Voltammetric Techniques for Determination of
Allura Red AC Synthetic Pigment in Beverages
ELENA DIACU1, ELEONORA-MIHAELA UNGUREANU1*, ANTON ALEXANDRU IVANOV2, MARIA MADALINA JURCOVAN1
1
University Politehnica of Bucharest, Faculty of Applied Chemistry and Materials Science, 1-7 Polizu,011061, Bucharest, Romania
2
National Institute for Research & Development in Environmental Protection, 294 Spl. Independentei Bucharest, Romania
The analysis of synthetic pigments has become a very important issue due to the suspicion that the presence
of these substances in food and beverages is harmful to human health. Among these is Allura Red AC (AR),
a food red pigment used to improve visual aspect of some food and drinks. Two votlammetric techniques,
cyclic voltammetry and differential pulse voltammetry were investigated for their practical application in
potential AR analysis from beverages. The electrochemical behaviour of AR in aqueous solutions was
established in experiments conducted at five pH values in the range of 3.0 - 7.0 on glassy carbon disk
electrode, at different scan rates and AR concentrations. The dependence of peak currents and peak potentials
upon electrochemical parameters was investigated and the linear response range and the detection limit
were established. For both voltammetric techniques good correlations of the parameters have been found
showing that the methods can be of practical importance in the quantitative determination of AR from
beverages.
Keywords: Allura Red AC, cyclic voltammetry, differential pulse voltammetry, glassy carbon disk electrode,
beverages
Natural and synthetic pigments fall into the category of
food additives, being used in food processing in order to
enhance sensory response and to be more aesthetically
attractive for consumers. Synthetic pigments are used with
preference by food manufacturers because they have some
advantageous properties in comparison with the natural
ones. Listing only a few of them, one can mention their
high colouring power and stability, provision of desired
colour in a wide variety, as well as a low price [1-3]. The
suspicion that the exposure to these substances is harmful
to humans, especially children, imposed conducting
toxicological and clinical studies on animals and human.
These studies have showed that almost all synthetic dyes
can cause serious diseases such as hyperactivity in
children, migraines, vomiting, asthma, allergies, and can
even develop cancer [4-5]. Therefore, the use of synthetic
pigments in food was strictly regulated and harmonized
across all European Union countries, the Directive 94/36/
EC being major document on food colours [6-8]. To check
the fulfilment of legal requirements and thus ensure food
safety, much attention has been given to the development
of advanced analytical methods for the identification and
quantitative determination of various synthetic colorants.
An impressive number of papers dealing with the analysis
of synthetic food pigments have been reported [9-25], the
majority of analytical methods for synthetic colorants being
in the range of chromatographic methods [11-15] and in
spectro-photo-metric ones [16-19]. Generally, the
spectrophotometric methods involve almost always a
sample pre-treatment step consisting in a chemical
separation procedure in order to avoid spectral
interferences, which is time consuming. However,
numerous studies revealed that the electrochemical
methods are selective and sensitive techniques suitable
for the analysis of common azo pigments, with very good
detection limits [20-23]. In addition, voltammetric
techniques have proven to be powerful tools in the synthetic
pigment investigation that offer analytical options not only
for their analysis, but also a good way to determine the
degradation products, because these techniques are able
to establish certain degradation processes that are likely.
Moreover, they can make distinction between synthetic
and natural pigments of the same colour because the last
do not posses azo linkage in their molecule, and therefore
are not electroactive compounds. Recently in our
laboratory, differential pulse voltammetry (DPV) and cyclic
voltammetry (CV) methods were developed for the
determination of two synthetic pigments Tartrazine and
Sunset Yellow [24-25].The present study aimed to
investigate the electrochemical behaviour of Allura Red
AC (AR) in view of its determination from soft drinks.
AR, a red by-petroleum product, is known by consumers
as E129 or as FD&C Red 40, in USA. AR is one of the most
common synthetic food pigment used in red colour
beverages (strawberry, raspberry, grapes, apple, etc.). In
food, this colour is a permitted colour by European law
only in a maximum concentration of 100μg/mL, either
when is alone, or in mixtures with other synthetic pigments
[6-8]. The determination of AR may by a difficult task,
because it is a complex chemical substance of relatively
low concentration in food, but also because it can be in
very complex food matrix samples. In view of the potential
health problems due to the presence of AR in food, such as
allergy, diarrhea, migraines, thyroid tumours, chromosomal
damage, hyperactive behaviour in children, the content of
AR in food and drinks must be monitored by applying
performant analytical methods allowing the identification
and quantification in food products.
