Food Research International 38 (2005) 11751188

www.elsevier.com/locate/foodres

Review

A review of the analytical methods to determine organic acids in

grape juices and wines
Ines Mato, Silvia Suarez-Luque, Jose F. Huidobro

Facultad de Farmacia, Departamento de Qumica Analtica, Nutricion y Bromatologa, Area de Nutricion y Bromatologa,
Universidad de Santiago, 15782 Santiago De Compostela (Galicia), Spain
Received 20 September 2004; accepted 11 April 2005

Abstract
The determination of low molecular weight organic acids in grape juices and wines is important because of their inuence in organoleptic properties (avour, colour and aroma) and in the stability and microbiologic control of these beverages. The analysis of
these acids allows to check the process of maturation of grapes and to control the evolution of the acidity of wines during the several
stages of their elaboration process (alcoholic fermentation, malolactic fermentation, aging process, etc).
In this work, a comprehensive review of the analytical methods developed for the determination of short-chain organic acids in
grape juices and wines has been shown. This review included non-enzymatic spectrophotometric, enzymatic, chromatographic and
electrophoretic methods. Details of the chromatographic and electrophoretic conditions of these methods have been collected in
tables.

2005 Elsevier Ltd. All rights reserved.
Keywords: Grape juice; Wine; Organic acids; Analytical methods; Review

Contents
1.
2.
3.
4.

5.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Spectrophotometric methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Enzymatic methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Chromatographic methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.1. Thin layer chromatography (TLC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.2. Gas chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3. Liquid chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3.1. High performance liquid chromatography. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
4.3.2. Ion chromatography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Electrophoretic methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Corresponding author. Tel.: +34 981 594626; fax: +34 981 594912.
E-mail address: qnhuidob@usc.es (J.F. Huidobro).

0963-9969/$ - see front matter
2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodres.2005.04.007

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I. Mato et al. / Food Research International 38 (2005) 11751188

1. Introduction
Low molecular weight organic acids are an important
group of compounds in grape juices and wines since
their inuence in some respects like organoleptic properties (avour, colour, and aroma) or in the stability and
microbiologic control of these beverages. These acids
come directly from the grape and/or from the processes
which are subjected, as alcoholic fermentation, malolactic fermentation, oxidation of the ethanol, etc.
(Peynaud, 1999).
In grape juice, tartaric and malic acids are the predominant organic acids and succinic and citric acids
are present in minor proportion. In the case of wine, a
common dierentiation is made between acids which
come directly from the grape (tartaric, malic and citric
acids) and those that are originated, fundamentally, in
the fermentation process (succinic, lactic and acetic
acids). Also, there are small amounts of other acids like
galacturonic, glucuronic, citramalic, dimethylglyceric,
pyruvic, ketoglutaric, etc. (Berlitz & Grosch, 1992;
Peynaud, 1999).
The evolution of tartaric and malic acids in grapes
are useful for checking their processes of maturation
(Lamikanra, Inyang, & Leong, 1995). In the case of
wines, the analysis of organic acids allows to control
the evolution of the acidity during the dierent steps
of the winemaking process (alcoholic fermentation,
malolactic fermentation, aging process, etc). The level
of tartaric acid is a parameter of critical control in the
stabilization of wine. These organic acids also have great
importance in the detection of wine alterations and/or
illnesses, because they suppose a modication of acids
content. For example, some wine alterations are related
to increase in the levels of acetic and lactic acids (acetic
or lactic sharpness, respectively).
For all these reasons, several methods have been
developed for identifying and quantifying these organic
acids in grape juices and wines, so much individually
(non-enzymatic spectrophotometric and enzymatic
methods) or as a group of them simultaneously (chromatographic and electrophoretic methods).
The aim of this work has been to carry out a revision
of methods for the determination of the more thoroughly
organic acids in grape juices and wines and to make easy
the approach for solving one specic problem.

2. Spectrophotometric methods
Spectrophotometric methods are based on the reaction of the organic acid with a certain substance, giving
rise to the formation of a compound or coloured
complex that is measured at a certain wavelength. In order to avoid interferences, organic acids are isolated by
precipitation, ionic exchange resins, etc.

Rebelein (1961) determined tartaric, malic and lactic

acids with a spectrophotometric method. These organic
acids were separated by means of a strongly basic ion
exchange resin. The portions of the eluent underwent
to several treatments in order to form the coloured compounds that were measured at several wavelengths (490
nm for tartaric acid, 420 nm for malic acid and 530 or
570 nm in the case of lactic acid).
Other methods for the determination of tartaric acid
have been developed having the grounds of the
described method by Rebelein (1961). Basically, these
methods dier in the way of claring the samples in
order to avoid the interferences owed to the colour of
wine and, also, in the automation of the process
(Vereda, Garca de, Torres, Rivero, & Cano, 1998).
These methods are tedious and there are no data
about the determination of important acids as succinic,
citric or acetic acids.

