Food Chemistry 136 (2013) 1183–1192

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Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem

Analytical Methods

Analysis of total polyphenols in wines by FIA with highly stable amperometric

detection using carbon nanotube-modified electrodes
Alberto Sánchez Arribas a,⇑, Marta Martínez-Fernández a, Mónica Moreno a, Esperanza Bermejo a,
Antonio Zapardiel b, Manuel Chicharro a
a
Dpto. Química Analítica y Análisis Instrumental, Universidad Autónoma de Madrid, C/Francisco Tomás y Valiente, 7, 28049 Madrid, Spain
b
Dpto. Ciencias Analíticas, Universidad Nacional de Educación a Distancia, P Senda del Rey, 9, 28040 Madrid, Spain

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

Article history: The use of glassy carbon electrodes (GCEs) modified with multi-walled carbon nanotube (CNT) films for
Received 6 November 2011 the continuous monitoring of polyphenols in flow systems has been examined. The performance of these
Received in revised form 6 May 2012 modified electrodes was evaluated and compared to bare GCE by cyclic voltammetry experiments and by
Accepted 2 September 2012
flow injection analysis (FIA) with amperometric detection monitoring the response of gallic, caffeic, feru-
Available online 16 September 2012
lic and p-coumaric acids in 0.050 M acetate buffer pH 4.5 containing 100 mM NaCl. The GCE modified
with CNT dispersions in polyethyleneimine (PEI) provided lower overpotentials, higher sensitivity and
Keywords:
much higher signal stability under a dynamic regime than bare GCEs. These properties allowed the esti-
Carbon nanotubes
Electrochemical detection
mation of the total polyphenol content in red and white wines with a remarkable long-term stability in
Flow injection analysis the measurements despite the presence of potential fouling substances in the wine matrix. In addition,
Polyphenols the versatility of the electrochemical methodology allowed the selective estimation of the easily oxidis-
Wine able polyphenol fraction as well as the total polyphenol content just by tuning the detection potential at
+0.30 or 0.70 V, respectively. The significance of the electrochemical results was demonstrated through
correlation studies with the results obtained with conventional spectrophotometric assays for polyphe-
nols (Folin–Ciocalteu, absorbance at 280 nm index and colour intensity index).
Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction components (Jackson, 2008). Polyphenols are found in lower con-

Polyphenols, especially those naturally found in foods, are sub-
stances of increasing interest as a result of their biological proper-
ties such as antioxidant, anti-thrombotic, anti-bacterial, anti-
allergic and anti-inflammatory activities (Hurtado-Fernández,
Gómez-Romero, Carrasco-Pancorbo, & Fernández-Gutiérrez, 2010).
As a consequence of their beneficial effects on the human health
(Cifuentes, 2006; Kris-Etherton et al., 2002), the intake of polyphe-
nol-rich foods is recommended to be included in diet habits and
new dietary supplements and functional foods containing polyphe-
nols have been released.
Among others, wine is one of the foods with higher polyphenol
content. Many studies correlate a moderate wine intake (mostly
red wine) with an increase in antioxidant power in plasma after
polyphenol absorption during digestion process which finally pro-
tects tissues from oxidative stress (Jackson, 2008, chap. 6, 7 and
12). Its composition is very complex and includes hundreds of
compounds present simultaneously in different concentrations,
with water, ethanol, glycerol, sugars and organic acids as the major
⇑ Corresponding author. Fax: +34 91 497 4931.
E-mail address: alberto.sanchez@uam.es (A. Sánchez Arribas).
centration levels and a large variety of these compounds has been
described in these matrixes (Jackson, 2008; Monagas, Bartolomé, &
Gómez-Cordovés, 2005). They have different structures and they
are mainly derived from simple benzoic and cinnamic acids, stilb-
enes and flavonoids. From these basic structures, more complex
compounds are formed by condensation, glucosylation and poly-
merisation. The precursors of all of these compounds are found
mainly in seeds, and in skin tissues and epidermal cells, of grapes.
Their content in grapes and wines depends on the variety of wine
grape, the weather, the soil, and the farming practices (Jackson,
2008).
The determination of the quantitative composition and the
investigation of the factors affecting the composition of these
bioactive substances, using robust, sensitive and reliable methods,
are considered a priority. There are two general approaches to
examine and quantify the polyphenolic content in foods and there-
fore in wines. The first one is the determination of a total index
(‘‘total polyphenolics’’) by spectrophotometric detection, usually
carried out using the Folin–Ciocalteu method or the absorbance
measurement at 280 nm (Luque de Castro, González-Rodríguez, &
Pérez-Juan, 2005). The second one consists of a separation of the
individual polyphenolic species, typically by HPLC (Kalili & de

