Critical Reviews in Analytical Chemistry

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Analysis and Antioxidant Capacity of Anthocyanin

Pigments. Part IV: Extraction of Anthocyanins

María José Navas , Ana María Jiménez-Moreno , Julia Martín Bueno ,

Purificación Sáez-Plaza & Agustin G. Asuero

To cite this article: María José Navas , Ana María Jiménez-Moreno , Julia Martín Bueno ,
Purificación Sáez-Plaza & Agustin G. Asuero (2012) Analysis and Antioxidant Capacity of
Anthocyanin Pigments. Part IV: Extraction of Anthocyanins, Critical Reviews in Analytical
Chemistry, 42:4, 313-342, DOI: 10.1080/10408347.2012.680343

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Critical Reviews in Analytical Chemistry, 42:313–342, 2012
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ISSN: 1040-8347 print / 1547-6510 online
DOI: 10.1080/10408347.2012.680343

Analysis and Antioxidant Capacity of Anthocyanin

Pigments. Part IV: Extraction of Anthocyanins
Marı́a José Navas,1 Ana Marı́a Jiménez-Moreno,1 Julia Martı́n Bueno,2
Purificación Sáez-Plaza,1 and Agustin G. Asuero1
1
Department of Analytical Chemistry, Faculty of Pharmacy, The University of Seville, Seville, Spain
2
Department of Analytical Chemistry, Higher Polytechnic School, The University of Seville, Seville, Spain

Anthocyanins are naturally occurring polyphenol compounds that impart orange, red, purple,
and blue color to many fruits, vegetables, grains, flowers, and plants. In recent decades, in-
terest in anthocyanin pigments has increased due to their possible utilization as natural food
colorants and especially because their consumption has been linked to protection against many
chronic diseases. It seems that anthocyanin posseses strong antioxidant properties leading to
a variety of health benefits. Coupled to increasing consumer demand, food manufacturers
have moved towards increased usage of approved natural colors. Despite the great potential
in applications that anthocyanins represent for food, pharmaceutical, and cosmetic industries,
their use has been limited because of their relative instability and low extraction percentage.
Growing demands have been made on sample pretreatment, and over time some novel extrac-
tion techniques have been developed. Solid phase extraction, countercurrent chromatography,
adsorption, pressurized liquid or fluid extraction, and microwave-assisted extraction are en-
vironmentally friendlier in terms of using smaller amount of solvents (often nontoxic) and
reducing working time. The past few years have been characterized by wide interest in these
techniques, and many contributions describing these methods have been published. The aim of
this article is to review the literature available on the most important procedures proposed for
the extraction of anthocyanins; the use of non-thermal technologies in the assisted extraction
of anthocyanins will be covered in a separate report.