3. Enzymatic methods
Enzymatic methods have been used, mainly, for the
quantication of malic, lactic and citric acids in grape
juices and wines. However, it would be possible the
determination of other acids like tartaric, acetic, L-ascorbic, dehydroascorbic/L-ascorbic, formic, D-gluconic/Dglucono-d-lactone, D-isocitric, oxalic and succinic acids
(Bergmeyer, 1985; Boehringer Mannheim GmbH,
1995). In the case of wines, the method for the determination of citric acid is a Final Action method in the
AOAC (Association of Ocial Analytical Chemists,
2000).
These methods are based on the measurement of the
increase or decrease in absorbance of the coenzymes
NADH (nicotinamideadenine dinucleotide, reduced
form) or NADPH (nicotinamideadenine dinucleotide
phosphate, reduced form) which absorb in the distance
wavelength region (Boehringer Mannheim GmbH,
1995). The measurement was usually made with a spectrophotometer at 340 nm.
The main advantage of these methods is the high
specicity because the determination of L- and D-isomers
of some acids is possible and easy when enzymatic methods were used. Nevertheless, only one organic acid is
determined in each assay, so this is the main disadvantage of these methods together time-consuming.
A way of these methods automatization, in order to
reduce their cost and analysis time, consists on the use
of ow injection analysis (FIA). Puchades, Herrero,
Maquieira, and Atienza (1991) determined L-malic and
L-lactic acids simultaneously in wine samples by FIA
with tubular open reactors with enzymes immobilized
in them. Lima and Rangel (1992) also determined these
two acids in wine by FIA with spectrophotometric
detection. In this case, the ow injection system had

I. Mato et al. / Food Research International 38 (2005) 11751188

an unit of dialysis in order to adjust the composition of

the solutions injected to the requirements of the system
of analysis.
Enzymatic methods have sometimes been used as reference methods in order to validate chromatographic
methods, fundamentally, high performance liquid chromatography (HPLC) and ion chromatography (IC)
(Frayne, 1986; Kupina, Pohl, & Gannotti, 1991; Polo,
Barahona, & Caceres, 1986) and also for the validation
of capillary electrophoresis methods (Kandl & Kupina,
1999).

1177

derivatization procedure, by gas chromatography. Yang

and Choong (2001) determined 13 volatile short chain
organic acids in 37 liquid food samples (some of them
were grape juices) obtaining good limits of detection.
The detectors more widely used were ame ionization
detector (FID) (Barden et al., 1997; Yang & Choong,
2001) and mass spectrometry (MS) (Barden et al., 1997).
In conclusion, the choice of this technique for the
determination of these compounds is very limited for
its cost and its complexity. Other alternatives like liquid
chromatography or capillary electrophoresis are more
suitable for the determination of organic acids in grape
juices and wines.

4. Chromatographic methods
4.3. Liquid chromatography
4.1. Thin layer chromatography (TLC)
Bourzeix, Guitraud, and Champagnol (1970) identied and separated tartaric, malic, lactic, succinic, citric
and citramalic acids in grape juices and wines by TLC.
These acids quantication in the samples was carried
out with photodensitometry of each one of the points
of the plate. Ryan and Dupont (1973) identied the
majority acids of grape juices and wines by TLC and
gas chromatography (GC). These acids quantication
were carried out by a gas chromatography method.
4.2. Gas chromatography
In spite of its high sensitivity and selectivity, GC has
been scarcely used for the determination of organic
acids. The majority of short-chain organic acids are
not volatile, so, it is necessary to carry out a derivatization procedure like the formation of trimethylsilyl
derivates (TMS) (Bertrand, 1974; Fantozzi & Bergeret,
1973; Ryan & Dupont, 1973; Tanner & Zanier, 1976),
methylic esters (Barden, Croft, Murby, & Wells,
1997;Bertrand, 1974), tert-butyldimethylsilyl derivates
(TBDMS) (Kim, Kim, & Park, 1990) or ethylic esters
(Deng, 1997).
The organic acids isolation before the derivatization
procedure is sometimes necessary, due to the complex
composition of grape juices and wines. This isolation
was usually carried out by lead salts precipitation (Ryan
& Dupont, 1973), ion exchange resins (Bertrand, 1974)
or solid phase extraction cartridges (Kim et al., 1990).
With all these steps, the sample preparation time increased considerably and these GC methods became
tedious.
In order to reduce the time of the sample treatment,
Deng (1997) unied the steps of separation and derivatization carrying out an esterication directly in the
ion exchange resin where organic acids were isolated
in 60 min at a temperature of 90 C.
Nowadays, some organic acids like acetic, lactic or
malic acids, could be determined directly, without a

Liquid chromatography is the technique most thoroughly used for the determination of organic acids in
grape juices and wines (Vereda et al., 1998). Separation
and quantication of organic acids have been usually
carried out by HPLC or IC. We are going to discuss rst
HPLC methods and then, IC methods.
4.3.1. High performance liquid chromatography
There are many HPLC methods to determine organic
acids in grape juices and wines which are summarized in
Table 1.
Regarding the previous treatment of the samples, there
is a great dierence among works. The sample pre-treatment procedures could be divided into two groups. In
the rst group, samples are only diluted and ltrated
and, in the second group, a more complex treatment
was applied to the samples. The majority of studies for
the determination of organic acids in grape juices and
wines carried out a previous treatment to avoid interferences from sugars or colourants which could co-elute
with organic acids. The more common previous treatments are an extraction with ion exchange resins or with
solid phase extraction cartridges and a derivatization
procedure (Table 1). Castellari, Versari, Spinabelli, Galassi, and Amati (2000) compared the direct injection
and the solid phase extraction procedure with a SAX
cartridge to remove organic acids from grape juices
and wines. They concluded that the direct injection with
previous dilution and ltration showed better precision
and recovery results than the solid phase extraction
procedure. Verette, Qian, and Mangani (1995) and
Linget, Netter, Heems, and Verette (1998) developed a
completely automatic system of sample preparation with
an on-line dialysis procedure before HPLC analysis that
removed macromolecular and microparticle interferences resulting from complex matrices. This system
was applied to grape juices and wines with good results
in repeatability and sensitivity. Regarding methods that
carried out a derivatization procedure they usually
determined organic acids in grape juices and wines as

