 2007) and a
photodiode array (Chirinos et  al., 2008), among others.
HPLCs coupled with mass spectroscopy (MS) are widely
Table 3.  Application of NMR spectroscopy for the analysis of plant polyphenolics.
Sample NMR method
Gentiana ottonis 1
H-NMR & 1H-1H-NMR
Rice 1
H, 13C, and HMBC NMR
Root bark of Cheiloclinium gHMQC and gHMBC
cognatum
Oregano vulgare L. ssp Hirtum, 1H-1H DQF-COSY),
Salvia fruticosa, & Satureja (1H-13C HMQC), & (1H-13C HMBC)
hortensis
Cider apple juice 1H NMR spectra
 Critical Reviews in Biotechnology
used for the identification and characterization of phe-
nolic compounds from plant extracts and food. There
have been MS systems with different ion sources such
as electrospray ionization mass-spectrometry-ESI-MS
(Barros et  al., 2009), fast atom bombardment mass
spectrometry-FAB-MS, atmospheric pressure chemical
ionization-APCI (Lai et  al., 2007), MS-MS (Gruz et  al.,
2008), matrix-assisted laser desorption ionization time-
of-flight mass spectrometry -MALDI- TOF-MS (Kool
et al., 2010), electron impact mass spectrometry - EI MS
coupled with HPLC for the identification and structural
elucidation of phenolic compounds derived from differ-
ent sources. ESI is one of the most versatile ionization
techniques and is mainly preferred for the detection of
polar compounds separated by liquid chromatography.
ESI is a gentle ionization method generating mainly
deprotonated molecules of the compounds analyzed in
the negative/ positive ion mode for rapid determination
of molecular mass (Gioacchini et  al., 1996). The main
advantage of MS detection is its ability to determine the
molecular formula and to obtain the structural informa-
tion of unknown compounds (Carrasco-Pancorbo et al.,
2007).
TOF- MS can provide better accuracy over a wide
range of molecular weights and allows measurements
of the isotopic pattern which can help to find out the
elemental composition of the compounds. The major
information obtained by MS-MS analysis for the elu-
cidation of unknown phenolic compounds structures
are the aglycone moiety, types of carbohydrates or their
substituents present, stereochemical assignment of ter-
minal monosaccharide units, the sequence of glycan
part, interglycosidic linkages and the attachment points
of the substituent to the aglycans (Cuyckens and Claeys,
2004). Quadrupole mass analyzers have been generally
used with EI sources. Quadrupoles offer three main
advantages: (a) they tolerate relatively high pressures
and have siginificant mass range; (b) they have capability
of analyzing up to an m/z of 4000; and (c) they are rela-
tively low cost mass analyzer instruments. In an ion trap,
the ions are trapped in a radio frequency quadrupole
field. The major advantage of an ion trap is the isolation
of single ion species by ejecting all others from the trap
and the isolated ions can be subsequently fragmented
by collision activation. Another major advantage of the
quadruple ion trap is that multiple collision induced dis-
sociation experiments can be performed quickly without
Compounds identified References
Xanthone and flavone Wolfender et al., 1998
Hydroxycinnamate sucrose esters Tian et al., 2004
22β-hydroxypristimer in & cognatine Jeller et al., 2004
Phenolic antioxidants Gerothanassis et al., 1998
(−)-epicatechin Termrntzi et al., 2009