Keywords Anthocyanins, extraction

INTRODUCTION subject of a previous report (Bueno et al., 2012b). The sugar

In the human diet, anthocyanins are found in red wine
(He et al., 2012a, 2012b), certain varieties of cereals (Abdel-
Aal et al., 2006, 2011), and certain leafy and root vegetables
(aubergines, cabbage, beans, onions, radishes), but they are most
abundant in fruits (Bueno et al., 2012a; Mercadante and Bobbio,
2008; Kuskoski et al., 2002). A high intake of anthocyanin-rich
food has been linked to health-protecting effects (Tsuda, 2012;
He and Giusti, 2010). The dietary consumption of grapes and
their products, for example, is associated with a lower incidence
of degenerative disease and certain types of cancer (Xia et al.,
2010). Anthocyanins are glycosylated derivatives of the 3,5,7,3 -
tetrahydroxyflavylium cation also known as anthocyanidin and
they comprise the largest group of water-soluble pigments in the
plant kingdom (Harborne, 1967; Harborne and Grayer, 1988).
Chemical structure, color, and intake of anthocyanins are the
Address correspondence to Agustin G. Asuero, Dept. of Analytical
Chemistry, Faculty of Pharmacy, The University of Seville, 41012
Seville, Spain. E-mail: asuero@us.es
313
compound increases the chemical stability of anthocyanidin,
therefore anthocyanins are the chemical form most prevalent in
berries, grapes, and wine. The number of hydroxyl groups, the
nature and number of sugars, and the position of these attach-
ments determine the difference between the individual antho-
cyanins (Bueno et al., 2012b; Kong et al., 2003). Sugars pro-
vide additional sites for structure modifications, as they may be
acilated with aromatic or aliphatic acids, e.g., p-coumaric, caf-
feic, ferulic, sinapic, acetic, malonic, or p-hydroxybenzoic acid.
Anthocyanins have an antioxidant activity (Kuskoski et al.,
2005, 2003), i.e., they act as reducing agents, hydrogen donors,
and single oxygen quenchers. This property depends to a large
extent upon the number and location of the OH groups present,
the extent of strutural conjugation, and the presence of electron-
donating and electron-withdrawing substituents in the ring struc-
ture (Lapornik et al., 2005; Kuskoski et al., 2004). The range
of color associated with anthocyanins is the result of varied
substitution of the parent C6 C3 C6 aglycone nucleus, acylations
patterns, and various environmental influences (Bueno et al.,
314 M. J. NAVAS ET AL.
2012b; Rodriguez-Saona and Wrolstad, 2001). It is possible
to extract colors from various plant sources, but for economic
reasons, grape skins, a byproduct of the wine industry, are the
common source of anthocyanins (Scotter, 2010; Francis and
Markakis, 1989). Because of their highly reactive nature antho-
cyanins readily degrade or react with other constituents to form
colorless or brown compounds (Cavalcanti et al., 2011). Loss of
anthocyanins also occurs in the presence of oxygen and various
enzymes and as a result of high-temperature processing (Bridle
and Timberlake, 1997).
Overall, cyanidin is the most common anthocyanidin found
in foods (Manach et al., 2004), followed by pelargonidin, peoni-
din, and delphinidin, then by petudinin and malvidin (Jackman
and Smith, 1992). The anthocyanin content in foods (fruits,
berries, etc.) is generally proportional to color intensity and
reaches values up to 2–4 g/kg fresh weight in blackcurrants and
blackberries, increasing as the fruit ripens (Basu et al., 2010).
Wine contains approximately 200–350 mg anthocyanins/L, and
involved anthocyanins are transformed into various complex
structures as the wine ages (He et al., 2012a, 2012b; Jordheim,
2007). There is also significant interest in the identification of
pyrranoanthocyanins, which are products formed from antho-
cyanins during processing of fruit juices and the aging of wines
(Jing and Giusti, 2011).
Documentation of the anthocyanin content of different botan-
ical sources is important for determination of structural diversity
as well as for food and health perspectives (Jordheim, 2007).
The anthocyanin composition in berries has been used as a
botanical tool for taxonomic classification of plants, as the an-
thocyanin profile tends to be characteristic of a plant, similar to
a fingerprint (Jing and Giusti, 2011). The pigmentation of plants
is rarely due to a single anthocyanin, and several food sources
of anthocyanins contain significant levels (Scotter, 2010, 2011a,
2011b). These include grapes, blackcurrants, elderberries, cran-
berries, and cherries and the aqueous extracts of roselle (the
calyces of hibiscus) and of red cabbage.
Among the food plants available in nature, grapes are
the major source of anthocyanins. Pigment content in grapes
ranges between 30 and 75 mg/100 g and varies greatly accord-
ing to cultivar, season, and environmental factors (Bridle and
Timberlake, 1997). Depending upon the grape variety, the sepa-
rate anthocyanin content usually goes from at least 4 to over 16
anthocyanins (Rein, 2005). The Concord variety has 31 antho-
cyanins, the greatest number in any simple cultivar (Mazza and
Francis, 1995). Some anthocyanin profiles are rather simple,
such as the cases of strawberries, where 80–90% of the pigment
composition is just one pigment, pelargonidin-3-glucoside (Jing
and Giusti, 2011). Blueberries have the most complex antho-
cyanin profile among common berries, containing over 25 in-
dividual anthocyanins, from five different aglycones (cyanidin,
peonidin, malvidin, petunidin, and delphinidin) and three differ-
ent glycosilating sugars and acetylated monoglycosides of these
anthocyanins (Howard et al., 2012; Jing and Giusti, 2011). Thus,
the preparation of a full range of reference materials is difficult
and expensive (Scotter, 2010), due to the potential number of
anthocyanins present in a given fruit or vegetable extract. How-
ever, if the main anthocyanin of the extract is known or can be
identified, the total anthocyanin content may then be expressed
in terms of the major component (Prodanov et al., 2005; Giusti
and Wrolstadt, 2001), and only one purified reference standard
is then required for quantitation.
Metabolites are the intermediates and end products of cel-
lular regulatory processes, and their levels can be regarded as
the ultimate response of biological systems to genetic or en-
vironmental changes (Bueno et al., 2012a; Traka and Mithen,
2011; Pereira et al., 2005). Differences due to volatility, polar-
ity, solubility, and chromatographic behavior mean that multiple
methods will need to be deployed to analyze different subsets
of metabolites (Jones and Kinghorn, 2005; Ward et al., 2003).
In this context (Seger and Sturm, 2007; Pereira et al., 2005)
high-performance liquid chromatography (HPLC), mass spec-
troscopy (MS), Fourier transform-infrared spectroscopy (FT-
IR), spectroscopy methods (UV, fluorescence), and coupled
gas chromatography-mass spectrometry (GC-MS) have already
been successfully applied to plant metabolite profiling. Another
potentially powerful tool for plant metabolite analysis is high-
resolution nuclear magnetic resonance (NMR) spectroscopy, in
particular, 1H NMR (Krishnan et al., 2005). The use of chemo-
metric methods on data obtained by spectrometer analyses to-
gether with recent advances in computer technology allows the
development of multivariate data analysis as a powerful tool
in the evaluation of food quality (Ferrari et al., 2011; Saurina,
2010; Winterhalter, 2007; Wrolstad and Durst, 2007; Deborde
et al., 2005).
Much of the literature on food colors is concerned not so
much with the role of natural pigments as food additives as with
the composition and analysis of those pigments (Schoefs, 2002,
2003, 2005) that are inherently present in foodstuffs. This is
certainly the case with anthocyanins (Scotter, 2011a, 2011b).
Extraction of anthocyanins from natural sources may be more
favorable than laboratory synthesis because of the labile nature
of the compounds (Castañeda-Ovando et al., 2009). For natu-
ral products, extraction of anthocyanins typically involves some
form of solid-liquid extraction, followed by solid phase and/or
liquid-liquid extraction to help remove unwanted species, such
as phenolic acids, sugars, proteins, and other flavonoids (Barnes,
2010; Barnes et al., 2009). In recent years, various novel extrac-
tion techniques have been developed (Wijngaard et al., 2012;
Ignat et al., 2011; Kataoka, 2010) for the extraction of nutraceu-
ticals from plants, including countercurrent chromatography,
ultrasound-assisted extraction, microwave-assisted extraction,
supercritical fluid extraction, and high hydrostatic pressure ex-
traction (HHP).
Polyphenol analytical chemistry has been the subject of var-
ious studies in this journal (Bueno et al., 2012a, 2012b; Navas
et al., in press; Escudero, 2011; Pyrzynska and Pekal, 2011;
Biesaga and Pyrzynska, 2009; Pappas and Schaich, 2009; Pohl
et al., 2009; Zima et al., 2009; Marszall and Kaliszan, 2007;
 ANTHOCYANIN PIGMENTS. PART IV: EXTRACTION
Escarpa and Gonzalez, 2001). This article aims to overview the
extraction approaches that have been either developed or applied
to anthocyanins. It brings together information from a scattered
literature to give an accessible account of assisted extraction
methods for anthocyanins. However, the use of non-thermal
technologies in the assisted extraction of anthocyanins will be
covered in a separate report.
ANTHOCYANIN SOLVENT EXTRACTION
The analysis of anthocyanins is complex as a result of their
ability to undergo structural transformations and complexation
reactions (Bueno et al., 2012a, 2012b). Anthocyanins may be
part of complexes, may occur in complex matrices, and may
exist in a variety of protonated, deprotonated, hydrated, and iso-
meric forms, and the relative proportion of these molecules is
strongly dependent on pH. Acid dissociation constants (Asuero
and Michalowski, 2011; Asuero, 1989, 2007; Asuero et al.,
1986a, 1986b; Herrador et al., 1987) are important physico-
chemical parameters that describe the extent of ionization of
functional groups as a function of pH (Bueno et al., 2011a,
2012b); they are of vital importance in the analysis of bioactive
compounds as well as in the interpretation of their mechanism
of action. Their antiradical properties, i.e., the ability to react
quickly and efficiently with electron-deficient radicals, depend
on the acidity of phenolic hydroxyl groups and on the stability of
the formed radical (Leopoldini et al., 2011; Estevez et al., 2010;
Musialik et al., 2009; Pina et al., 2012a; Pina et al., 2012b).
Typical procedures for isolation and characterization of pure
anthocyanins consist of several steps: (i) extraction of the plant
material, followed by a preliminary purification step, (ii) frac-
tionation of the mixture followed by isolation of pure pigments,
and finally (iii), characterization and identification of pure an-
thocyanins (Strack et al., 1989). Methods such as solid-phase
extraction (SPE), as shown later, use solid adsorbents to extract
phytochemicals from liquid matrixes such as juices. It is easy,
rapid, and economical compared to solvent extraction. However,
SPE is perhaps more often used in sample cleanup, purification
or pre-concentration than in extraction because of the selectivity
and saturation of the adsorbents (de la Rosa et al., 2009; Tsao
and Deng, 2004).
Extraction is a very important stage in the isolation, iden-
tification, and use of anthocyanins (Andersen and Markham,
2006). The recovery of anthocyanins is commonly performed
through a solvent-extraction procedure and the solvent type, sol-
vent concentration, liquid-to-solid ratio, temperature, and time
are important parameters to be optimized (Zou et al., 2011).
The extracting solution should be slightly acidic to maintain
the flavylium cation form, which is red and stable in highly
acidic medium, but not so acidic to cause partial hydrolysis of
the acyl moieties in acylated anthocyanins (Welch et al., 2008).
The structural diversity, together with the susceptibility of an-
thocyanins to heat, pH, metal complexing, and copigmentation,
complicates the protocols of extraction and analysis from both
plant material and biological fluids (Mazza et al., 2004). Antho-
315
cyanins are heat-sensitive, so high temperatures must be avoided
during extraction and concentration, i.e., <30◦ C (Andersen and
Markham, 2006).
Anthocyanins, like flavonoids in general, have aromatic rings
containing polar substituent groups (hydroxyl, carboxyl, and
methoxyl) and glucosil residues that altogether produce a polar
molecule (Bueno et al., 2012a, 2012b; Delgado-Vargas et al.,
2000). While flavonoid glycosides are more polar, aglycones
are extracted with alcohols or alcohol-water mixtures. Antho-
cyanins are extracted with cold acidified solvents (polar organic
solvents, water) under mild conditions (Giusti and Jing, 2008).
The organic solvent is usually methanol, but many other sol-
vents may be used such as acetone, ethanol, or acetonitrile. This
solvent system denatures the cell membranes, simultaneously
dissolving the anthocyanins and stabilizing them (Naczk and
Shahidi, 2006). The acid employed is usually acetic acid (about
7%) or trifluoroacetic acid (TFA; about 3%), whereas the or-
ganic solvent content varies from 50% to 100% of the mixture
(Nicoue et al., 2007; Robards, 2003). The use of mineral acid
can lead to the loss of attached acyl group (Dai and Mumper,
2010; Barnes et al., 2009). Sulfured water has also been used as
extraction solvent in seeking a reduction of the use of organic
solvents as well as the cost of extraction (Cacae and Mazza,
2002).
Anthocyanins are normally extracted with methanol contain-
ing 0.5% trifluoracetic acid (TFA) (v/v). Black beans (Phase-
olus vulgaris) were also presoaked in water containing 0.5%
TFA (Jordheim, 2007; Jordheim et al., 2006). In this case this
was done to improve the extraction yields of anthocyanins be-
cause direct methanolic extractions provide very poor yield.
The extraction was performed in a refrigerator (5◦ C) at low
temperatures to avoid hydrolysis of potential acyl groups in the
anthocyanin structure and degradation. After extraction the ex-
tract was filtered, and the methanol was removed by evaporation
under reduced pressure at relatively low temperatures (<30◦ C).
Anthocyanins are also extracted under cold conditions with
methanol, if it contains 2–10% formic acid. This process is very
good for grape extraction despite the fact that the final water-
alcohol extract will include some non-polyphenolic substances
such as sugars, amino acids, proteins, and pigments (Shi et al.,
2005). Thus, the extract must be purified. If appreciable amounts
of lipids, chlorophylls, or unwanted polyphenols are suspected
to be present in anthocyanin-containing extracts, they may be
removed by washing with ethyl acetate, petroleum ether, ethyl
ether, or diethyl ether (Rodriguez-Saona and Wrolstadt, 2001).
A procedure used acetone as the extracting solvent followed
by separation of the aqueous phase by addition of chloroform,
isolating and partially purifying the pigments in this way. The
aqueous portion contains the anthocyanin, phenolics, sugar, or-
ganic acids, and other water-soluble compounds, whereas the
choroform phase contains the lipids, carotenoids, chorophyll
pigments, and other nonpolar compounds. High recoveries of
anthocyanins are obtained requiring little concentration or fur-
ther purification (Rodriguez-Saona and Wrolstad, 2001).
316 M. J. NAVAS ET AL.
Column chromatography has been also employed for frac-
tionation of phenolic extracts. This method, though often labor
intensive and solvent consuming, ensures that greater amounts
of fractions can be obtained for use in subsequent isolation
and identification of pure substances (Dai, 2009; Andersen and
Markham, 2006).
The composition of anthocaynins and polyphenols is highly
dependent on the extraction temperature (Holtung et al., 2011;
Ruenroengklin et al, 2008), which reflects the conflicting actions
of solubilization and analyte degradation. Under cold extraction,
color degradation was significantly lower and extraction times
were 15-fold longer (Ramirez Rodrı́gues et al., 2011). A 50%
methanol extract shows maximum recovery for anthocyanins
bunga kantan (Etlingera elatior Jack.) inflorescence (Wijekoon
et al., 2011). Eight different solvent mixtures containing acetone
or methanol, pure or combined with an acid (acetic, formic, hy-
drochloric), were tested for their efficiency for extraction of phe-
nolic compounds from strawberries belonging to five groups of
polyphenols including anthocyanins (Kajdžanoska et al., 2011).
Solvent extraction offers good recovery of antioxidant phy-
tochemicals from various samples. The primary drawback of
the traditional extraction procedure is that the obtained final
extracts often require subsequent concentration and cleanup
prior to analysis. Furthermore, when considering the extraction
of bioactive compounds, which are unstable and thermolabile
and are found in low concentrations, traditional extraction tech-
niques would not be the most suitable option (Routray and Orsat,
2012). However, the use of large amounts of organic solvents
poses health and safety risks and is environmentally unfriendly.
There are many alternative methods that either eliminate or re-
duce significantly the use of organic solvents, e.g., microwave-
assisted extraction (MAE), supercritical fluid extraction (SFE),
and pressurized liquid extraction (PLC), whose use is increas-
ingly popular in the extraction of antioxidant phytochemicals.
Some of these methods offer identical, if no better, extrac-
tion efficiency and cost effectiveness (Tsao and Deng, 2004).
Table 1 shows the advantages of limitations of these extraction
procedures.
SOLID PHASE EXTRACTION
Solid phase extraction (SPE) was developed in the 1980s and
has emerged as a powerful tool for chemical isolation and pu-
rification (Santana et al., 2009). The goals of SPE are usually
retention and elution of an analyte from a sample, removal of
contaminants and interfering substances, and sample concen-
tration (Kole et al., 2011). It is based on the same principle of
affinity-based separation as liquid chromatography and over-
comes the limitations of liquid-liquid extraction (Zwir-Ferenc
and Bizink, 2006). It requires application of samples in a liquid
state, a proper extraction being the first preparation step of solid
samples. Simple filtration or centrifugation and then a filtrate or
supernatant is applied to the SPE cartridge.
SPE on C18 cartridges (because polyphenol molecules have
hydrophobic groups, nonpolar absorbents should be used) or
Sephadex is commonly used for the initial purification of the
crude anthocyanin extracts (Giusti and Jing, 2008; Rodriguez-
Saona and Wrolstad, 2001). Although they are available in nor-
mal phase, reverse-phase, and ion-exchange modes, reverse-
phase adsorbents are used as the main adsorption materials
in SPE (McDonald and Bouvier, 2001). The anthocyanins are
bound strongly to these adsorbents through their unsubstituted
hydroxyl groups and are separated from unrelated compounds
by using a series of solvents or increasing polarity (Mazza et al.,
2004; Kong et al., 2003).
Currently used methods for anthocyanin extraction are nons-
elective and result in solutions with large amounts of undesirable
products such as sugars, acids, amino acids, and proteins that re-
quire removal. Crude extracts have been purified by removal of
sugars, acids, and water-soluble compounds with C18 cartridges
previously activated with methanol, followed by water or 0.01%
aqueous HCl or 3% formic acid (Mazza et al., 2004; Wang and
Sporns, 1999). Partitioning of extracts with ethylacetate has
been shown to remove interferences prior to LC-MS analysis
(Giusti et al., 1999). Anthocyanins were recovered from diluted
fruit juice or wine (after removal of ethanol) by elution from
a C18 cartridge with an aqueous eluent at a low pH (Corradini
et al., 2011). However, SPE was used to obtain anthocyanin-
rich extracts from berry species (Denev et al., 2010) and to
obtain a pigment-rich fraction in anthocyanins from grape skins
(Kneknopoulos et al., 2011) as well as in the anthocyanin anal-
ysis of cranberry fruit and cranberry fruit products (Brown and
Shipley, 2011), prior to the HPLC final determination. Shah and
Chapman (2009) used mixed-mode cation exchange SPE to pu-
rify anthocyanins from tulip extracts in 50:50 methanol:water
with 0.1% formic acid, and Ling et al. (2009) have used
hydrophilic-lipophilic balanced (HLB) SPE to extract antho-
cyanins from human tissue homogenates. Automated off-line
SPE can be used in combination with LC-MS-MS to meet
specific needs (Dumont et al., 2010). Applications of SPE to
anthocyanins are compiled in Table 2.
COUNTERCURRENT CHROMATOGRAPHY
Separation by countercurrent chromatography (CCC) is
based on the partition of a phytochemical (Sarker et al., 2005;
Tsao and Deng, 2004). CCC is a technique that allows the frac-
tionation and isolation of pure compounds to yield the large
amounts required for identification by MS and NMR method-
ologies (Yao et al., 2012; Andersen and Markham, 2006), or
for further utilization as standards in analytical methods, or as
bioactive compounds for biological studies (Hillebrand et al.,
2009). In certain cases CCC can replace traditional techniques
like low-pressure columns or semi-preparative and preparative
HPLC, since CCC can provide great resolution and greater
yields at lower costs (Valls et al., 2009).
High-speed countercurrent chromatography (HSCCC), a rel-
atively new technique, is the most advanced CCC form in terms
of partition efficiency and separation time (Du, 2011). Un-
like other chromatographic techniques, HSCCC does not use
 ANTHOCYANIN PIGMENTS. PART IV: EXTRACTION 317