1178

Matrix

Organic acidsa

Sample preparation

Separation
mechanism

Columns

Detection

Analysis
time
(min)

Reference

Grape juice

acet., asc., citram., cit.,

fum., glucuron.,
mal., shyk., succ., tart.
cit., mal., tart.
acet., cit., fum., lact.,
mal., oxal., succ., tart.
acet., cit., lact., mal.,
succ., tart.
acet., cit., lact., tart.
lact., mal.
acet., lact., mal.,
tann., tart.
acet., cit., lact., succ.,
tart.
cit., lact., mal., oxal.,
tart.
acet., cit., fum., galact.,
lact., mal., shyk., succ.,
tart.
lact., mal., male., malon.,
succ., tart.

Dilution + ltration

Reversed-phase

UV spectrophotometry

40

Llorente et al. (1991)

Filtration + precipitation
Ion exchange resins

Ion exclusion
Ion exchange

1
1

UV spectrophotometry
UV spectrophotometry

11
18

Soyer et al. (2003)

Hunter et al. (1991)

Filtration

Ion exchange

25

Vonach et al. (1998)

Filtration
Dilution + ltration
Dilution + ltration

Ion exchange
Reversed-phase
Reversed-phase

1
1
1

Real-time Fouriertransform IR
Diamond ATR-FTIR
UV spectrophotometry
UV spectrophotometry

20
10
20

Edelmann et al. (2003)

Buglass and Lee (2001)
Radin et al. (1994)

Dilution + ltration

Reversed-phase

Conductimetry

16

Gennaro and Bertolo (1989)

Dilution + ltration

Reversed-phase

30

Perez-Ruz et al. (2004)

Dilution + ltration

Reversed-phase

2 in series

Photochemically induced
chemiluminescent
UV spectrophotometry

25

Pazo et al. (1999)

Dilution + ltration

UV spectrophotometry

20

Jun et al. (1996)

Dilution + ltration

Reversed phase
(coated with
n-cetylpyridinium
chloride)
Ion exclusion

Electrochemical

15

Casella and Gatta (2002)

Solid phase extraction

Reversed-phase

UV spectrophotometry

Marce et al. (1990)

Solid phase extraction

Reversed-phase

UV spectrophotometry

26

Marce et al. (1991)

Solid phase extraction

Solid phase extraction

Reversed-phase
Ion exchange

1
1

UV spectrophotometry
Conductimetry

4
13

Olalla-Herrera et al. (1993)

Mongay et al. (1996)

Grape juice
Grape juice
Wine
Wine
Wine
Wine
Wine
Wine
Wine

Wine

Wine
Wine
Wine
Wine
Wine

acet., cit., form., lact.,

mal., tart.
acet., cit., fum., lact.,
mal., shyk., succ., tart.
acet., cit., lact., mal.,
succ., tart.
lact., mal., tart.
acet., cit., lact., mal.,
succ., tart.

I. Mato et al. / Food Research International 38 (2005) 11751188

Table 1
Studies of the determination of the organic acids in grape juices and wines by high performance liquid chromatography (HPLC)

Wine
Wine
Wine
Grape juice, Wine
Grape juice, Wine
Grape juice, Wine
Grape juice, Wine
Grape juice, Wine
Grape juice, Wine

Grape juice, Wine

Grape juice, Wine

Grape juice, Wine

Grape juice, Wine

Grape juice, Wine

Grape juice, Wine
Grape juice, Wine
a

Organic acid abbreviation. Full name in Table 3.

Ion exchange resins

Reversed-phase

UV spectrophotometry

12

Schneider et al. (1987)

Ion exchange resins

Reversed-phase

3 in series

UV spectrophotometry

40

Goion et al. (1985)

Derivatization with
phenacyl esters
Filtration
Filtration

Reversed-phase

2 in series

UV spectrophotometry

17

Caccamo et al. (1986)

Reversed-phase
Ion exclusion

1
1

UV spectrophotometry
Electrochemical

7
20

Escobal et al. (1997)

Kotani et al. (2004)

Dilution + ltration
Dilution + ltration
Dilution + ltration

Reversed-phase
Reversed-phase
Ion exclusion

1
1
1

UV spectrophotometry
UV spectrophotometry
Refractive index

12
4
15

Billingsley et al. (1996)

Kerem et al. (2004)
Lopez-Tamames et al. (1996)

Dilution + ltration

Ion exchange

30

Dilution + ltration

Ion exclusion

20

Falque-Lopez and
Fernandez-Gomez (1996)
Chinnici et al. (2002)

Solid phase extraction

Reversed-phase

2 in series

UV and RI connected
in series
UV and RI connected
in series
UV spectrophotometry

20

Tusseau and Benoit (1987)

Solid phase extraction

Reversed-phase

2 in series

UV spectrophotometry

32

Garca-Romero et al. (1993)

Solid phase extraction

Ion exchange

Refractive index

25

Calull et al. (1992).