According with IUPAC, AR is the disodium 2-hydroxy-1(2-methoxy-5-methyl-4-sulphonatophenylazo)
naphthalene-6-sulphonate azo-dye, with the chemical
structure shown in figure 1.
Due to the presence of the azo-group in its molecule, AR
possess electroactive properties, which have been
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Fig. 1. Chemical
structure of Allura
Red AC
investigated by voltammetric methods, such as cyclic
voltammetry (CV) and differential pulse voltammetry
(DPV) at a glassy carbon disk electrode.
Experimental part
Reagents, equipment and methods
All reagents were of analytical purity and the aqueous
solutions were prepared in purified bi-distilled water. AR
was purchased from SIGMA-Aldrich (Germany, purity 90%).
Standard buffer solutions (citrate for pH = 3.0 and 4.0,
acetate for pH = 5.0 and 6.0, and phosphate for pH 7.0)
were prepared according to usual procedures. Bidistilled
water was further purified using an Elgastat system (5 ΩW
cm). Glassware bottles were soaked in KMnO4-H2SO 4
mixture, rinsed with a H 2SO 4-H 2O 2 solution, and then
carefully cleaned with purified water before use to avoid
contamination.
In order to establish the electrochemical behaviour of
AR and evaluate the optimal analytical parameters for the
determination of this food colour, voltammetric
experiments were performed on glassy carbon disk
electrode at 25°C in buffered aqueous solutions of AR at
different pH values (3.0-7.0), AR concentrations (0.52.0mM) and scan rates, according to the previous reported
procedures [23, 24].
Electrochemical experiments were carried out using a
Princeton Applied Research 283 (PAR283) potentiostat in
conventional three-electrode cells on glassy carbon
electrode disk (3 mm diameter, from CH Instruments)
with a platinum wire as counter electrode and Ag/AgCl/
aqueous saturated KCl as reference electrode. All potentials
were referred to this reference potential (0.197V at 25oC).
The glassy carbon disk electrodes were polished with
0.2μm diamond paste before each experiment, then rinsed
in absolute ethanol and distilled water. Solutions were
degassed with a stream of argon for 20 min before each
measurement and kept under argon atmosphere during
the entire experiment.
DPV experiments were usually performed with pulses
of 25 mV for 50 ms, step height of 2 mV, step time
100 ms, and scan rate of 20 mV s-1. CV experiments were
recorded at 0.1 V s-1, and with different scan rates (0.1–
1.0 V s-1), for investigation of the scan rate influence. To
measure the pH Denver Instrument Model 220 pHconductivity meter was used.
Results and discussions
The CV and DPV experiments were conducted in 0.1 M
aqueous buffer solutions as supporting electrolytes in
milimolar concentrations of AR, at different pH values
within the pH range similar to the real soft drinks samples
(3.0 – 7.0). The concentrations were varied between 0 2.0 mM. In order to establish the reversible character of
the involved electrochemical processes of AR the CV
curves have been recorded at different scan rates and
potential ranges. The results concerning the behaviour of
AR at pH 5 are presented further as examples.
Electrochemical characterization of AR at pH 5
Figure 2 shows the DPV curves for AR solutions at
different concentrations. Anodic and cathodic curves are
shown together on the same voltammogram. A pair of
770
anodic and cathodic peaks can be observed. They have
been attributed to the oxidative and reductive cleavage of
– N = N – group. The linear dependences of peak potentials
with the logarithm of the AR concentration are displayed in
the upper inset of figure 2, and for the peak currents with
AR concentration in the lower inset of figure 2. The two
insets from figure 2 show the slopes of these linear
dependences in accordance with equations given in table
1. The direct proportionality of the peaks currents with AR
concentration could be used in analytical determination of
AR by DPV method.
Fig. 2. DPV curves for AR at different concentrations (mM) on
glassy carbon electrode (3 mm in diameter) at pH 5; upper inset Linear dependences of the anodic (Ea) and cathodic (Ec) peaks
potentials on AR concentration logarithm (log c); lower inset Linear dependences of the anodic (ia) and cathodic (ic) peaks
currents on AR concentration (c)
The anodic peak identified in the DPV curves could be
attributed to the oxidation of AR to its stable radical cation.