TABLE 1
Advantages and disadvantages of pretreatment techniques (Ignat et al., 2011; Kivilompolo, 2009; Tsao and Deng, 2004; Wang
and Weller, 2006; Jakubowska et al., 2005)

Technique Advantages Limitations

SFE (CO2 ) CO2 is chemically inactive, has low toxicity, and is CO2 is nonpolar and addition of modifier is often
environmentally friendly, nonflammable, and needed
cheap Expensive instrumentation
Reduces use of organic solvents Optimizing the method is demanding and often
Low operation temperature possible matrix dependent
Rapid, automatable, selective High concentrations of organic modifiers lead to
reduced selectivity
PHWE (pressurized Water is environmentally friendly, nontoxic, High temperature and pressure make demands on
hot water nonflammable, and cheap energy and material
extraction) Reduces use of organic solvents No commercial instrument available
Also suited to wet samples Not suitable for labile compounds
Dirty extracts (if high temperature)
MAE (microwave- Reduced extraction time It is necessary to remove the solid residue during
assisted Reduced solvent usage MAE
extraction) Improved extraction yield The efficiency of microwaves can be very poor
Process simplicity and low cost when either the target compounds or the solvents
are nonpolar, or when they are volatile
SPE (solid phase Reduced lab time Channeling
extraction) Easy manipulation Limited flow rates
Higher concentration factor Insufficient equilibration time for quantitative
No problem with the miscibility of solvents uptake
Easy adaptable for very selective extraction Incomplete elution
Easy automation Memory effects from previous extraction (though
these disadvantages are more pronounced only in
chelating resins)

a solid support as the stationary phase (Sethi et al., 2009) and
therefore has many advantages over conventional chromatogra-
phy (Berthod et al., 2009). It has been used for separation of
anthocyanins in the pigment mixtures extracted from various
samples. Anthocyanins were successfully fractionated based on
their polarities into the biphasic mixture of tert-butyl methyl
ether/n-butanol/acetonitrile/water acidified with trifluoroacetic
acid (TFA) (Dai and Mumper, 2010). Wine is an important
source of dietary antioxidants because of its phenolic compound
content (Noguer et al., 2008). CCC, because of its gentle sep-
aration conditions, is ideally suited to the analysis of various
groups of wine constituents (Winterhalter, 2009). Selected ap-
plications of the CCC method applied to anthocyanins are shown
in Table 3.
ADSORPTION
Processing of solutions containing phenolics by adsorption-
desorption enables the recovery and purification of bioactive
compounds or fractions from plants (Soto et al., 2011). Although
most reported information deals with experiments at the lab
scale, studies conducted in pilot plants and industrial facilities
are also available (El-Shafey et al., 2005). Commercial resins
are widely used at the pilot scale, e.g. recovery of anthocyanins
from grape pomace (Kammerer et al., 2005). The versatility,
simplicity, low cost, high efficiency, and ease in upscaling of
adsorption processes make them an attractive possibility for the
selective removal/recovery of phenolic compounds (Soto et al.,
2011). Adsorption shows a number advantages over separations
carried out by partition with organic solvents, such as selectivity,
environmental impact, and toxicological effects.
Adsorption onto polymeric resins of anthocyanins from grape
pomace, Ficus racemosa L. fruit, mulberries, eggplant peel, and
red cabbage were reported as well as selective recovery from
citrus byproducts containing anthocyanins. Cation-exchange
resins and clays have been used for anthocyanin recovery from
pomegranate juice and red cabbage extracts, respectively. Ta-
ble 4 contains a number of applications of adsorbent and eluting
agents proposed for the selective recovery of anthocyanins.
PRESSURIZED FLUID EXTRACTION
Supercritical fluid extraction (SFE) represents an interest-
ing alternative technique to conventional solid-liquid extraction
318
Type of matrix/analyte
Archaeological
samples/malvidin
Queensland red
wines/phenolic profiles
Grape skins of pinot
noir/anthocyanins and
related compounds
Different biological
tissues/procyanidins,
anthocyanins, and alkaloids
Five berry
species/anthocyanins
Wine matrices and grape
juice/anthocyanins and
other phytochemicals
Wine/anthocyanins
Red kiwifruit/anthocyanin
components
Human saliva, plasma, and
oral tissue/cyanidin
3-glucoside and other
3-substituted cyanidins
from bioadhesive black
raspberry gel
Human plasma/anthocyanins
Selected applications of solid phase extraction (SPE) applied to anthocyanins
Extraction/cleanup
technique
Sonication and
centrifugation
previous/RP SPE
Non-anthocyanin
phenolics previously
extracted/SPE
Extraction with
MeOH/SPE
chromatography
Off-line liquid-solid
extraction (LSE)-SPE
and off-line LSE-µSPE
Extraction with citric acid
and centrifugation/SPE
Liquid-liquid
extraction/automated
off-line SPE
SPE
Extraction with EtOH and
HCOOH/SPE
Centrifugation and
supernadant dilution
(saliva). Samples spiked
with internal standard
(tissue, plasma)
SPE with a centrifugation
previous
TABLE 2
Sorbent Final analysis Reference
SPE cartridge (Agilent LC-MS/MS Barnard et al., 2011
AccuBond ODS-C18)
C18 reversed-phase packing HPLC-DAD-MS Ginjom et al., 2011
(Alltech Associates,
Deerfield, Ill.)
Amberlite XAD-7 resin Multilayer coil Kneknopoulos et al.,
countercurrent 2011
chromatography
(MLCCC) and ESI-MS
OASIS HLB cartridges UPLC-EI-MS/MS Serra et al., 2011b
(60 mg, Waters Corp.,
Milford, Mass.)
Amberlite XAD-7 Spectrophotometry Denev et al., 2010
LC-MS-MS Dumont et al., 2010
Polymer phase Strata SDB-L, LC/MS Hartmanova et al., 2010
(Phenomenex, Torrance,
USA)
XAD-7 HPLC-DAD, LC-MS/MS, Comeskey et al., 2009
and GC-MS
HLB cartridge LC-MS/MS Ling et al., 2009
Oasis HLB 1 cm3 (10 mg) LC/MS Nakamura et al., 2009
extraction cartridges
 Red tulip blooms (Tulipa 50/50 methanol/water DSC-MCAX SPE cartridge, HPLC/DAD/ESI-MS/MS Shah and Chapman,
darwin hybrid with 0.1% formic acid, 100 mg/3 mL (52783-U), 2009
‘Apeldoorn’)/anthocyanins sonication and filtration
and flavonol glycosides
Acerola/pulps and SPE Waters C18 cartridges HPLC-DAD Mezadri et al., 2008
juices/antioxidant
compounds
Violet cauliflower and red SPE C18 HPLC-MS/MS Lo Scalzo et al., 2008
cabbage/anthocyanin
profiles
Red wines/phenolic SPE with prior HLB with HPLC Pérez-Magariño et al.,
compounds centrifugation N-vinylpyrrolidone- 2008
divinylbenzene
copolymer
Hibiscus/anthocyanins SPE Amberlite XAD-2 (pore size CE-ESI-MS Segura-Carretero et al.,
9 nm, particle size 2008
0.3–1.2 mm)
Greek red wines/phenolic SPE under vacuum C18 (Bakerbond J.T. Baker UV-vis and mid-IR with Tarantilis et al., 2008
fingerprint SPE C18) multivariate data analysis
“Vinho Verde” Solid-liquid Chromabond C18 non HPLC/DAD Dopico-Garcı́a et al.,
grapes/phenolic extraction/SPE end-capped and end-capped 2007
compounds and organic columns
acids
Wild mulberry (Morus nigra Extraction with Polyamide (CC-6, HPLC/DAD and MS Hassimotto et al., 2007
L.)/anthocyanin pigments MeOH/water/acetic Macherey-Nagel,
acid/polyamid SPF Germany)
Commercial juices/food SPE Sep-Pak Vac (6 cm3, 1 g) C18 Total reflectance IR He et al., 2007
authenticity cartridge (Waters, Milford,
Mass.)
Defatted bran of dark blue Extraction with AccuBond II ODS-C18 HPLC and ESI-MS Hu et al., 2007
grained EtOH/SPE (500 mg, Agilent)
wheat/anthocyanins
Red cabbage/anthocyanins SPE C18 LC-MS McDougall et al., 2007
(Continued on the next page)