0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.foodchem.2012.09.027
1184 A. Sánchez Arribas et al. / Food Chemistry 136 (2013) 1183–1192
Villiers, 2011) or capillary electrophoresis (Hurtado-Fernández
et al., 2010), and their subsequent detection by UV, fluorimetric,
mass spectrometry or electrochemical detectors. Regarding the
determination of the total index, it is important to remark that
these assays provide results from the general contribution of all
the compounds present in the sample (polyphenolic and non-
polyphenololic) and they actually constitute an approach to know
the real ‘‘total polyphenolic’’ content (usually also employed to
estimate the ‘‘antioxidant capacity’’). The chemical complexity of
these compounds, the interferences present in the wine matrix
and their problematic extraction from the sample are the main
problems that hinder an accurate estimation of this index (Blasco,
Rogerio, González, & Escarpa, 2005). The Folin–Ciocalteau is the
reference method of the International Organization of Vine and
Wine (OIV) and it is used in the European Union as the official
method of analysis because of its simplicity and good repeatability
values whereas the absorbance measurement at 280 nm is also
used as a quite simpler but less sensitive method (Luque de Castro
et al., 2005). The main drawbacks of these methods are the overes-
timation of the polyphenol content due to the contribution of non-
polyphenolic substances to the spectrophotometric signal, the dif-
ference in the contribution of the different types of polyphenol that
generate the total signal and the lack of information that these
methods provide about the reducing strength of the polyphenols
present in the sample (Blasco et al., 2005; Escarpa & González
2001; Everette et al., 2010); they do not distinguish between cate-
chol and galloyl-containing polyphenols that are easier to oxidise
(e.g., gallic and caffeic acids, (+)-catechin, quercetin, etc.), and poly-
phenols that are more difficult to oxidise (e.g., coumaric acids and
malvidin-3-glucoside).
Electroanalytical techniques, more precisely the voltammetric
ones, are especially well suited for the analysis of polyphenols.
The antioxidant properties of these compounds are related to their
ability to donate electrons. Thus, most present native electroactiv-
ity and their electrochemical oxidation at moderate potentials has
been exploited for their detection. These characteristics allow a
selective detection of polyphenols with good sensitivity, even in
very complex samples such as wine, and responses are indepen-
dent from the optical path-length or sample turbidity (Blasco,
González Crevillén, González, & Escarpa, 2007). These advantages
have led to the consideration of electroanalytical techniques as
attractive alternatives to the spectrophotometric ones for the
polyphenol analysis in this complex matrix.
The electroanalytical evaluation of the ‘‘total polyphenolic’’ con-
tent has been carried out by employing both the potential sweep
and the amperometric modes. The first approach is exemplified
by the Kilmartin’s works (Kilmartin, Zou, & Waterhouse, 2001;
Makhotkina & Kilmartin, 2010) where cyclic voltammetry using
glassy carbon electrodes was exploited and the characteristic sig-
nals at relevant potentials of different types of polyphenols were
employed to estimate the content of some polyphenols groups in
wines. In this way, the presence of voltammetric signals at low
overpotentials correlate with the presence of polyphenolics of high
antioxidant activity, whereas those compounds with low antioxi-
dant power show electrochemical activity at more positive poten-
tials. Indeed different polyphenol classes can be quantified
separately by processing the signals at different potential intervals.
This concept has been used by other authors for the analysis of dif-
ferent polyphenol fractions in wines (Aguirre et al., 2010; Dhroso,
Laschi, Marrazza, & Mascini, 2010; Ksenzhek, Petrova, &
Kolodyazhny, 2007; Seruga, Novak, & Jakobek, 2011) using
carbon-based electrodes. The second approach is described in
Mannino’s (Mannino, Brenna, Buratti, & Cosio, 1998) and Escarpa’s
(Blasco et al., 2005) works and it is based on the amperometric
detection at fixed selective potentials using glassy carbon elec-
trodes coupled to a flow injection analysis (FIA) device. Mannino
et al. (1998) estimated the antioxidant power of wine samples
using the signals at +0.40 V of easily oxidizable polyphenols
whereas Blasco et al. (2005) proposed the electrochemical index