Dilution + ltration/solid
phase extraction

Ion exchange

UV and RI connected
in series

20

Castellari et al. (2000)

Derivatization with
p-nitrobenzyl esters
On-line dialysis

Ion exchange

UV spectrophotometry

30

Cunha et al. (2002)

Ion exchange

Refractive index

25

Verette et al. (1995)

On-line dialysis

Ion exchange

Refractive index

35

Linget et al. (1998)

I. Mato et al. / Food Research International 38 (2005) 11751188

Grape juice, Wine

acet., cit., fum., lact.,

mal., succ., tart.
acet., citram., cit., lact.,
mal., shyk., succ., tart.
acet., cit., lact., mal.,
succ., tart.
acet., cit., mal., tart.
acet., cit., lact., mal.,
succ., tart.
mal., tart.
cit., tart., mal., lact., acet.
cit., galact., lact., mal.,
tart., succ.
acet., cit., lact., mal.,
succ., tart.
acet., cit., fum., lact.,
mal., shyk., succ., tart.
acet., cit., lact., fum.,
mal., prop., shyk.,
succ., tart.
acet., citram., cit.,
fum., lact., mal.,
shyk., succ., tart.
acet., cit., lact.,
mal., succ., tart.
acet., asc., ketoglut.,
cit., fum.,
lact., mal., pyr.,
shyk., succ., tart.
acet., cit., lact.,
mal., succ., tart.
acet., cit., lact.,
mal., succ., tart.
acet., cit., lact.,
mal., succ., tart.

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I. Mato et al. / Food Research International 38 (2005) 11751188

p-nitrobenzyl esters (Cunha, Fernandes, Faria, Ferreira,

& Ferreira, 2002). Caccamo, Carfagnini, Di Corcia, and
Samperi (1986) reported a method for the determination
of ve organic acids as phenacyl esters. The sample was
buered, mixed with a solution of phenacyl bromide and
crown ether in acetone and heated for 40 min. This
method is accurate, with recoveries higher than 95%,
precise and the resolution of chromatograms obtained
is good. But, in general, organic acids extraction and
isolation procedures are tedious, expensive and timeconsuming.
On the other hand, methods with dierent separation
mechanisms have been developed, for instance, reversed
phase HPLC, ion exchange HPLC or ion exclusion
HPLC (Table 1). Reversed phase is shown as the separation mechanism most widely used. Llorente, Villarroya,
and Gomez-Cordoves (1991) determined organic acids
in grape juices by reversed phase HPLC with UV detection and only a dilution and ltration. It is a simple
method but the time of analysis is long (40 min).
Lopez-Tamames, Puig-Deu, Teixeira, and Buxaderas
(1996) and Casella and Gatta (2002) developed ion
exclusion HPLC methods with refractive index and electrochemical detection, respectively, which allow the direct determination of majority organic acids in grape
juices and wines. Although the sample treatment of both
methods is very simple, chromatograms have worse resolution than other methods, as we can see in Fig. 1.
Falque-Lopez and Fernandez-Gomez (1996) developed
an ion exchange HPLC method with two detectors con-

Fig. 1. Chromatogram of a red wine (Aglianico, 2000) diluted 1:900

with distilled water: (1) citric acid; (2) tartaric acid; (3) malic acid;
(4) lactic acid; (5) acetic acid; (6) ethanol. Reprinted with permission
from Casella and Gatta (2002). Copyright (2002) American Chemical
Society.

nected in series (UV and RI) for the direct determination

of organic acids in grape juices and white wines. Subsequently, Castellari et al. (2000) also used an ion exchange HPLC method with two detectors connected in
series (UV and RI). In this method direct injection allowed decreasing the time and the cost of analysis and
organic acids, sugars and alcohols were determined
simultaneously with an acceptable resolution. Tusseau
and Benoit (1986) compared the analysis of organic
acids in grape juices and wines by HPLC using reversed
phase and ion exchange columns. They concluded that
reversed phase is recommended for the determination
of tartaric, malic and lactic acids but better results were
obtained with ion exchange columns for citric and acetic
acids. Ion exchange as separation mechanism implies
that organic acids should be in their ionic form, so a
severe control of pH is required. Ding, Koizumi, and
Suzuki (1995) compared the three chromatographic
systems (ion exchange, ion exclusion and reversed
phase) and they concluded that narrow peaks were obtained with ion exclusion methods but reversed phase
methods carried out the fastest analysis. The advantages
of ion exchange methods are the lack of interferences
and a simpler sample treatment. Pazo, Traveso, Cisneros, and Montero (1999) used a reversed phase HPLC
method with two columns connected in series and UV
detection. A lot of wine samples have been analyzed
with this method by injecting samples directly without
a previous treatment. The use of two columns connected
in series had been already described by Tusseau and
Benoit (1987) and Garca-Romero, Sanchez-Munoz,
lvarez, and Cabezudo-Ibanez (1993) but in
Martn-A
these cases a previous solid phase extraction procedure
was carried out to remove organic acids. Two columns
connected in series improve the resolution but the
analysis time gets longer, as we can see in Fig. 2, which
shows a chromatogram of a method developed by GarcaRomero et al. (1993). Jun, Lima, and Montenegro
(1996) developed a HPLC method with UV detection
and a reversed phase column which was permanent
coated with n-cetylpyridinium chloride. The wine previous treatment was only dilution and ltration.
Finally, several types of detectors have also been used
like UV spectrophotometry, the most used, refractive index detector (RI), conductivity detector, electrochemical
detector, or photochemically induced chemiluminescence detector (Table 1). Vonach, Lendl, and Kellmer
(1998) developed a method using a coupling of HPLC
with Fourier-transform infrared spectroscopy. This type
of detection provides the possibility of identifying substances that cannot be determined by UVVis methods,
but the detection limits (0.2 g/l) made the technique not
much sensitive for practical applications. Edelmann,
Diewok, Baena, and Lendl (2003) incorporated a horizontal diamond attenuated total reection (ATR) in a
ow-through cell with low dead volume and they used