319
320
Type of matrix/analyte
Berries/anthocyanins
Human plasma and
urine/anthocyanins
Aqueous extract of red
radish/anthocyanins
Red wine/nonpolymeric and
polymeric phenols
Aged red wine/polyphenols
Urine samples/anthocyanin
metabolites
Human serum and urine/total
anthocyanins
Human urine and
serum/anthocyanin
metabolites
Berries of Smilax aspera
L/anthocyanins
Olive fruits/phenolic
compounds
TABLE 2
Selected applications of solid phase extraction (SPE) applied to anthocyanins (Continued)
Extraction/cleanup
technique Sorbent Final analysis Reference
Extraction using EtOH C-18 solid-phase extraction HPLC-ESI-MS/MS) Nicoue et al., 2007
alone or EtOH cartridge
acidified/SPE
SPE Oasis HLB SPE cartridges HPLC and MS Cooke et al., 2006
(1 mL; Waters, Elstree,
U K)
SPE Oasis HLB-cartridge (1 mL, HPLC/DAD/MS and Fleschhut et al., 2006
30 mg; Waters, Milford, GC/MS
Mass.)
SPE C18 Sep-Pak cartridge HPLC-UV-vis Pinelo et al., 2006
(Fisher Scientific, 1 g) spectroscopy
SPE LiChroprep RP 18 (particular HPLC-DAD, HPL-ESI-MS Sun et al., 2006
size 25–40 µm) and multistage MS
fragment analysis
SPE Sep-Pak C18 Plus cartridge HPLC-ESI-MS-MS and Felgines et al., 2005
(Waters, Milford, Mass.) HPLC with UV-vis
SPE C18 cartridges (Supelclean RP-HPLC-DAD Kay et al., 2005
ENVI-18 6 mL 2000 mg;
Sigma; lot # SP2419C)
SPE C18 cartridges (Supelclean HPLC-DAD, MS, GC, and Kay et al., 2004
ENVI-18 6 mL 500 mg; enzymic techniques
Sigma, Oakville, Ont.,
Canada)
MeOH extraction/SPE C-18 Sep-Pak cartridge HPLC-DAD-MS Longo and Vasapollo,
(Waters Corporation, 2006
Milford, Mass.)
MeOH extraction/SPE ISOLUTE C18 RP HPLC/DAD and/or Vinha et al., 2004
non-end-capped (NEC) HPLC-DAD/ESI-
MS/MS
 Urine samples/strawberry SPE Sep-Pak C18 Plus (Waters, HPLC-ESI-MS-MS and Felgines et al., 2003
anthocyanins Milford, Mass.) HPLC-UV-visible
Red wine/pigments SPE C18 HPTLC Lambri et al., 2003
Plasma and SPE Octadecylsilane cartridge HPLC/DAD Cao et al., 2001
urine/anthocyanins (Sep-Pak C18)
Botanical supplement raw SPE Sep-pak Plus C-18 cartridge HPLC and LC/ES-MS Chandra et al., 2001
materials/anthocyanins (1 mL) (Waters, Milford,
Mass.).
Austrian red wines/phenolic SPE C-18 columns Mid-IR and UV-vis Edelmann et al., 2001
compounds spectroscopy
Foods/anthocyanins Extraction with methanol C 18 (SPE) cartridges or LC-UV, LC-MS, and CE Da Costa et al., 2000
or acetone Sephadex (capillary
electrophoresis)
Grape skins/anthocyanins Extraction with Sep-Pak C-18 cartridge ESI-MS Favretto and Flamini,
EtOH-water-HCl (Waters) 2000
solution/SPE
Berries of the hybrid grape Extraction with Sep-Pak C-18 cartridge HPLC-UV-vis Flamini and Tomasi,
cultivars/anthocyanins EtOH-water-HCl (Millipore-Waters) 2000
solution/SPE
Finnish SPE CH sorbent100 mg, SAX Spectrophotometry and Huopalahti et al., 2000
cranberry/anthocyanin and sorbent, 100 mg HPLC
organic acids
Hungarian red SPE Silica, octadecylsilica, Multiwavelengh Kiss et al., 2000
wines/pigments cyanopropyl, alumina spectrophotometry with a
(acidic, basic, neutral), diol, spectral mapping
aminopropyl, florisil, accell technique and by RP
plus QMA, carbon black HPLC
Black bean and Extraction with ethyl C18 SPE cartridge HPLC-UV-vis Dao et al., 1998
blackberry/anthocyanidins acetate/SPE spectroscopy
Black chokeberry (Aronia SPE Serdolit PAD IV, Amberlite HPLC-UV-vis Kraemer-Schafhalter
melanocarpa var XAD-7 et al., 1998
Nero)/anthocyanins
Black beans (Phaseolus Extraction with HCl in Extrelut 20 cartridge (Merck, HPLC-DAD, TLC, GC, Takeoka et al., 1997
vulgaris L.)/anthocyanins MeOH/SPE Darmstadt, Germany) UV-vis, MS, and NMR