The positive charge is mainly delocalized along the
molecule after ionization. The oxidation potential of anodic
peak is in the range characteristic for azo compounds. The
cathodic peak identified in the DPV curves could be
attributed to the reduction of AR to its stable radical anion.
After ionization the negative charge is also delocalized on
the molecule. The reduction potential values are in the range
characteristic for these azo compounds. It is very likely
that both oxidation and reduction of AR involve
electrochemical and chemical steps.
The same electrode processes are identified for AR in
the CV curves (fig. 3) having the values of peak potential
sensitively equal to those obtained by the DPV. The
processes are irreversible, showing the electrochemical
degradation of AR during the potential scanning. The peak
potentials depend linearly with the logarithm of the AR
concentration (upper inset in fig. 3) and the peak currents
vary linearly with AR concentration (lower inset in fig. 3).
The insets from figure 3 show the slopes of these
dependences. Their equations are also given in table 2.
These linear dependences of the peaks currents could be
used in analytical determination of AR by CV method.
Figure 4 shows the CV curves on different scan rates in
the potential domains of the anodic and cathodic
processes. Both anodic and cathodic scans are plotted on
the same graph. The reversibility of the anodic and cathodic
processes has been carefully evaluated from the CV curves
obtained at different scan rates. As it can be seen these
processes are all irreversible. Linear dependences of the
peak potentials on the logarithm of the scan rate have been
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REV. CHIM. (Bucharest) ♦ 66 ♦ No. 6 ♦ 2015
Fig. 3. CV curves (v = 0.1 V s-1) for different AR concentrations
(mM) on glassy carbon electrode (3 mm in diameter) at pH5;
upper inset: - linear dependences of the anodic (Ea) and cathodic
(Ec) peaks potentials on the logarithm of AR concentration; lower
inset - linear dependences of the anodic (ia) and cathodic (ic)
peak currents on AR concentration (c)
Fig. 4. CV curves in the first anodic and cathodic scans at different
scan rates (V s-1) on glassy carbon electrode (3 mm in diameter) at
pH 5 in AR aqueous solutions (2 mM); upper inset - linear
dependence of the anodic (Ea) and cathodic (Ec) peak potentials
on the logarithm of the scan rate (log v); lower inset - linear
dependence of the anodic (ia) and cathodic (ic) peak currents on
the square root of the scan rate (sqrt v).
obtained and presented in the upper inset of figure 4. Linear
dependences of the peak currents anodic and cathodic on
the square root of the scan rate have also been obtained
and presented in the lower inset of figure 4, proving
diffusion controlled processes. The values of ip/v1/2c ratio
of around 175 and 200 μA(V s-1) -1/2(mM) -1 have been
calculated for the anodic and cathodic peaks, respectively.
Clearly, in the range of investigated scan rates, and AR
concentrations the processes are irreversible. The diffusion
coefficients that vary between 10-5 and 2 . 10-5 cm2s-1 for
AR species have been calculated using Randless-Sevcik
equation by supposing a single electron transfer. However,
the lower inset in figure 4 shows that the slope of peak
current dependence on the scan rate is higher (1.25 times)
for the cathodic peak than the slope for the anodic peak.
The pH influence
The DPV and CV curves were recorded at pH values
between 3 and 7. All curves recorded at pH 3 - 6 have only
a single cathodic peak. At pH 7 the cathodic peak is spitted
in two very close peaks due to the fact that the colour
molecule can be either in the fenolic or fenoxy forms. The
influence of pH on the peak potentials and on the peak
currents of the anodic and cathodic processes has been
evidenced from the DPV and CV curves. The linear
dependences obtained in AR buffer solutions of different
pHs are shown in figure 5. These dependences from the
DPV curves show better correlation coefficients than
dependences from CV curves, as it can be seen from their
parameters given in tables 1 - 4.
From table 1 it can be seen that, in DPV, Ea peak
dependence has an absolute slope approximately two
times smaller than for Ec peak (around 52 mV compared
Table 1
PARAMETERS OF THE LINEAR DEPENDENCES OF THE PEAK
POTENTIAL E (IN V) FOR THE ANODIC PEAK Ea AND THE CATHODIC
PEAK Ec ON AR CONCENTRATION LOGARITHM (log c) FROM DPV
AND CV EXPERIMENTS. (R2 IS THE CORRELATION COEFFICIENT).