I. Mato et al. / Food Research International 38 (2005) 11751188

1181

that allows the separation of organic anions on Dionex

OMNI PAC PAX-500 column by NaOH gradient elution and conductometric detection. They analyzed more
than 500 samples of fruit juices. Finally, Masson (2000)
used a Dionex As11 column, a sodium hydroxide gradient elution and a suppressed conductivity detection to
determine organic acids and inorganic anions in grape
musts. He studied the inuence of three dierent solvents (methanol, ethanol and acetonitrile) on the eciency of column. The best separation was achieved
with an eluent containing 13% (v/v) methanol and
13% (v/v) ethanol in water. Sample preparation was a
simple 20-fold dilution and a ltration. This kind of
chromatography is a real option for the determination
of samples with very low quantity of organic acids and
for routine analysis.

5. Electrophoretic methods

Fig. 2. Chromatogram of a red wine submitted to malolactic fermentation. Isocratic elution using two 3-lm ODS-2 columns (250 4.6 mm
ID). Mobile phase, 0.02 M diammonium hydrogenphosphatephosphoric acid (adjusted to pH 2.35) containing 3% of methanol as polar
modier; ow-rate, 0.5 ml/min. Peaks of acid: 1, galacturonic; 2,
gluconic; 3, tartaric; 4, unknown 1; 5, malic; 6, shikimic; 7, lactic; 8,
acetic; 9, unknown 2; 10, citric; 11, succinic; 12, citramalic; 13, fumaric.
Reprinted from Garca-Romero et al. (1993), with permission from
Elsevier.

for online mid-RI detection. Although it is a new system

of detection, the method showed is not better than the
others with regards to the time of analysis or the number
of acids determined. It is also, not much sensitive, because of the detection limits are about 0.2 g/l. Kotani,
Muyaguchi, Tomita, Takamura, and Kusu (2004) made
a HPLC system with electrochemical detection for determining organic acids during wine brewing by means of
voltammetric reduction of quinone.
4.3.2. Ion chromatography
Ion chromatography with suppressed conductivity
detection allows the separation and quantication of organic acids in samples as grape juices and wines. This
technique has advantages like its specicity and sensibility in the determination of organic acids due to the use
of, for instance, a conductivity detector and the minimization of the sugar interferences. In these methods the
complex previous sample treatments such as extraction
and/or derivatization were not necessary. This system
was evaluated by Kupina et al. (1991) for the separation
and quantication of major organic acids in grape juice
and wine without prior sample clean-up by using a Dionex OmniPac PAX-500 column. Saccani et al. (1995)
also developed a gradient ion chromatographic method

In recent years, liquid chromatography has widely replaced by capillary electrophoresis for the determination
of organic acids in very dierent matrix as clinical samples (Barbas et al., 1998; Garca & Barbas, 2003; Garca,
Barbas, Aguilar, & Castro, 1998), environmental samples (Ahumada, Mendoza, Escudero, Mossert, & Ascar,
2001; Li, Huang, & Shan, 2003; Naidu & Chen, 2001;
Wang, Qu, Shan, & Lin, 2003; Xu, Chen, Yu, & Tang,
2001) or food samples (Buiarelli, Cartoni, Coccioli, &
Jasionowska, 2003; Cortacero-Ramrez, Segura-Carretero, Hernaiz-Bermudez de Castro, & Fernandez-Gutierrez, 2005; Galli & Barbas, 2004; Izco, Tormo, &
Jimenez-Flores, 2002a, Izco, Tormo, & Jimenez-Flores,
2002b; Klamp, 1999; Rosello, Galiana-Balaguer,
Herrero-Martnez, Maquieira, & Nuez, 2002; Saavedra,
Garca, & Barbas, 2000; Saavedra, Ruperez, & Barbas,
2001; Xiao-Yun, Jian-De, & Yao-Zu, 1997). This great
development is because capillary electrophoresis is a
technique with a lot of advantages like high resolution,
simplicity and automation, short analysis times, low
consumption of reagents and samples, and minimum
preparation of sample even in complex matrices.
In regard to grape juices and wines, multiple methods
for determination of organic acids by capillary zone
electrophoresis (CZE) have appeared in a few years.
They are summarized in Table 2.
The simplicity of the previous treatment of the samples
is one of the advantages of CE because this technique
has the ability to separate small molecules in complexes
matrices with simple sample pre-treatment. Therefore,
only dilution and ltration were required in most of
the developed methods. Only Levi, Wehr, Talmadge,
and Zhu (1993) described a dierent sample treatment
which consists of claried wine by centrifugation and solid phase extraction with a C18 cartridge to remove some
compounds as the anthocyanins. Nevertheless, there are

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I. Mato et al. / Food Research International 38 (2005) 11751188

Table 2
Studies of the determination of the organic acids in grape juices and wines by capillary zone electrophoresis (CZE)
Matrix

Organic acidsa

Electrophoretic conditions

Grape juice

mal., tart., cit.