321
322
Selected examples of countercurrent chromatographic methods applied to anthocyanins
Sample/analyte
Grape skins from Vitis vinifera
cv. pinot noir/anthocyanins
Black carrot/pigment
composition
Different Bolivian purple corn
(Zea mays L.)
varieties/phenolic
compounds
Strawberry/antioxidant activity
of anthocyanin compounds
Japanese purple sweet potato
(Ipomoea batatas L.)
varieties/anthocyanins
Corozo (Bactris guineensis)
fruit/anthocyanin pigments
Berries (blackberries, black
chokeberries, and
grapes)/anthocyanins,
stilbenes, and
proanthocyanins
Fruits and vegetables
Purple sweet
potato/anthocyanins
TABLE 3
Solvent system
Tert-butyl methyl ether/n
butanol/acetonitrile/water
acidified with 0.01% TFA
(2:2:0.1–1.8:5 v/v/v/v). MLCCC
Tert-butyl methyl ether/n-butanol/
acetonitrile/water (2/2/1/5
v/v/v/v), acidified with 0.1% TFA
Tert-butyl methyl ether/n-butanol/
acetonitrile/water (2:2:1:5
v/v/v/v), acidified with 0.1% TFA
Tert-butyl methyl ether/n-butanol/
acetonitrile/water (2.75:1.25:1:5),
acidified with 0.1% TFA acid
Tert-butyl methyl ether/n-butanol/
acetonitrile/ water (1:3:1:5,
v/v/v/v), acidified with 0.1% TFA
Tert-butyl methyl ether/n butanol/
acetonitrile/water (2:2.1:5,
v/v/v/v), acidified with 0.1% TFA
Tert-butyl methyl ether/n-butanol/
acetonitrile/ water/TFA
(1:4:1:5:0.01, v/v).
Elution mode/comment Reference
Aqueous layer (lower) as stationary Kneknopoulos et al., 2011
phase.
Step gradient elution to separate
anthocyanin oligomers from grape
anthocyanins
Stationary phase: less dense layer Montilla et al., 2011a
Isolation of pure anthocyanin standards
by HSCCC in high amounts
Stationary phase: less dense layer. Montilla et al., 2011b
HSCCC separated five fractions as
well as the coil residue
Stationary phase: the lighter (organic) Cerezo et al., 2010
phase. Two fractions:
pelargonidin-3-glucoside and
pelargonidin-3-rutinoside
Stationary phase: less dense layer. Montilla et al., 2010
HSCCC shows advantages with
respect to HPLC
Stationary phase: less dense layer. Osorio et al., 2010
HSCCC allows the identification of
cyanidin-3-O-(malonyl)
glucopyranoside
Different types of the Winterhalter et al., 2010
all-chromatographic techniques of
countercurrent chromatography as well
as semi-synthetic strategies
Isolation of different non-acylated and Hillebrand et al., 2009
acylated cyaniding derivatives
Stationary phase: upper organic phase. Qiu et al., 2009
Four acylated cyanidins and peonidins
obtained by HSCCC
 Red wine (Monastrell)
(antioxidant
activity)/polyphenolic
fractions (including
polymers)
Potato (Solanum tuberosum L.)
Varieties/anthocyanins
Red wine (cabernet sauvignon
(60%) and tannat
(40%)/pigments
Camu-camu (Myrciaria
dubia)/anthocyanins
Tea, shallot and,
endive/flavonol glycosides;
fruit juices, purple
carrot/anthocyanins
Bilberry fruit crude extract
(Vaccinium myrtillus,
Ericaceae)/anthocyanins
Blood orange (Citrus sinensis
(L.) Osbeck)
juice/anthocyanins and
pyranoanthocyanins
Wine from South African
grape variety pinotage (pinot
noir ×)
Grape skins/oligomeric
anthocyanins
323
Tert-butyl methyl ether/n-butanol/
acetonitrile/water (2/2/1/5)
acidified with 0.1% TFA. HSCCC
Tert-butyl methyl ether/n-butanol/
acetonitrile/water (2:2:1:5
v/v/v/v), acidified with 0.1% TFA
acid
Tert-butylmethyl ether/n-butanol/
acetonitrile/water (2/2/1/5),
acidified with 0.1% TFA
Tert-butyl methyl ether (MTBE)/n-
butanol/acetonitrile/water
(2:2:1:5, v/v/v/v) acidified with
0.1% TFA
Methyl tert-butyl ether–n-
butanol–aceto-nitrile–water–TFA
(1:4:1:5:0.01, v/v). HSCCC
Methyl tert-butyl ether/n-butanol/
acetonitrile/water (1:3:1:5,
v/v/v/v), acidified with 0.1% TFA
Adsorption on Amberlite XAD-7
resin and purification by HSCCC
Tert-butyl methyl ether/n-butanol/
acetonitrile/water acidified with
TFA (2/2/ x/5); MLCCC with
gradient elution
Organic phase as stationary. Noguer et al., 2008
Four fractions: polymeric compounds,
malvidin-3-gluco-side,
peonidin-3-glucoside, and visitin A
Mobile phase: the lower layer. Eichhorn and Winterhalter, 2005
Four major fractions: anthocyanins and
3-chlorogenic acid (1–3); and
5-chlorogenic acid (4)
Stationary phase: the organic phase. Salas et al., 2005
Separation of anthocyanin-derived
pigments by HSCCC
Stationary phase: less dense layer. Zanatta et al., 2005
Anthocyanins were isolated by HSCCC
HSCCC and MLCCC techniques applied Degenhardt et al., 2004
to the isolation of flavonol glycosides
Upper organic phase as stationary. Du et al., 2004
Preparation of pure anthocyanins
(delphinidin and
cyanidin-3-O-sambubiosides)
Mobile phase: denser layer Eight Hillebrand et al., 2004
fractions obtained
New derivatives of malvidin-3-glucoside, Schwarz and Winterhalter, 2004
malvidin-3-(6 -acetylglucoside) and
malvidin-3-(6-coumaroyl-glucoside)
Fractionation of anthocyanins as Vidal et al., 2004a
glucosides and the corresponding
acetylated, coumaroylated, and
caffeoylated derivatives
(Continued on the next page)
324
Selected examples of countercurrent chromatographic methods applied to anthocyanins (Continued)
Sample/analyte
Grape skin extract/oligomeric
anthocyanins
Phytosterol, chlorinated
s-triazines, flavonoids,
theaflavins, anthocyanins,
sporaviridins, etc.
Tradescantia pallida leaves,
purple corn, elderberry juice,
red wine, and
blackberries/anthocyanins
Red wine, red beet, Gardenia
jasminoides, black
tea/natural pigments
(anthocyanins. . .)
Red onions (Allium cepa) and
flowers of tulip cultivar “Los
Angelos”/anthocyanins
Red wine, grape skin extracts,
and other fruit and juice
extracts/polymeric
anthocyanins; acetylated
anthocyanins
Red wine and grape skin
extracts/malvidin-3-
glucoside
Champagne vintage
byproducts/malvidin-3 and
peonidin-3-glucosides
CCC: countercurrent chromatography; HSCCC: high-speed countercurrent chromatography; MLCCC: multilayer coil countercurrent chromatography; TFA: trifluoroacetic acid.
TABLE 3
Solvent system
Tert-butyl methyl ether/n-butanol/
acetonitrile/water with TFA
(2/2/x/5)
Tert-butyl methyl
ether/n-butanol/acetonitrile/water
(2:2:1:5), acidified with 0.01%
TFA
n-butanol/tert-butyl methyl
ether/acetonitrile/water. I: 2:2:1:5
v/v/v/ v plus 0.1% TFA. II: The
same plus 0.1% TFA. III: 3:1:1:5
v/v/v/v with 0.1% TFA
HSCCC
Tert-butyl methyl
ether/n-butanol/acetonitrile/water
(2/2/1/5), plus 0.01% TFA
Tert-butyl methyl ether/n-butanol/
acetonitrile/water (2/2/1/5), plus
0.1% TFA. Ethyl
acetate-n-butanol-water (4/1/5)
acidified with 0.1% TFA
Red pigments first cleaned up on an
Amberlite XAD-7 column.
HSCCC
Ethylacetate/n-butanol/water
acidified with TFA
Elution mode/comment Reference
MLCCC coupled with step gradient Vidal et al., 2004b
elution. Identification by mass
spectrometry
Aqueous phase as mobile. Chen et al., 2003
Five analogs of anthocyanins
successfully separated.
Several hundred milligrams of pure Schwarz et al., 2003
anthocyanins were obtained within a
single CCC run.
HSCCC offers several advantages over
HPLC.
Isolation of hydrophilic thearubigins Degenhardt and Winterhalter, 2001
from black tea
Fragile anthocyanins with aliphatic Torskangerpoll et al., 2001
acilation in semipreparative scale
HSCCC provides a tool for the chemist Degenhardt et al., 2000b
to isolate large quantities of pure
standards
Purity and identity of isolated Degenhardt et al., 2000a
compounds checked by HPLC-DAD,
NMR, and MS
Centrifugal partition chromatography Renault et al., 1995
was applied for the first time