having multiple analyzers. There has been lot of literature
published on the isolation, purification and characteriza-
tion of phenolic compounds using HPLC and the related
detection methods. Some of these are summarized in
Table 4.
High-speed countercurrent chromatography (HSCCC;
centrifugal partitioning chromatography) is an all-liquid
chromatographic technique mainly used for the prepara-
tive isolation of pure compounds (Naczk and Shahidi,
2006; Marston and Hostettmann, 2007). In high speed
countercurrent chromatography, the sample is parti-
tioned between two non-miscible liquid phases. In this
case, one phase is held stationery by centrifugal force
and the second phase is pumped through the equipment.
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by Inrs Inst Armand Frappier on 11/12/10
Extraction and Analysis of Polyphenols: Recent trends  13
of highly concentrated rectangular peaks with minimum
overlap and elute the separated compounds based on
their pKa values and its hydrophobic nature (Wang et al.,
2006). The pH zone refining CPC has more advantages
over conventional CPC, such as a 10 fold increase in
sample loading capacity, high purity with high concen-
tration, among others (Maurya and Srivastava, 2009). The
method has been successfully used for the separation of
a wide variety of natural products, especially alkaloids
(Wang et al., 2006; Maurya and Srivastava, 2009).
Gas chromatography methods alone and coupled with
mass spectrometric methods have been also reported
for the separation, quantification, and structural eluci-
dation of different phenolic compounds, such as phe-

In HSCCC, the stationary phase occupies 5–7% and the
mobile phase occupies 75% of the column, so a large
amount of sample can be loaded compared to HPLC.
High-speed counter-current chromatography (HSCCC)
has been used for the separation of grape seed procya-
nidins (Köhler et al., 2008), anthocyanins from red wines
and grape skins (Degenhart et al., 2000).
Centrifugal partition chromatography (CPC) is
another method utilized for separation and purification
of bioactive compounds, such as alkaloids and other
organic compounds. The method uses centrifugal forces
to enhance phase separation and is based on liquid-
liquid partitioning (Foucault and Chevolot, 1998). In a
For personal use only.
nolic acids (Proestos et  al., 2006), isoflavones (Liggins
et al., 1998), flavonoides (Liu et al., 2007), anthocyanins
(Nakabayashi et  al., 2009), among others. Compared to
HPLC, GC needs preparation of samples which includes
removal of lipid from the extract and release of phenolics
by cleaving ester and glycosidic bonds by acid, alkali or
enzymatic hydrolysis (Liggins et al., 1998; Proestos et al.,
2006). Volatile derivatization of phenolic compounds,
such as methylation and acetylation (Chassagne et  al.,
1998) are also required for the sample for GC analysis.
Silylation is a common procedure for the GC analysis of
the non-volatile and thermolabile polyphenols trimeth-
ylsilyl derivatives are more volatile, less polar, and more

biphasic solvent system, the liquid phase is mobile and
the solid phase is stationary inside the column affected
by a centrifugal force field. The column is in the form of a
multiple disk stack in which partition cells are engraved
(Renault et al., 1997).
The technique of pH zone refining centrifugal par-
tition chromatography is a preparative purification
method for the separation of compounds whose electric
charges depend on its pH value (Wang et al., 2006). In this
method, the molecules are separated into a succession
themostable. During the silylation process, one active
hydrogen group is replaced by a trimethylsilyl group (Pan
and Pawliszyn, 1997). Polyphenolic compounds, such as
lignins and tannins were analyzed by GC coupled with
direct chemical ionization (DCI) MS by the methylation/
thermally assisted hydrolysis by tetramethylammonium
hydroxide and trimethylsulfonium hydroxide (Shadkami
et al., 2009).
Development of high temperature chromato-
graphic columns, electronic pressure controllers, and

Table 4.  Application of HPLC for the analysis of plant phenolics.

Source Analyte Stationary phase Mobile phase Detection Reference
Melicoccus bijugatus Jacq. Phenolics Vydac C18 0.2% formic acid in UV/ Vis/ LC-MS-MS Bystrom et al., 2008
(5 μm/300 water (A) and
Å/10 × 150 mm) 0.2% formic acid in
column methanol ( B)
Sorbus domestica fruits Phenolics 125 × 2 mm 2 % AcOH (A) and LC-DAD–MS (ESI+) Termentzi et al., 2008
Superspher MeOH (B)
100-4 RP-18 column
Helianthus annuus L Polyphenols C18 Hydro-Synergi 2% (v/v) acetic acid in HPLC-DAD/ESI-MS Weisz et al., 2009
water (eluent A) and
of 0.5% acetic acid in
water and acetonitrile
(50:50, v/v; eluent B)
Apple Polyphenols Nova-Pak C18 0.5% acetic acid in LC/MS/APCI Alonso-Salces et al.,
column water (A) & 2004
methanol (B)
Solanum tuberosum Polyphenols Nucleosil C18 1.5% H3PO 4 in H20 FAB- MS Keller et al., 1996
column (A)80% aq. MeCN (B)
Rubus loganbaccus Ellagitannin Zorbax rapid formic acid:H2O LC–ESI-MS/MS, Kool et al., 2010
resolution SB–C18 (5:95; A) & 100% MALDI-TOF-MS
acetonitrile (B).