Wine

tart., mal., cit., succ.,

acet., lact.

Wine

tart., mal.

Wine

tart., mal., lact.

Wine

mal., lact., acet., succ.,

cit.

Wine

tart., mal., cit., succ.,

pyr., acet., lact.

Wine

tart., mal., succ, adip.,

glut., acet., lact., shyk.

Wine

tart., mal., succ., acet.,

lact.

180 mM phosphate; 1 mM CTAB; 15 % (v/v)

methanol (pH 7.2)
Fused silica capillary, 50 cm 50 lm ID;
V = 15 kV
Inj.: Hydrodynamic pressure (15 psi s)
Det.: Direct UV (200 nm)
7 mM MES/His; 0.5 mM TTAB; 30 %
methanol (pH 6)
Fused silica capillary, 67 cm 75 lm ID;
V = 25 kV
Inj.: Hydrodynamic siphoning (1020 s)
Det.: Conductivity
5 mM phthalate; 0.5 mM TTAB; 50 mM
MES (pH 5.2)
Fused silica capillary, 70 cm 75 lm ID;
V = 30 kV; Ta = 20 C
Inj.: Electrokinetic ( 10 kV, 1 s)
Det.: Indirect UV (205 nm)
5 mM phthalate; 0.5 mM EOF modier
(pH 5.6)
Fused silica capillary, 100 cm 75 lm ID;
V = 20 kV; Ta = 20 C
Inj.: Electrokinetic ( 10 kV, 5 s)
Det.: Indirect UV (254 nm)
5 mM chromate; 0.154 % (p/v) PDDPi
chromate (pH 8)
Fused silica capillary, 60 cm 77 lm ID;
V = 30 kV
Inj.: Hydrodynamic siphoning (15 s)
Det.: Indirect UV (254 nm)
5 mM PDC; 0.5 mM CTAB (pH 5.6)
Fused silica capillary, 80.5 cm 75 lm ID;
V = 25 kV; Ta = 20 C
Inj.: Hydrodynamic pressure (50 mbar, 2 s)
Det.: Indirect UV (200 nm)
7.5 mM PAB; 10.5 mM BIS-Tris; 0.1 mM
TTAB (pH 7.0)
Fused silica capillaries, 48 cm (UV) and 60
cm (Cond.) 50 lm ID; V = 30 kV
Inj.: Hydrodynamic pressure (25 mbar, 0.2 s)
Det.: Conductivity and Indirect UV (254 nm)
3 mM phosphate; 0.5 mM MTAB (pH 6.5)

Wine

tart., mal., succ., cit.,

acet., lact.

Wine

acet., cit., fum., lact.,

mal., oxal., succ., tart.

Wine

tart., mal., succ., cit.,

acet., lact.

Fused silica capillary, 60 cm 75 lm ID;

V = 20 kV
Inj.: Hydrodynamic siphoning (30 s)
Det.: Direct UV (185 nm)
3 mM BTA; 15 mM Tris; 1.5 mM TEPA
(pH 8.4)
Fused silica capillary, 65 cm 50 lm ID;
V = 25 kV
Inj.: Hydrodynamic siphoning (20 s)
Det.: Indirect UV (240 nm)
200 mM phosphate buer (pH 7.50)
Polyacrylamide coated capillary, 50 cm 50
lm ID;
V = 14 kV; T a = 20 C
Inj.: Hydrodynamic pressure (0.035 mbar,
20 s)
Det.: Direct UV (200 nm)
7.5 mM PDC; 0.5 mM CTAB; 0.5 mM
EDTA (pH 5.6)

Analysis time
(min)

Reference

Vorarat et al. (2002)

12

Huang et al. (1989)

20

Kelly and Nelson (1993)

Levi et al. (1993)

Stathakis and Cassidy

(1995)

Soga (1996)

7 (Cond.),
8 (UV)

Klamp et al. (1998)

Castineira et al. (2000,

2002)

Sing Fung and Man Lau

(2003)

13

Saavedra and Barbas

(2003)

18

De Villiers et al. (2003)

I. Mato et al. / Food Research International 38 (2005) 11751188

1183

Table 2 (continued)
Matrix

Organic acidsa

Wine

tart., mal., succ., acet.,

lact.

Grape juice, Wine

cit., tart., mal., succ.,

acet., lact.

Grape juice, Wine

tart., mal., succ., cit.,

acet., lact.

Grape juice, Wine

tart., mal., cit., succ.,

acet., lact.

Grape juice, Wine

succ., mal., tart., acet.

Grape juice, Wine

tart., mal., succ., acet.,

cit., lact.