THE AR CONCENTRATIONS: 0.5; 1.0; 1.5 AND 2.0 mM
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Table 2
PARAMETERS OF THE LINEAR DEPENDENCES OF THE PEAK
CURRENTS (IN A) FOR THE ANODIC PEAK (ia) AND THE CATHODIC
PEAK (ic) ON AR CONCENTRATION C (IN mmole L-1) FROM DPV
AND CV EXPERIMENTS (R2 IS THE CORRELATION COEFFICIENT).
THE AR CONCENTRATIONS: 0.5; 1.0; 1.5 AND 2.0 mM
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Table 4
PARAMETERS OF THE LINEAR DEPENDENCES OF THE PEAK
CURRENTS (IN A) FOR THE ANODIC PEAK ia AND THE CATHODIC
PEAK ie ON pH AT DIFFERENT AR CONCENTRATION (c) FROM DPV
AND CV EXPERIMENTS. (R2 IS THE CORRELATION COEFFICIENT)
Fig. 5. Dependences on pH of peak parameters for different
concentrations of AR from DPV curves:
(A) -the anodic (Ea) and cathodic (Ec) peak potentials; (B) - the
anodic (ia) and cathodic (ic) peak currents
Table 3
PARAMETERS OF THE LINEAR DEPENDENCES OF THE PEAK
POTENTIAL E (IN V) FOR THE ANODIC PEAK Ea AND THE
CATHODIC PEAK Ec ON pH AT DIFFERENT AR CONCENTRATIOn (c)
FROM DPV AND CV EXPERIMENTS. (R2 IS THE CORRELATION
COEFFICIENT)
to 93 mV, respectively). The corresponding values from
CV are about 30 mV, higher than the DPV values (both
slopes of about 89 mV, respectively). These differences
could be attributed to the ECE processes, consisting in
successive electron transfer (E) and chemical (C) steps.
From table 2 it can be seen that, in DPV, the ia peak
current has an absolute slope similar as magnitude to that
of ic peak current (around 8.7μA and 8.6μA, respectively).
The corresponding values from CV experiments are about
3.6μA and 3.8μA, respectively, being lower than the DPV
values, at each studied pH. The slope of ic curves has about
the same value as the slope of ia. These data confirm that
772
there is no difference between the numbers of electrons
involved in the corresponding complex electrode
processes. From analytical point of view, the CV method
offer similar possibilities with DPV for the determination of
AR. However, in the case of colour mixtures the DPV
method could be a preferred choice due to the higher
selectivity.
Figure 5 and also table 3 show that, in DPV experiments,
the slope of Ea peak is almost constant while the slope of
Ec peak has a bigger value (about 100 mV/decade)
meaning that the reduction process is more influenced by
the pH than the oxidation process. However, as table 4
indicates, the anodic currents have absolute slopes of about
two times bigger than the cathodic ones.
Analyzing the data from table 3 it can be observed that
for both Ea and Ec dependences the two voltammetric
methods display similar dependences on the pH of the
solution. However, to determine AR content, the anodic
DPV peak potential is recommended as analytical signal,
because the anodic potential is more stable in respect to
pH variations than the cathodic one.
Table 4 displays the results concerning pH influence on
peak currents for the anodic and cathodic peaks. It can be
seen that there are some variations with pH, for instance,
in DPV, the absolute slope is of around 1.52μA and 6.9μA
for ia and ic, respectively. The corresponding values from
CV are of about 2.4μA and 4.4μA, respectively. This leads
to the conclusion that ia values obtained from CV can be
used as analytical signals for AR analysis.
The experimental data regarding the effect of pH,
concentration of active species, scan rate on cathodic and
anodic peak potentials and peak currents showed that the
processes are diffusion controlled.
Conclusions
Differential pulse voltammetry (DPV) and cyclic
voltammetry (CV) on glassy carbon disk electrode were
carried out to establish the electrochemical properties of
synthetic colorant Allura Red AC at different pH values.
The specific parameters which can be further employed in
the analysis of this colour were determined. The
experiments revealed that Allura Red AC colour is
electrochemically active in both anodic and cathodic
potential domains, displaying linear dependences of the
anodic and cathodic peaks currents and potentials on
concentration. All results allow the conclusion that both
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REV. CHIM. (Bucharest) ♦ 66 ♦ No. 6 ♦ 2015
voltammetric methods are of practical importance for
Allura Red AC determination in soft drinks at mM
concentrations.
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Manuscript received: 22.01.2015
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