© 2010 Informa Healthcare USA, Inc.

 14  C.M. Ajila et al.
detectors led to large scale advancement in the analysis
and ­structural elucidation of phenolics. Supercritical
fluid chromatography (SFC) is a relatively recent chro-
matographic technique. The difference of supercritical
fluid chromatography from other chromatographic tech-
niques such as gas chromatography (GC) and high per-
formance liquid chromatography (HPLC) is the use of a
supercritical fluid as the mobile phase. Supercritical fluid
chromatography has several advantages over other con-
ventional chromatographic techniques (GC and HPLC),
such as better separation of compounds with reduced
amount of solvents, is faster, and also can analyze ther-
molabile compounds. A supercritical fluid chromato-
graphic method for isolation and identification of various
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by Inrs Inst Armand Frappier on 11/12/10
polyphenols from grape seeds have also been developed
(Kamangerpour et al., 2002).
In view of the recent trend to incorporate green
chemistry as an important approach in most of the eco-
friendly techniques, recently a method based on ther-
mal desorption, such as laser diode thermal desorption
(LDTD) has been developed. LDTD is another ion source
for mass spectrometry. It is mainly based on a shotgun
approach, and the ion source works without any LC
equipment, which will reduce the solvent consumption.
The ionization of the samples can be carried out without
solvent, mobile phase, and/or external matrix (www.
ldtd-ionsource.com). The LDTD method works well with
For personal use only.
a very small volume of samples (2 µL) and it can carry out
simultaneous analysis of 90 samples. There have been
some reports on the use of this method for the analysis
of hormones from wastewater, environmental pollut-
ants, and drug metabolites (Wu et al., 2007; Fayad et al.,
2010). Meanwhile, this method can also be adapted to
­polyphenol extraction.
Capillary electrophoresis
Capillary electrophoresis is an important novel and
versatile separation technique for many classes of
compounds (Cikalo et  al., 1998). It has advantage over
other separation techniques due to its higher efficiency,
reproducibility, minute sample volume, speed, less
stringent purification of sample, higher resolution and
sensitivity, simultaneous separation and identification
of complex multi-component mixture of structurally dif-
ferent chemical compounds, and minimal consumption
of solvent (Frazier and Papadopolou, 2003). In capillary
electrophoresis, the separation is based on the electro-
phoretic migration of charged analytes and also the dif-
ference between the charge/mass ratio of the solutes and
the degree of ionization of polyphenols (pKa values of
–OH groups ranging between ≈7 and 12, depending on
the structure of polyphenol molecule). Capillary zone
electrophoresis (CZE) and micellar electrokinetic chro-
matography (MEKC) are the two general modes of elec-
tromigration methods, which are used regularly for the
determination of phenolic compounds. CZE separation
is mainly based on the differences in the migration of
charged solutes in a conductive liquid phase in a capillary
 Critical Reviews in Biotechnology
under the influence of high voltage electric field. MEKC
is mainly used for the separation of neutral analytes. In
MEKC, background analytes (BGE) contain a charged
surfactant, such as sodium dodecyl sulfate and the
micelles formed act as a “pseudostationary phase” and
the analytes undergo partitioning between the micelles
(hydrophobic) and BGE (hydrophilic phase). The mecha-
nism of separation is mainly based on the differences in
lipophilicity of the analytes. In CE, acidity and concentra-
tion of the running buffer has an important effect on zeta
potential (f ), the electro osmotic flow (EOF), as well as
the overall charge of all the analytes, which can affect the
migration time and the separation of the analytes.
Various separation techniques, such as capillary
zone electrophoresis, micellar electrokinetic chroma-
tography, capillary isoelectric focusing, and capillary
iso-octachophoresis, have been used for the analysis of
polyphenolic compounds (Zeece, 1992). Capillary elec-
trophoresis has been applied for the analysis of phenolic
compounds present in food, grapes, wines, olives, spices,
medicinal herbs, tea, oil seeds, and different types of fruits
(Arce et al., 1998a,b; Cao et al., 2001a; Cao et al., 2001b;
Vaher and Koel., 2003; Ehala et al., 2005). Capillary zone
electrophoresis has been used to evaluate the effect of
grape variety and the aging of wine (Andrade et al, 1998).
Peng et al. (2005) reported simultaneous determination
of phloridzin, epicatechin, chlorogenic acid, and myrice-
tin in apple juices and ciders by capillary electrophoresis
with electrochemical detection (CE-ED). A simultaneous
determination of catechin, epicatechin, and tras-resver-
atrol in red wine has also been developed by Peng et al.
(2004) using capillary electrophoresis.
Arce et  al. (1998a) developed a capillary electropho-
resis method for the simultaneous determination of caf-
feine, adenine, theophylline, epigallocatechin-3 gallate,
epigallocatechin, epicatechin-3 gallate, (2)-epicatechin,
(1)-catechin, gallic acid, quercetin, and caffeic acid
from green tea. Separation was performed with a fused
capillary column with 0.15M borate as buffer at a pH of
8.5, UV detection at 210 nm and 20 kV of 3.3 voltages.
A simultaneous separation of catechins with caffeine,
theanine, and ascorbic acid in green tea infusion has
been developed by capillary zone electrophoresis (Horie
et al., 1998). Later, Bonoli et al. (2003) have determined
catechins in green tea, namely (+)-catechin, (−)-epigal-
locatechin, (−)- gallocatechin, (−)-gallocatechingallate,
(−)-epigallocatechin- 3-gallate, (−)-epicatechingallate,
and (−)-epigallocatechin gallate by micellar electrochro-
matography. Pazourek et al. (2000) developed a method
for the separation and determination of polyphenols in
canary wines without a pre-concentration step with a
higher sensitivity and higher limits of detection. Direct
detection of phenolic acids in beer has been reported by
Moane et  al. (1998) by capillary electrophoresis with a
fused silica capillary in 25mM phosphate buffer and pH
7.2 at 25kV. Capillary electrophoresis with an amperomet-
ric detection method has been developed for the separa-
tion of protocatechuic aldehyde and protocatechuic acid