Electrophoretic conditions

Analysis time
(min)

Fused silica capillary, 110 cm 75 lm ID

V = ramp from 0 to 22 kV in 0.5 min;
Ta = 15 C
Inj.: Electrokinetic ( 10 kV, 2 s)
Det.: Indirect UV (210 nm)
5 mM PDC; 0.5 mM CTAB (pH 5.6)
Fused silica capillary, 78 cm 75 lm ID;
V = 25 kV; Ta = 18 C
Inj.: Hydrodynamic pressure (0.3 psi, 2 s)
Det.: Indirect UV (200 nm)
5 mM phthalate; 0.5 mM OFM-Anion BT
(pH 7)
Fused silica capillary, 100 cm 75 lm ID;
V = 20 kV
Inj.: Hydrodynamic siphoning (45 s)
Det.: Indirect UV (254 nm)
3 mM PMA; 3 mM EDTA (pH 7.5)
Fused silica capillary, 44 cm 75 lm ID;
V = 20 kV; Ta = 30 C
Inj.: Hydrodynamic pressure (1.5 psi, 2 s)
Det.: Indirect UV (220 nm)
5 mM PDC; 0.5 mM CTAB (pH 5.6)
Fused silica capillary, 80.5 cm 75 lm ID
V = 25 kV; Ta = 18 C
Inj.: Hydrodynamic pressure (50 mbar, 2 s)
Det.: Indirect UV (200 nm)
10 mM tetraborate; 0.5 mM TTAOH;
10 ppm Ca2+ and Mg2+ (pH 9.3)
Fused silica capillary, 60 cm 75 lm ID;
V = 7 kV; Ta = 20 C
Inj.: Hydrodynamic siphoning (30 s)
Det.: Direct UV (185 nm)
7.5 mM NaH2 PO4 ; 2.5 mM Na2 HPO4 ;
2.5 mM OFM-OH; 0.24 mM CaCl2 (pH 6.40)
Fused silica capillary, 60 cm 75 lm ID;
V = 25 kV; Ta = 25 C
Inj.: Hydrodynamic siphoning (30 s)
Det.: Direct UV (185 nm)

7.5

Reference

Esteves et al. (2004)

15

Kenney (1991)

12

Arellano et al. (1997) and

Arellano et al. (1997)

7.5

20

3.5

Kandl and Kupina (1999)

Garca-Moreno et al. (2001)

and Moreno et al. (2003)

Mato et al. (submitted)

Organic acid abbreviation. Full name in Table 3.

no dierences between separation obtained by this

method and separations obtained by methods with a
simple sample treatment (dilution and ltration).
Regarding the sample injection mode, two types of
injection were used for the determination of organic
acids in grape juices and wines: hydrodynamic and electrokinetic injection. As we can see in Table 2, hydrodynamic sample injection is the most widely used method.
It can be accomplished by application of pressure (at the
injection end of the capillary), vacuum (at the detection
end of the capillary) or by siphoning action obtained by
elevating the injection reservoir relative to the exit reservoir. In this type, the loaded sample volume is nearly
independent of the sample matrix, although it depends
on the viscosity. Few methods employed the electrokinetic injection (De Villiers, Lynen, Crouch, & Sandra,

2003; Kelly & Nelson, 1993; Levi et al., 1993) which is

performed by replacing the injection-end reservoir with
the sample vial and applying voltage. Therefore, the
amount loaded is dependent on the EOF, conductivity
and viscosity of background electrolyte, and sample
and electrophoretic mobilities of the analytes. The use
of electrokinetic injection enhances CE sensitivity but
it suers from matrix and poorer precision and,
therefore, it is not recommended for quantication.
Tacking into account electrophoretic conditions, the
electrolyte composition was extremely important to
obtain a good CE separation. In grape juices and wines,
several types of running buer like bis(2-hydroxyethyl)imino-tris(hydroxymethyl)aminomethane (BIS
Tris), boric acid, 1,3,5-benzenetricarboxylic acid
(BTA), 2-(N-morpholino) ethanesulfonic acid (MES),

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chromate, 4-aminobenzoic acid (PAB), phosphate,

phthalate, pyridinedicarboxylic acid (PDC), pyromellitic
acid (PMA) or tetraborate were used.
Furthermore, several types of electrosmotic ow
modiers (cationic surfactants) like cetyltrimethylammonium bromide (CTAB), ethylenediaminetetracarboxylic acid (EDTA), myristyltrimethylammonium
bromide (MTAB), tetradecyltrimethylammonium bromide (TTAB) or tetradecyltrimethylammonium hydroxide (TTAOH) were added to the background electrolyte
(BGE) to reverse the electroosmotic ow. Most of the
methods employed uncoated fused silica capillaries with
a cationic surfactant added, but the adsorption of compounds to the wall causes problems of reproducibility.
Therefore, coated capillaries are employed to avoid the
electroosmotic ow and the addition of cationic surfactant is not necessary. Saavedra and Barbas (2003) developed a method for routine quality control with a neutral
coated capillary (polyacrylamide) which present higher
reproducibility and precision than other methods with
uncoated capillaries (Fig. 3). In some methods, organic
modiers, as methanol (Huang, Luckey, Gordon, &
Zare, 1989; Vorarat, Aromdee, & Podokmai, 2002) or
complexion agent, as Ca2+ and/or Mg2+ salts (GarcaMoreno, Jurado-Campoy, & Garca-Barroso, 2001;
Mato, Huidobro, Simal-Lozano, & Sancho, submitted;
Moreno, Jurado, & Barroso, 2003) were added to electrolyte to alter the relative order of solute migration or
selectivity.
Only two types of detection systems were used for
determination of organic acids in grape juices and wines:
conductivity and UV spectrophotometry. As we can see