Examples of adsorbents and eluting agents proposed for the selective recovery of phenolic compounds
Solution (target phenolic
compounds)
Grape skins of pinot noir
(anthocyanins)
Raw juice (flavonoids and
anthocyanins)
Blood oranges (anthocyanins)
Muscadine grape pomace
(polyphenolics)
Ficus racemosa fruit
(anthocyanins)
Eggplant peel (anthocyanins)
Red cabbage (anthocyanins)
Orange juice (anthocyanins,
hesperidin, hydroxycynnamates,
nariturin, limonin)
Cassis, commercial powder
obtained from blackcurrant juice
(anthocyanins)
Grape pomace extracts
(anthocyanins)
Red wine from Vitis vinifera L. cv
Graciano grapes (anthocyanins)
Orange juice
(cyanidin-3-glucoside, hesperin)
Brassica oleraceae (anthocyanins)
Wines from the South African
grape variety pinotage (pinot
noir × cinsault) (acetylated and
coumaroylated anthocyanins)
Different cultivars of mulberry
(anthocyanins)
Wastewater of orange juice
processing (hesperidin,
anthocyanins, hydroxycinnamic
acids)
Pomegranate juices (antioxidant
activity, anthocyanins)
Hungarian red wines (pigments
and anthocyanins)
ANTHOCYANIN PIGMENTS. PART IV: EXTRACTION
TABLE 4
Adsorbent
XAD-7 resin
Macroporous adsorptive resins
D141
NKA-9 macroporous resin
Amberlite XAD-4 resins
Amberlite XAD-4 resin
Methacrylic resin Relite
EXA-31
Tonsil Terrana 580 FF clay and
Amberlite XAD-7 resin
Relite EXA 118
Previously cleaned C18
material or sea sand
Nonpolar SDVB resin
Cell walls of different strains
of Saccharomyces
Relite EXA 118
Amberlite XAD-7
Amberlite XAD-7 resin
D3520, D4020, X-5, NKA-9,
D101A, and AB-8 resins
Kastell S-112
Bio-Rad cation-exchange resin
AG50W-X8
Silica, octadecylsilica,
cyanopropyl, alumina, diol,
aminopropyl, florisil, accell
plus QMA, carbon black.
325
Eluting and/or regenerating
agent References
50% aqueous methanol Kneknopoulos et al.,
2011
Anthocyanins eluted with Li et al., 2011
ethanol-H2 O (30:70, v/v),
and flavonoids with
ethanol
50% ethanol with citric acid Cao et al., 2010
(pH 2.5)
100% methanol Cardona et al., 2009
Acidified methanol (1% Sarpate et al., 2009
HCl)
Acidified H2 O ethanol 1:1; Todaro et al., 2009
1% w/v of citric acid
70:30:5 v/v/w ethanol, water Lopes et al., 2007
and citric acid
Ethanol, 75–100% Scordino et al., 2007
MeOH/H2 O (1:1, v/v) Manhita et al., 2006
Methanol, ethanol, propanol Kammerer et al.,
2005
10 mL formic acid: Morata et al., 2005
methanol (10:90)
Methanol, 75–100% Scordino et al., 2005
70% Ethanol Coutinho et al., 2004
Purification by high-speed Schwarz and
countercurrent Witnterhalter,
chromatography and 2004
semi-preparative
high-performance liquid
chromatography
Acidified ethanol (0.5% Liu et al., 2004
(v/v) of hydrochloric acid)
96% Ethanol NaOH/10% Di Mauro et al.,
ethanol 2000, 2002
Water and methanol Gil et al., 2000
Acetonitrile, methanol, Kiss et al., 2000
tetrahydrofuran, and
dioxane, each containing
10% (v/v) concentrated
formic acid.
326 M. J. NAVAS ET AL.
with lower solvent consumption and lower working temperature
(Temelli and Seifried, 2011; Garcı́a-Salas et al., 2010; Pereira
and Meireles, 2010). It is a form of liquid extraction where the
usual liquid solvent phase has been replaced by a supercritical
fluid; i.e., a substance that is above its critical point. Among
a wide variety of supercritical fluids, carbon dioxide is essen-
tially the only convenient supercritical extraction solvent used
because of its comparatively low critical temperature and pres-
sure (Sticher, 2008; Nahar et al., 2005; Handa et al., 2008). This
method provides an alternative to the pretreatment of the plant
materials, replacing toxic organic solvents (e.g., hexane).
The extraction of anthocyanins by using supercritical CO2
(SC-CO2 ) methods requires high pressures and the presence
of an organic co-solvent (methanol, ethanol, water) in high
percentage due to the polarity of anthocyanins (Ignat et al.,
2011; Serra et al., 2010). High concentrations of polar organic
modifiers added to obtain the best extraction yield lead, however,
to reduced selectivity. Bleve et al. (2008) described a method for
the purification of anthocyanins from grape skin extracts as liq-
uid matrix, by using CO2 under liquid and subcritical conditions.
The solid residues generated from blueberries, cranberries, and
raspberries after pressing were extracted by conventional sol-
vent extraction or by SC-CO2 extraction (Laroze et al., 2010).
SFE of bioactive compounds from grape peel by using 6–7%
ethanol as modifier has been carried out (Ghafoor et al., 2010).
Fractionated high-pressure extraction is performed in order
to obtain anthocyanin-rich extracts. A first step with SC-CO2
followed by a second step with mixtures of CO2 and ethanol
were applied to cherries (Serra et al., 2010, 2011a); the product
derived from the second CO2 -EtOH (90:10 V/V) step extraction
exhibited activity against colon cancer (Serra et al., 2011a). SC-
CO2 fluid extraction followed by enhanced solvent extraction
with CO2 -EtOH-H2 O mixtures allows obtaining anthocyanin-
rich fractions in the second step (Seabra et al., 2010b), from
elderberry pomace. Supercritical fluid extraction, with 6–7%
ethanol as modifier, was applied for the extraction of valuable
compounds, e.g., total anthocyanins, from grape peel (Ghafoor
et al., 2010).
Pressurized liquid extraction (PLE), also known under the
trade names of accelerated solvent extraction (ASE), pressurized
fluid extraction (PFE), pressurized solvent extraction (PSE), or
enhanced solvent extraction (ESE), is a relatively new technol-
ogy for extraction of phytochemicals under high temperature
and pressure and is partly derived from SFE (Sticher, 2008).
In PLE, pressure is applied to allow the use as extraction sol-
vents of liquids at temperatures greater than their normal boiling
point. The method was first described in 1995 (Richter et al.,
1995, 1996). The combined use of high pressures and tempera-
tures provides faster extraction processes (generally completed
within a few minutes, owing to increased solubility, better des-
orption, and enhanced diffusion) that require small amounts of
solvents (Dai and Mumper, 2010; Wibisono et al., 2009; Buldini
et al., 2002; Ramos et al., 2002). There are two ways to per-
form PLE, either in the static or dynamic mode. In both cases,
under conditions of elevated pressure and temperature, the mass
transfer rates are accelerated according to Fick’s law of dif-
fusion. Both commercially available and laboratory-assembled
PLE systems are used (Kivilompolo, 2009).
Therefore, extraction solvents, including water, that show
low efficiency in extracting phytochemicals at low tempera-
tures may be much more efficient at elevated PLE temperatures
(Dai and Mumper, 2010). The use of water as an extraction
solvent in PLE is the so-called pressurized hot water extrac-
tion (PHWE) (Teo et al., 2010). PHWE is also referred to as
subcritical water extraction, superheated water extraction, high-
temperature water extraction, extraction using hot compressed
water, and extraction with water at elevated temperatures and
pressures.
Subcritical water appears to be an excellent alternative to or-
ganic solvents to extract anthocyanins and other phenolics from
dried grape skin and possibly other grape byproducts (Ju and
Howard, 2003), and from industrially generated apple pomace
(Wijngaard and Brunton, 2009). PHWE is usually performed in
dynamic mode with water flowing constantly through the sam-
ple, but static extraction is also possible (Kivilompolo, 2009).
Water is heated up to 200◦ C and the change in dielectric con-
stant of the water with the temperature leads water to behave
like an organic solvent. For example, the dielectric constant of
water at 200◦ C is equal to 36, which is close to that of methanol
(Dai and Mumper, 2010; Dai, 2009).
A dynamic subcritical fluid extraction system (80% aque-
ous ethanol) has been used for extraction of flavonoids (an-
thocyanins) from food processing waste such as grape pomace
(Srinivas et al., 2011). Anthocyanins have been extracted from
red grape pigments by PLE in a static model (Hohnova et al.,
2008). A static bath reactor has also been used for the extraction
of anthocyanins from red onion (Pettersson et al., 2010). Pres-
surized hot water containing 5% ethanol was used to extract
anthocyanins from red cabbage (Arapitsas and Turner, 2008;
Arapitsas et al., 2008). Anthocyanins were extracted from red
pomace in acidified aqueous methanolic and aqueous ethanolic
solvents and identified and quantified in the extracts by HPLC-
MS and HPLC (Monrad et al., 2010).
Residence time of the extracted solute must be minimized in
order to prevent possible degradation of anthocyanin moieties
or their possible reaction with sugar and other products. There
is some evidence that side reactions generating antioxidant moi-
eties may occur in pressurized hot water (Bravi et al., 2012; King
and Srinivas, 2009). At first, the extraction effect dominates, but
degradation effects soon take over (Pettersson et al., 2010), as
occurs with anthocyanins from red onion. Anthocyanins present
in black carrot were extracted with pressurized acidified water;
anthocyanin degradation became significant above 100◦ C (Gizir
et al., 2008).
At the present, no commercial instrument is available and,
thus, the equipment has been self-built or modified from other
instruments. The difference from commercial instruments for
SFE and PLE or ASE is that PHWE equipment can tolerate
 ANTHOCYANIN PIGMENTS. PART IV: EXTRACTION
TABLE 5
Factors influencing pressurized liquid extraction
Pressurized liquid extraction
Matrix characteristics Nature of the analyte
Position and bonding degree of
the analyte
Particle size
Water content
Extraction kind Controlled by solubility
Controlled by diffusion
Extraction operation Dynamic
Static
Factors influencing the Solvent type
extraction Temperature
Time
Extraction enhancers Drying agent
Dispersing agents
Other additives
temperatures over 300◦ C (Kivilompolo, 2009; Hyötylainen,
2009). The high temperature makes demands on the material
of the extraction vessels and sealing rings. In pressurized hot
water extraction the most important parameter affecting extrac-
tion is the temperature.
The other significant parameters are pressure, flow rate of
the water, and extraction time. In addition, the matrix needs to
be taken into consideration, and choosing the proper analyte
collection system can increase selectivity of the extraction. The
most important parameters affecting the extraction in PLE are
shown in Table 5 (Dai and Mumper, 2010; Mustafa and Turner,
2011; Kivilompolo, 2009). Response surface methodology was
usually used to optimize the response values in the extraction
of anthocyanins from different sources (Ghafoor et al., 2010;
Wijngaard and Brunton, 2009; Arapitsas and Turner, 2008).
Recent applications of PLE to anthocyanin extractions are listed
in Table 6.
MICROWAVE-ASSISTED EXTRACTION
Microwave-assisted extraction (MAE), or simply microwave
extraction, is a relatively new technology for extracting solu-
ble products into a fluid from a wide range of materials using
microwave energy (Dai and Mumper, 2010; Garcı́a-Salas et al.,
2010; Jain et al., 2009). The use of MAE in natural product
extraction started in the late 1980s (Delazar et al., 2012). This
technique allows one to extract compounds more selectively
and more rapidly (usually in less than 30 minutes) with similar
or better recovery than traditional extraction processes (Garcı́a-
Salas et al., 2010; Escribano-Bailón and Santos-Buelga, 2003).
Microwaves directly heat the solvent or solvent mixture, thus
accelerating the speed of heating. Besides the advantage of high
extraction speed, MAE also enables a significant reduction in
327
the consumption of organic solvent (Mandal et al., 2007; Wang
and Weller, 2006).
The application of microwave energy to the samples may be
performed using two technologies: closed vessels under con-
trolled pressure and temperature (PMAE with a multimode
cavity) or open vessels at atmospheric pressure (FMAE using
the waveguide as a single-mode cavity) (Kivilompolo, 2009;
Sticher, 2008). The closed-vessel MAE system is quite similar
to PLE technology, as the solvent is heated and pressurized in
both systems. The main difference is in the means of heating,
either by microwave energy or by conventional oven heating.
Consequently, as for PLE, the number of influential parameters
is reduced, thus making the application of this technique quite
simple to use (Camel, 2001).
Microwaves are a non-ionizing electromagnetic radiation
with a frequency from 300 to 300000 MHz. In order to avoid
interference with radio communications, domestic and com-
mercial systems generally operate at 2450 MHz. Even though
microwave energy as a source of heat has been used in analytical
laboratories since the late 1970s, their application to enhance
extraction is very recent (Handa, 2008; Sticher, 2008). Exten-
sive use began around 10 years ago, with the commercialization
of several extraction instruments.
In MAE, it is important that the extraction solvent has good
polarity because solvents with high dielectric constants (polar)
can absorb more microwave energy and therefore result in better
extraction efficiency (Dai and Mumper, 2010; Jain et al., 2009;
Rawa-Adkonis et al., 2003). Water or other polar solvent is
therefore often added as a modifier in order to achieve an optimal
dielectric constant of the extraction solvent. However, differing
opinions also exist: when solvents of low dielectric constants are
used, all the microwave energy may be directed to the sample
material, and the moisture inside the cellular structure absorbs
the energy so quickly that it erupts and breaks the cell wall,
releasing the phytochemicals to the surrounding solvent.
Due to the particular effects of microwaves on matter (namely
dipole rotation and ionic conductance), heating with microwaves
is instantaneous and occurs in the heart of the sample, leading
to very fast extractions (Sticher, 2008). The results obtained so
far have concluded that microwave radiation causes no degra-
dation of the extracted compounds, unless the temperature in
the vessel rises too high. At the same time, a specific effect of
microwaves on plant material has been found (Sticher, 2008).
They interact selectively with the free water molecules present
in the solid matrix, leading to rapid heating and temperature
increase, resulting in rupture of the plant tissue and release of
solutes into the solvent (Mandal et al., 2007; Wang and Weller,
2006).
MAE (especially PMAE) is used in analytical protocols, very
often to investigate extraction parameters including pressure and
temperature, extraction time, microwave power, solvent nature,
and volume, or in comparative studies of this and other re-
cent techniques (such as SFE or PLE) with classical extraction
methods for particular applications (de la Rosa et al., 2009).
328
TABLE 6
Application of pressurized fluid extraction to the analysis of anthocyanins