with a detection limit of 0.10 g/ mL and 0.25 µg/ mL for
protocatechuic aldehyde and protocatechuic acid under
optimal conditions (Pan et al., 2001).
Micellar electrokinetic capillary chromatography has
been used for the analysis of a range of tea types includ-
ing its potential in the analysis of polyphenols in instant
green, Darjeeling and black Assam tea with a system-
atically optimized condition of buffer, micelle, borate,
and organic solvent concentration (Larger et  al., 1998).
Polyphenolic mixture of scopoletin, rutin, esculetin,
chlorogenic acid, and caffeic acid from herbal medicines
of Cortex fraxini has been separated by a simple and con-
venient method of micellar electrokinetic capillary chro-
matography by using Tween-20 to form single micelle
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by Inrs Inst Armand Frappier on 11/12/10
and methanol as a buffer additive (Wang et al., 2007).
Nowadays, capillary electrophoresis is found to be
a better analytical tool compared to the conventional
methods. It has many advantages over other alternative
methods, such as easy and predictable selectivity, high
separation efficiency (105 to 106 theoretical plates), small
sample sizes (1–10 µl), and minimal sample preparations,
minimal consumption of sample, and reagents, speedy
separations (1 to 45 min), and can be automated and
linearly quantified. It can be easily coupled with MS for
further structural characterization studies. Capillary elec-
trophoresis cannot be used for preparative scale prepara-
tions, and sticky compounds and also compounds that
For personal use only.
are difficult to dissolve. The joule heat generation during
the electrophoresis may also lead to the destruction of
the sample. It also has reproducibility problems and poor
limits of detection.
Biosensors
Biosensors are an important alternative tool to classi-
cal analytical techniques with marked advantages due
to potential of miniaturization, low production cost,
specificity, fast response, capacity to incorporate with
integrated systems, facility of automation, capacity to
work in real time, versatility precision, and high sensitiv-
ity (Rasooly, 2001; Velasco-García and Mottram 2003). A
biosensor is a compact device for analysis, which incor-
porates a biological, or biomimetic recognition element,
such as nucleic acid, enzymes, antibody, receptor tissue,
cell with a transduction system that allows for process-
ing the signals produced by the interaction between
the recognition element and the analyte. A schematic
representation of biosensors is presented in Figure 7.
The detection of the biosensor is based on the interac-
tion between the analyte and the recognition element
and this specific interaction produces single or several
physico-chemical effects, such as pH, electrochemical
transference, heat transference, change of potential or
mass, optical properties, among others. These changes
are detected and coupled to a chemical or physical trans-
ductor and the signal indicative of the presence of analyte
can be measured proportional to its concentration in the
sample (Velasco-García and Mottram, 2003). Biosensors
have been used in different applications, such as clinical,
© 2010 Informa Healthcare USA, Inc.
Extraction and Analysis of Polyphenols: Recent trends  15
Recognition
Analyte
eg: polyphenols Biocomponent
eg: tyrosinase, laccase etc
Transducer
(Physiochemical
signaling)
Electric signaling
(Signal processing
and analyzing the data)
Figure 7.  Schematic representation of biosensors for polyphenols.
(See colour version of this figure online at www.informahealthcare.