in Table 2, UV detection is the most widely used detection method mainly due to its nearly universal detection
nature (Heiger, 1992). It can be carried out according to
two modes, direct (the electrolyte absorption in the UV
region is lower than the absorption of organic acids, so
when the analyte passes through the detector the absorbance increases) and indirect (the electrolyte absorption
in the UV region is greater than the absorption of organic acids, so when the analyte passes through the
detector, the absorbance decreases and a negative peak
occurs). The use of low UV detection wavelength, which
ranged between 185 and 254 nm, allows to achieve high
sensitivities in the determination of organic acids. Klamp, Katzmayr, and Buchberger (1998) combined two
detection systems: conductivity and indirect UV and
they concluded that only the combination of this both
detection techniques allowed the quantication of most
of the organic acids of interest in a single CE run.
The short analysis time is other advantage of these
CE methods for the determination of organic acids in
grape juices and wines. This analysis time ranged between
3.5 and 20 min, although the majority of these methods
had an analysis time lower than 15 min (Table 2). Levi
et al. (1993) used both CE and HPLC to monitor the
extent of malolactic fermentation in wine samples by
the determination of malic and lactic acids levels. They
concluded that both techniques provided rapid analysis
and could be used for the automated processing of multiple samples, but CE had additional advantages as low
solvent consumption, use of nonhazardous solvents and
operation near ambient temperature. Kandl and Kupina
(1999) compared quantitative CE data of organic acids

Fig. 3. Electropherogram for standards and medium red wine (lower level) and white wine (upper level) with direct detection. Peaks: 1, nitrate; 2,
oxalate; 3, sulte; 4, fumarate; 5, succinate; 6, tartrate; 7, malate; 8, citrate; 9, phthalate (IS); 10, acetate; 11, lactate. Conditions: polyacrylamidecoated capillary, 200 mM phosphate buer at pH 7.50; 14 kV; direct detection at 200 nm. Reprinted from Saavedra and Barbas (2003), with
permission from Wiley-VCH Verlag GmbH & Co.

I. Mato et al. / Food Research International 38 (2005) 11751188

1185

Fig. 4. Red wine (diluted 1:40). Reprinted from Kandl and Kupina (1999), with permission from the American Society for Enology and Viticulture.

used dierent methods: an IC method for tartaric, malic

and citric acids, a distillation method for acetic acid, a
colourimetric method for tartaric acid and, an enzymatic method for malic, lactic and, citric acids. They
concluded that CE is capable of replacing ve current
methods for organic acid analysis HPLC, IC, enzymatic,
distillation and colourimetric methods. The CE method
results were in satisfactory agreement with those obtained from IC, colourimetric and, distillation methods.
Enzymatic methods are biased low compared to the CE
method. These dierences can be explained by the fact
that the enzymatic methods used were specic for Dand L-isomers, while the CE method determines both
D- and L-isomers as one peak. Fig. 4 shows an electropherogram of a CE method for determination of organic
acids developed by Kandl and Kupina (1999).
Most CE methods allow the determination of main
organic acids in grape juices (tartaric, malic and citric
acids) and in wines (tartaric, malic, citric, succinic, acetic
and lactic acids). Only two methods determined other
acids as adipic, glutaric and shikimic acids (Klamp
et al., 1998) or fumaric acid (Saavedra & Barbas, 2003).
Fumaric acid can prevent malolactic fermentation, but
its addition is forbidden in some countries and therefore,
its measurement is of great interest for quality control.
Finally, capillary electrophoresis main disadvantage
is its lower reproducibility, if compared with enzymatic
and chromatographic methods, so some authors use
internal standards or reference compounds to improve
migration time reproducibilities. These internal standards were butyric acid (Kenney, 1991), formic acid
(De Villiers et al., 2003) or glyoxylic acid (Esteves, Lima,

Lima, & Duarte, 2004; Kandl & Kupina, 1999). Oxalic

acid was used as the reference compound for the calculation of relative migration times of organic acids by
Mato et al. (submitted).
Table 3 shows a list of organic acids analyzed in
grape juices and wines by HPLC and CE methods with
their abbreviations.

Table 3
Full names and abbreviations of organic acids analyzed in grape juices
and wines by HPLC and CE methods
Abbreviation

Full name

acet.
adip.
asc.
cit.
citram.
form.
fum.
galact.
glucuron.
glut.
ketoglut.
lact.
mal.
male.
malon.
oxal.
prop.
pyr.
shyk.
succ.
tann.
tart.

Acetic acid
Adipic acid
Ascorbic acid
Citric acid
Citramalic acid
Formic acid
Fumaric acid
Galacturonic acid
Glucuronic acid
Glutaric acid
Ketoglutaric acid
Lactic acid
Malic acid
Maleic acid
Malonic acid
Oxalic acid
Propionic acid
Pyruvic acid
Shykimic acid
Succinic acid
Tannic acid
Tartaric acid

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I. Mato et al. / Food Research International 38 (2005) 11751188

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