Extraction
Matrix/compounds Solvent T Pressure/cycles time Technique Reference

Cherries (Prunus Supercritical CO2 , 50◦ C 25 ± 1 MPa 15, 60, TLC, HPLC-DAD Serra et al., 2011a
avium)/anticancer activity CO2 :ethanol (90:10, v/v) 90 min
Freeze-dried sunbelt (Vitis Hydroethanolic acidified 60◦ –140◦ C 10.3 MPa Srinivas et al., 2011
labrusca L.) grape with organic acids
pomace/flavonoids
Lyophilized grape CO2 , CO2 -methanol 40◦ C 110 bar HPLC-DAD, Floris et al., 2010
residues/polyphenols and LC-MS
anthocyanins
Grape peel/total phenols, Supercritical CO2 /6–7% 45◦ –46◦ C 160–165 kg 30 min UV-visible Ghafoor et al., 2010
antioxidants, and total ethanol as modifier cm−2
anthocyanins
Berries of crowberry/flavonols Supercritical carbon dioxide HPLC-DAD, GC Laaksonen et al., 2010
and other phenolic compounds
Solid residues generated from Supercritical CO2 ethanol as 60◦ C 80–300 bar 2h UV-visible, Laroze et al., 2010
blueberries, cranberries, and co-solvent GC-MS
raspberries
Dried red grape Methanol/water/formic acid 40◦ , 60◦ , 80◦ , 6.8 Mpa/1 HPLC-MS Monrad et al., 2010
pomace/anthocyanins (60:37:3 v/v/ v); 10, 30, 100◦ , 120◦ ,
50, and 70% ethanol in and 140◦ C
water, v/v
Red onion/anthocyanins Ultrapure 110◦ C 1–3 bar 10 min HPLC-DAD Pettersson et al., 2010
water-ethanol-formic acid
Grape skin extracts/polyphenols, Methanol and ethanol 40◦ –120◦ C 15 Mpa/3 5 min HPLC, EPR Polovka et al., 2010
total phenolic spectroscopy
Elderberry (Sambucus nigra L.) Supercritical CO2 with 313 K 20 MPa Static and TLC, HPLC-UV Seabra et al., 2010b
pomace/anthocyanins ethanol-H2 O mixtures dynamic
Elderberry pomace/anthocyanins CO2 /ethanol/H2 O 313 K 21 MPa 45 min UV-vis and Seabra et al., 2010a
HPLC-DAD
Sweet cherry variety from Supercritical CO2 , CO2 and 50◦ C 25 MPa 1 plus 1.5 h HPLC-DAD, LC- Serra et al., 2010
Portugal (“Saco”)/anthocyanin ethanol (10–100%, v/v) DAD-MS/MS
phenolic content, antioxidant
activity
 Grape pomace/anthocyanins, CO2 with water or ethanol 80◦ C 300 bar Casas et al., 2009
resveratrol, catechin, and as cosolvents
epicatechin
Black currant (Ribes nigrum) Supercritical CO2 HPLC-DAD Sandell et al., 2009
juice/phenolic compounds,
anthocyanins
Grape pomace/solubility of Water and ethanol 25◦ and 75◦ C /1 6 min Hansen solubility Srinivas et al., 2009
anthocyanins parameter
Red cabbage/anthocyanins Dionex accelerated solvent 100◦ C 50 bar/1 6 min HPLC/DAD- Arapitsas et al., 2008
ESI/Qtrap
MS
Red cabbage/anthocyanins Water/ethanol/formic acid 99◦ C 50 bar/1 7 min HPLC/DAD Arapitsas and Turner, 2008
(94/5/1, v/v/v)
Grape skin extracts/ CO2 30◦ –40◦ C 100–130 bar HPLC-DAD, MS Bleve et al., 2008
anthocyanins
Black carrot/anthocyanins Water with sulfuric, citric, 50◦ –150◦ C 50 bar 10 min HPLC/UV-vis Gizir et al., 2008
and lactic acids
Red grape skin/anthocyanins Methanol and ethanol 40◦ –120◦ C 15 Mpa/3 5 min HPLC-DAD Hohnova et al., 2008
Red grape marc (variety of CO2 Ambient 10–18 MPa Spectrophotometry Vatai et al., 2008
Refosk)/anthocyanins temperature
Apple and peach CO2 + ethanol (20%) 55.7◦ –58.4◦ C; 54.– 57 MPa; 40 min Spectrophotometry Hasbay Adil et al., 2007
pomaces/polyphenols 50.9◦ –52.3◦ C 50.6–51 MPa
Apple juice/anthocyanins Water, ethanol (25–100%, 40◦ –180◦ C /3 2 min Spectrophotometry, Kammerer et al., 2007
v/v), and methanol HPLC-MS
(25–100%, v/v)
Malvidin 3,5-diglucoside/ Carbon dioxide and Validity of Diffusion Supercritical chro- Mantell et al., 2004
determination of diffusion methanol as cosolvent different coefficient matography/UV
coefficient equations detector
(Continued on the next page)

329
330
TABLE 6
Application of pressurized fluid extraction to the analysis of anthocyanins (Continued)

Extraction
Matrix/compounds Solvent T Pressure/cycles time Technique Reference

Fruitberry Subcritical deionized and 120◦ –160◦ C 4.0 MPa 40 min Spectrophoto-metry, King et al., 2003
substrates/anthocyanins Milli-Q-purified neat HPLC
water; acidified water
Red grape CO2 modified with methanol 560 bar 15 min Mantell et al., 2003a
pomace/anthocyanins
Diffusion coefficient of CO2 -methanol in near 40◦ , 50◦ , and 100, 200, 300, Supercritical chro- Mantell et al., 2003b
malvidin 3,5-diglucoside critical region 60◦ C and 400 bar matography/UV
detector
Dried red grape skin/ Acidified water Acidified 80◦ –100◦ C 60◦ C 10.1 Mpa/ 3 5 min HPLC-DAD Ju and Howard, 2003
anthocyanins and total 60% methanol
phenolics
Berries/anthocyanins Subcritical water with 110◦ –160◦ C 40 bar 40 min HPLC King, 2002
ethanol
Golden Delicious apple peel Hydromethanolic mixtures 20◦ –140◦ C 1000 p.s.i/2 5 min HPLC-DAD Alonso-Salces et al., 2001
and pulp/polyphenols
Anthocyanins and others Pressurized carbon dioxide 70◦ C 400 bar 24 h TLC, UV-vis, NMR, Jay et al., 1991
MS