com/bty)
environmental, agricultural, and biotechnology (Scheper
et  al., 1999; Wang, 1999; Tothill, 2001; Bourgeois et  al.,
2001). The biosensors require mild conditions of tem-
perature and pH to maintain the activity of the biological
element (Gibson, 1999). It is also recommended to carry
out pre-treatment of the sample to remove interfering
species, such as ascorbic acid, tyrosine and others by
different methods, such as neutralization, dilution or
extraction depending on the nature of samples.
Tyrosinase, peroxidase, and laccase biosensors have
been used for detecting phenolic compounds (Mello
and Kubota, 2007). Generally, phenol oxidase (PPO) and
peroxidase (POD) have different enzymatic mechanisms
in the electrochemical biosensors. On the surface of elec-
trodes, enzyme molecules are oxidized by oxygen (PPO)
or by hydrogen peroxide (for POD) followed by their
­re-reduction by phenolic compounds. The tyrosinase
biosensors are restricted to the phenolic compounds
with at least one free ortho-position whereas the laccase
biosensors are used for the detection of phenolic com-
pounds with free para- and meta- positions (Munteanu
et al., 1998). Peroxidase can be used for the detection of
polyphenols with certain selectivity and sensitivity since it
exhibits low specificity. A laccase based biosensor, which
was immobilized on polyether sulphone membranes and
applied on an amperometric biosensor detector with caf-
feic acid as a substrate was developed for the analysis of
polyphenols in red wines (Fernandes and Rebelo, 2009).
A tyrosinase-based biosensor operating in an organic
solvent, hexane using amperometric oxygen probe as a
transducer has been used for the evaluation of the phe-
nolic content of an extra-virgin olive oil with varying stor-
age time and storage conditions (Capannesi et al., 2000).
Some of the applications of biosensors for polyphenols
have been listed in Table 5. Despite the specificity and
sensitivity of biosensors, they have limited utilization
as they can be rapid detection methods, but for precise
 16  C.M. Ajila et al.
quantification and analysis, a comprehensive method,
such as a chromatographic technique is required.
Conclusions and Future Prospects
There has been an increasing interest in plant polyphe-
nols due to their antioxidant, anticancer and numerous
other human health benefits and industrial applica-
tions. Given their important health effects, the iden-
tification, characterization and structural elucidation
of phenolic compounds warrants a great attention in
their analytical science. This review discussed different
types of phenolic compounds and various methods of
extraction, analysis, and structural characterization of
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by Inrs Inst Armand Frappier on 11/12/10
and structural elucidation of plant polyphenolics as
each method encompasses advantages and drawbacks.
Most of the techniques reported in the literature such as
SLE, LLE, microwave, ultrasonication, and accelerated
solvent extraction rely on the use of solvents which may
be sometimes deemed toxic, especially if the extracted
component finds use in food and pharmaceutical appli-
cations. Most of the solvents used for the extraction of
polyphenolic compounds have been found to have many
health effects and toxicity. Due to this drawback, most
of solvents are listed in FDA and EPA rules with resid-
ual limit. (EPA 2009, revised list). The Environmental
Protection Agency (EPA) has removed acetone from the
list of toxic chemicals maintained under Section 313 of

the phenolic compounds. High extraction yield and
purification and proper characterization of phenolic
compounds remain an enigma for researchers owing to
the vast diversity of phenolic compounds and sources.
Some case studies have been summarized in Table 6.
These have utilized the different methods of extraction
and identification for various phenolic compounds.
These studies provide evidence to the fact that each
specific polyphenolic compound imposes a different
set of extraction conditions, consequently followed
by different methods. In spite of the specificity of each
method in analyzing the polyphenolic compounds,
each method encompasses some limitations. Some of
For personal use only.
the Emergency Planning and Community Right to Know
Act (EPCRA). In 1995, the EPA concluded that acetone
does not have any carcinogenicity and neurotoxicity in
very low doses based on animal studies. Hexane is a neu-
rotoxic petrochemical solvent which is listed as a haz-
ardous air pollutant with the EPA. A tolerance of 30 parts
per million is established for acetone in spice oleoresins
when present therein as a residue from the extraction of
spice according to a revised FDA 2009. According to the
FDA 2009 list, a tolerance limit of 30 parts per million
is established for ethylene dichloride in spice oleoresins
when present therein as a residue from the extraction of
spices.

the advantages and disadvantages of different extrac-
tion methods generally used for ­extraction of phenolics
are also discussed in Table 7.
There have been several publications already
reported in the literature, on extraction and analysis of
polyphenols, still there is a need of a standardized pro-
cedure for the preparation of samples, quantification,
At this juncture, the use of membrane separation
methods based on osmosis or ultrafiltration may be a
better strategy for the separation and purification of
polyphenolic compounds. These methods will conserve
the structural and nutritional integrity of the polyphenol
and at the same time score positively on different counts
of solvent toxicity, extraction efficiency, and percentage

Table 5.  Application of biosensors for analysis of polyphenols.

Analyte Application Biocomponent Transducer Detection range Reference
Polyphenols Green tea, grape & Tyrosinase Amp 10–100m M Romani et al., 2000
olive extracts
Polyphenols Olive oil Tyrosinase Amp 1–37 mM (hexane) Campanella, et al.,
10–350 mM (chloroform) 1999
Polyphenols Olive oil Tyrosinase Amp 0.3–30.0 mM Dall’Orto et al, 1999.
Polyphenols Wine Horse radish peroxidase Amp <25 mM Imabayashi et al., 2001
Polyphenols Olive oil Tyrosinase Amp 0.1-0.2 ppm Capannesi et al., 2000
Polyphenols Tea Laccase Amp Ghindilis et al., 1992
Kulys &Vidziunaite,
2003

Table 6.  Case studies on extraction and identification of phenolic compounds.