Selected applications of microwave-assisted extraction (MAE) techniques for anthocyanins
Sample/analyte
Wine lees/anthocyanin:
malvidin-3-glucoside
Grape skins (Tintilla de
Rota)/anthocyanins
(acylderivatives)
Blackcurrant pulp/vitamin C
and anthocyanin content
Two anthocyanin extracts
from purple sweet potato
Purple corn (Zea mays L.)
cob/anthocyanins
Dried cranberry/cyanidin-3-
and peonidin-3
galactosides and
arabinosides
Fresh and dried
raspberries/anthocyanin
content, total antioxidant
capacity, and other
properties
Colorado potato breeding
program/anthocyanins and
other compounds;
antioxidant properties
Red grape
skins/anthocyanins
Red
raspberries/anthocyanins
MAE: microwave-assisted extraction; MAFM: microwave-assisted foam mat drying; VMD: vacuum-microwave drying; MB: microwave baking;
MIVAC: microwave-vacuum; HAD/MIVAC: combination of hot-air drying and microwave-vacuum.
ANTHOCYANIN PIGMENTS. PART IV: EXTRACTION 331
TABLE 7
Comment Result Technique Reference
Ethanol 75%, HCl 1% in Better extraction efficiency HPLC-DAD Pérez-Serradilla
water 1:10 (w/v) ratio. in a much shorter time and de Castro,
MAE 200 W for 17 min than the conventional 2011
method
40% methanol, T = Extraction solvent is the HPLC-MS Liazid et al., 2011
100◦ C, 500 W power, most important variable
and 25 mL and 5 min as for the recovery. Notable
extraction volume and reduction in the extraction
time time from 5 h to 5 min
MAFM: 560 W power, Positive effects up to certain UV-vis Zheng at al., 2011
pulp load of 65 g, level and then a negative
drying time of 8 min, effect on both vitamin C
and pulp thickness of and anthocyanin content
4.46 mm
Microwave baking and Extraction yield by UV-vis Lu et al., 2010
acidified electrolyzed MB-AEW was 35.0%,
water (MB-AEW) much higher than that by
MB-EtOH technology
(14.2%)
MAE: 19 min extraction Cyanidin, pelargonidin, and HPLC-MS Yang and Zhai,
time; solid-liquid ratio peonidin-3-glucosides, 2010
1:20, 555 W and malonated
counterparts
VMD VMD and freezing-drying HPLC Leusink et al., 2010
preserved better
antioxidant activity as
well as anthocyanin
content than hot air-drying
MIVAC, HAD/MIVAC Anthocyanins shown to be RP-HPLC Mejia-Meza et al.,
90 min at 3000 W and a less heat stable than 2010
vacuum pressure of 20 polyphenols
torr (2.6 KPa).
Microwave cooking: Major phenolic LC/MS Stushnoff et al.,
1.0 min/30 g FW at compounds,anthocyanins, 2008
700 W full power and glycoalkaloid content
were investigated
Sample weight-to-solvent Recoveries of MAE (faster), LC-MS/MS Ghassempour et al.,
volume ratio equal to UAE, and maceration 2008
0.06 and T = 50◦ C with methods were 90, 82, and
MeOH 98%, respectively
Ratio of solvents to 43.42 mg of Acys/100 g of HPLC-MS Sun et al., 2007
materials 4:1 (mL/g), fresh fruits expressed as
extraction time 12 min, cyanidin-3-glucoside.
and power 366 W About 98.33% red
pigments
332 M. J. NAVAS ET AL.
Comparisons of extraction efficiency and susceptibility to ma-
trix effects, selectivity, time and solvent reduction, level of au-
tomation, and simplicity of the operating procedures are dis-
cussed in the literature (Sticher, 2008). MAE may also be used
in isolation protocols.
Less polar solvents are used for extraction of flavonoid
aglycones and more polar solvents are used for extraction of
flavonoid glycosides and anthocyanins, as flavonoids vary in
polarity. The conventional extraction of flavanoids often in-
volves the addition of an acid (Routray and Orsat, 2010). Using
microwave extraction can, in most cases, reduce the need for
an acid (Kothari and Seshadri, 2010; Xiao et al., 2008; Gao
et al., 2006). However, in cases where acids are used, such as
for the extraction of anthocyanins (Sun et al., 2007; Yang and
Zhai, 2010), less concentrated acids can be used with microwave
as compared with conventional methods of extraction without
affecting the final yield (Routray and Orsat, 2010).
The optimal conditions for the extraction of anthocyanins in
red raspberries (Sun et al., 2007), purple corn (Yang and Zhai,
2010), red grape skin (Ghassempour et al., 2008), and grapes
(Liazid et al., 2011) with MAE have been devised with the
aid of statistical designs, e.g., central composite rotate design,
Box-Behnken design, to factorial designs at three level, and
fractional factorial experiment, respectively. The applications of
microwave-assisted extraction to the recovery of anthocyanins
are summarized in Table 7.
CONCLUDING REMARKS
Phenolics can be extracted from fresh, frozen (Kuskoski,
2006a, 2006b), or dried plant samples. Usually, before extrac-
tion plant samples are treated by milling, grinding, and homoge-
nization, which may be preceded by air-drying or freeze-drying
(Dai, 2009). Solubility of phenolics is governed by such param-
eters as chemical nature of the plant sample and polarity of the
solvent used. There is no universal extraction procedure suit-
able for extraction of all plant phenolics, as plant materials may
contain different quantities of phenolics with varying natures,
associated, in addition, with other components such as carbo-
hydrates and proteins. A mixture of phenolics, which may also
contain non-phenolic substances such as sugar, organic acids,
and fats, will be extracted from plant materials, depending on
the solvent extraction system used. This means that additional
steps may be required to remove those unwanted components.
In preparing anthocyanin-rich phenolic extracts from plant
materials, the most commonly used solvents are methanol,
ethanol, and acetone, at a composition of 70–80% in water
(Mazza et al., 2004). Acidic (pH of approximately 1.5) condi-
tions are usually preferred for the extraction of anthocyanins
in order to minimize pH effects on the flavylium equilib-
ria. The concentrated extract can be then extracted with hex-
ane, petroleum ether, or diethyl ether to remove unwanted
lipophilic compounds after vacuum evaporation of the sol-
vent (Jackman and Smith, 1992). An advantage of the ace-
tone/chloroform method over the methanol method is that the
aqueous anthocyanin is not contaminated with lipophilic com-
ponents (Rodriguez-Saona and Wrolstad, 2001).
Conventional solvent extraction methods often have limited
applications because of the long extraction times and precau-
tions needed to protect the highly reactive phenol species from
degradation processes (Robards, 2003; Tura and Robards, 2002;
Antolovich et al., 2000). A feature of conventional extraction
is that it influences the integrity of flavonoid glycosides during
prolonged extraction, thus affecting reproducibility (Routray
and Orsat, 2010; Stalikas, 2007). The growing demand for new
extraction techniques amenable to automation with reduced sol-
vent consumption and analysis times has seen increasing use in
the extraction of anthocyanins of SPE, CCC, adsorption, PLE
(ASE), and MAE (Scotter, 2011a, 2011b; Dai and Mumper,
2010).
The applications of many of these developments have been
summarized in tabular form (Tables 2–6); the authors intend
to condense some of the applications of anthocyanin separa-
tion. Optimization experiments were performed using surface
response methodology. The significance of anthocyanin extrac-
tion is not limited to the analytical context, for it has important
practical applications in the food industry (Shi et al., 2005).
Grape peel and seed are good sources of important bioactive
components such as phenolics, anthocyanins, and antioxidants.
Recovery of these components and their proper utilization is im-
portant in the development of functional foods (Ghafoor et al.,
2011). It is not the purpose of this review to go into the theory
of the separation methods, which is adequately covered in other
reviews, but to describe the application of the methods.
The SPE process is usually carried out to remove polar non-
phenolic compounds (sugar, organic acids) from plant crude
extracts. SPE is becoming popular since it is rapid, economical,
and sensitive and because different cartridges with a great variety
of sorbents may be used (Tsao and Deng, 2004). Anthocyanis
are usually isolated in a C-18 semi-preparative cartridge, while
other phenolics are eluted using ethyl acetate. Anthocyanins can
then be recovered for futher analysis using acidified methanol
(Takeoka and Dao, 2008). The extracts can also be purified
using ion exchange resins. Processing of solutions containing
phenolics by adsorption-desorption enables the recovery and
purification of bioactive compounds or fractions from plants
(Soto et al., 2011).
Conventional methods such as low-pressure chromatogra-
phy and preparative reversed phase chromatography are used to
fractionate or isolate pure products from plants. However, both
are tedious and time and solvent consuming and require mul-
tiple chromatographic steps (Di et al., 2011). Countercurrent
chromatography (CCC) can be an excellent alternative. The big
advantage of CCC is that it uses no solid matrix and the role
of the liquid phases, namely, liquid stationary phase and mobile
phase, can be switched during a run. Thus, there is no irreversible
sample adsorption and recovery is 100% (Dai, 2009).
Dissolving power is dependent on the density of the fluid,
and by changing pressure and temperature this property can be
 ANTHOCYANIN PIGMENTS. PART IV: EXTRACTION
controlled (Kivilompolo, 2009). The unique properties of super-
critical fluids, liquid-like (high density and dissolving power)
and gas-like (low viscosity, low surface tension, and high diffu-
sion rate), make the extraction faster than conventional extrac-
tion processes (Brunner, 2010; Sekhon, 2010). Carbon dioxide
is nonpolar, but with small additions of a more polar modifier, it
can also be used as an extraction fluid for polar compounds, like
anthocyanins (Bravi et al., 2012). Supercritical carbon dioxide
(SC-CO2 ) methods are ideal for the extraction of natural prod-
ucts from plant materials; they are especially recommended for
the extraction of thermolabile compounds when low tempera-
tures are required (Ignat et al., 2011; Maróstica Junior et al.,
2010; Wang and Weller, 2006). Industrial applications of SFE
preceded analytical use of this technique.
Pressurized liquid extraction (PLE) is a solid-liquid extrac-
tion process using organic solvents at an elevated temperature
and applying higher pressure to maintain the solvent in its liq-
uid state, even at temperatures above its boiling point (Wang
and Weller, 2006). In addition, pressure helps to force the sol-
vent into the matrix pores, improving in this way the efficiency
of the extraction process (Ramos et al., 2002). Extraction at
elevated temperatures increases solubility, diffusion rate, and
mass transfer, coupled with the ability of the solvent to disrupt
the analyte-matrix interactions. PLE thus allows fast extraction
owing to increased solubility, better desorption, and enhanced
diffusion, and the extraction is generally completed within a
few minutes. The extraction of value-added products in food
and nutraceutical industries using pressurized liquids involves
a careful selection of solvent and optimal temperature condi-
tions to achieve maximum yield (Srinivas et al., 2009). PLE is
a green technique that can enhance extraction rates of bioactive
compounds (Wijngaard and Brunton, 2009).
Water, an inexpensive and environmentally friendly solvent,
is an ideal solvent for industrial extraction of phenolics, but
its use is limited due to poor extraction efficiency at low tem-
peratures (Ju and Howard, 2003). At room temperature wa-
ter has a high boiling point, dielectric constant, and polarity,
but when the temperature is increased, the properties of wa-
ter change significantly. At higher temperatures, the diffusion
rate increases and the dielectric constant, viscosity, and surface
tension decrease. The permittivity of heated water resembles
that of an organic solvent, e.g., the dielectric constants of water
and methanol are 30 (at 220◦ C) and 33 (at ambient tempera-
ture), respectively. This leads water to behave like an organic
solvent, dissolving a wide range of medium- and low-polarity
compounds (Dai and Mumper, 2010; Teo et al., 2010). Pres-
surized hot water extraction above and below the boiling point
of water has been demonstrated to be an affective analytical
technique (King, 2006) to recovery anthocyanins from various
sources (Arapitsas and Turner, 2008).
Microwave-assisted extraction (MAE) is a relatively new ex-
traction technique that combines microwave and the use of tra-
ditional solvent (Delazar et al., 2012; Santana et al., 2009). Ex-
traction using microwave energy is largely an unexplored area
333
(Ignat et al., 2011), although by using microwaves to mediate
the extraction, it is possible to maintain mild conditions and
achieve superior extraction (Routray and Orsat, 2010). For ther-
molabile compounds, reduced extraction times should minimize
analyte degradation, although the effects of temperature are not
always intuitive.
Although many traditional sample-preparation methods for
flavonoids are still in use (Stalikas, 2007; Andersen and
Markham, 2006), there are trends in recent years towards: (1)
use of smaller initial sample sizes, small volumes, or no or-
ganic solvents; (2) greater specificity or greater selectivity in
extraction; (3) higher recoveries or better reproducibility; and
(4) increased potential of automation (Liu et al., 2008), as shown
in this review. The use of non-thermal technologies in the as-
sisted extraction of anthocyanins will be covered in a separate
report.
ACKNOWLEDGMENTS
This work was supported by the Junta de Andalucia (Spain)
through Grant Excellence Research Project P06-FQM-02029,
for which the authors are grateful.
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