Polyphenolic Type Extraction method Specific conditions Analytical/ identification method Reference
Phenolic acids Solid phase extraction Extract-cleanTM cartridges HPLC-DAD–ESI-MS Liu et al., 2007
Phenolic acids Solid phase extraction Diol cartridge CE–ESI-MS Nevado et al., 2009
Flavonoid Ultrasonication HPLC-ESI-MS Shi et al., 2007
Flavonoid Solid phase extraction Pretreatment AB-8 resin ESI-MS Liu & Zhu, 2007
Anthocyanins Utrasonication HPLC-MS Chen et al., 2007
Tannin Solid phase extraction Solid phase purification MALDI-TOF- MS &CP-MAS 13C NMR Hoong et al., 2010
Lignan Ultrasonication HPLC, GC, GC-MS Elfahmi et al., 2007

 Critical Reviews in Biotechnology

 Extraction and Analysis of Polyphenols: Recent trends  17

Table 7.  Advantages and disadvantages of the extraction methods mentioned.

Method
1) Solid Liquid Extraction
2) Pressurized Liquid
Extraction
3) Super Critical Extraction
4) Ultrasonic assisted
Extraction
Critical Reviews in Biotechnology Downloaded from informahealthcare.com by Inrs Inst Armand Frappier on 11/12/10
Advantages Disadvantages Suggestions
Commonly used *Chances of impurities, *Can improve with better
Easiest method *Introduction of Analytical errors choice of solvents
Less solvent, *Not suitable for thermo labile *Setting of optimum solvent
Less time of extraction compounds ratio, temperature, pressure
and extraction time can
improve the method
Eco friendly method, *High Capital investment *Setting of optimum pressure
Can be used for thermo *Requirement of high pressure for extraction may reduce the
Labile compounds cost of operation
*Eco friendly, *Lack of uniformity in the distribution *Selection of varied
*Can replace the solvents of ultrasound energy frequency/power for
with GRAS solvents *Decline of power with time ultrasonic equipment for
the uniform distribution of

* High extraction efficiency

*Reduced extraction time,
*Good for thermo labile
compounds
5) Microwave Assisted *Reduced solvent usage
Extraction *Higher extraction rate
*Improved extraction yield
energy may reduce the energy
requirements
*High capital cost *Optimization of particle size
of the samples (by crushing/
grinding), which may offset
extra cost for filtration after the
sample extraction

recovery of polyphenols. This technology can be used Declaration of interest

to fractionate molecules of similar molecular weight at
a much lower operating temperature compared to con- The authors are sincerely thankful to the Natural Sciences
and Engineering Research Council of Canada (Discovery
For personal use only.

ventional processing operations. A single isolated study

has reported the extraction of polyphenols from grapes
by using ultrafiltration; however the technique was used
sequentially with liquid-liquid extraction reporting
recovery yields of 30–50% (Nawaz et al., 2006). Another
approach could be the use of hydrothermal treatment
where the sample is treated with hot water, at a tempera-
ture of 180 to 240°C, in a closed autoclave-type reactor, at
a pressure that permits maintaining the water in liquid
phase. In fact, this application does not use either sol-
vents and/or acids, or will not result in abrupt depres-
surization as in the steam explosion treatment making it
highly eco-friendly and at the same time conserving any
nutritional losses from thermal degradation. Likewise,
the analytical techniques used for the quantification
of the polyphenols which traditionally use solvents
and/or buffers can be replaced by the new emerging
laser diode-thermal desorption technology which can
employ moderate operating conditions without use of
solvent, resulting in precise quantification of the poly-
phenols. With further advances in analytical sciences,
the extraction methods using mild operating conditions
with increased recovery are desired, centrifugal ultrafil-
tration can be a typical process in this regard. With the
advent of many separation technologies and analytical
tools, there will be promising research in the area of
polyphenolic compounds by proper implementation of
these techniques. Globally, no single unique method can
aid in the characterization and analysis of polyphenolic
compounds, thus an ensemble of methods is required as
each analyte type exerts varied process conditions.
Grant 355254, Canada Research Chair), FQRNT (ENC
125216) and MAPAQ (No. 809051) for financial support.
The views or opinions expressed in this article are those
of the authors.
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