(EI) and the high antioxidant fraction (HA) from the non selective
signals at +0.80 V, corresponding total polyphenols, and from the
more selective +0.50 V signals, corresponding to easily oxidizable
phenols, respectively. Other interesting electroanalytical estima-
tion of ‘‘total polyphenolics’’ was based on the use of electrochem-
ical biosensors, typically using the amperometric mode, which
incorporated polyphenol oxidase or peroxidase enzymes (Granero,
Fernández, Agostini, & Zon, 2010). These biosensors provide selec-
tive and sensitive responses towards many polyphenols with min-
imal sample preparation but the enzymatic activity is different
depending on the polyphenol structure. The last has led to
introduce the bioelectrochemical polyphenol index in wines by
Carralero Sanz, Mena, González-Cortés, Yáñez-Sedeño, and
Pingarrón (2005). The results obtained using all the approaches
described within this paragraph usually were well-correlated with
those from the Folin–Ciocalteu assay.
In this work we present the advantages provided by the incor-
poration of carbon nanotube films onto glassy carbon electrodes
used for the electrochemical detection of ‘‘total polyphenols’’ in
wine samples using a FIA system. This carbon nanotubes layer
not only provides electrocatalytic properties (typically resulting
in diminution of overpotential) but also an enhanced signal stabil-
ity and increased resistance to passivation, as it has been reported
for analytes such as NADH (Banks & Compton, 2005; Kachoosangi
et al., 2009) or phenol (Arribas et al., 2007) that produce rapid sur-
face fouling in common electrodes when monitoring their electro-
chemical oxidation during long periods of time. This improvement
in electrochemical signals is related to the presence of a high den-
sity of edge-plane defects in CNT surface (Ambrosi, Sasaki, &
Pumera, 2010; Banks and Compton, 2005) which enhances the
electron-transfer kinetics and impedes the apparition of fouling
symptoms. In this way, a remarkable improvement in stability
and reproducibility of signals during the analysis of wine samples
is obtained and high sample throughput is possible.
2. Experimental
2.1. Reagents and samples
Caffeic, p-coumaric, ferulic and gallic acids were purchased
from Sigma–Aldrich (Madrid, Spain). The polyphenol stock solu-
tions were prepared by dissolving an appropriate amount of the
compound in methanol (Environmental grade, Alfa Aesar, Barce-
lona, Spain). All the stock solutions were kept away from the light
and stored under refrigeration. Diluted solutions were prepared
daily from these stock solutions. Folin–Ciocalteu reagent and poly-
ethyleneimine (PEI; Cat. No. P3143) were purchased from Sigma
(Madrid, Spain) whereas Nafion (Cat. No. 527084) was purchased
from Aldrich (Madrid, Spain). All the other chemicals were analyt-
ical-reagent grade and they were used without further purification.
Ultrapure water (q > 18 MX cm) from an Elga Purelab Option Q
system (ELGA LabWater, UK) was used for preparing all solutions.
All buffers were prepared from the corresponding 0.50 M acid solu-
tion and the desired pH was set by adding 1.0 M NaOH and made
up to their final concentration.
Red (Izadi, Heredad de Torresano, Alcorta and Pueblo Viejo) and
white (Marqués de Riscal, Martín Codax, Alectum, Viñas del Vero
and Viña Esmeralda) wine samples were purchased in different lo-
cal markets. Physicochemical characterisation was carried out fol-
lowing the regulations of the Office Internationale de la Vigne et du
Vin (OIV, 2012) and the enological parameters are listed in the
Supplementary information Section, Table 1. All wine samples
 A. Sánchez Arribas et al. / Food Chemistry 136 (2013) 1183–1192
were analysed within the first week after opening. Before being
analysed, samples were filtered through ‘‘Osmonics’’ 0.45 lm ny-
lon filters (Micron Separations Inc, MA, USA).
Model wine solution was prepared according to Gamella et al.
(2010) and contained tartaric acid (5.0 g L 1), D-glucose
(2.0 g L 1), D-fructose (2.0 g L 1), sodium chloride (0.2 g L 1),
ammonium sulphate (1.0 g L 1), disodium phosphate (2.0 g L 1),
magnesium sulphate heptahydrate (0.2 g L 1), manganese sul-
phate (0.05 g L 1), acetic acid (0.05 g L 1) and ethanol (12% v/v).
The pH of this solution was adjusted to 3.5 with KOH and filtered
through ‘‘Osmonics’’ 0.45 lm nylon filters (Micron Separations Inc,
MA, USA).
Multiwalled carbon nanotubes powder (CNT; 30 ± 15 nm diam-
eter, 5–20 lm length, purity >95%, ‘‘hollow tube’’ type, prepared by
chemical vapour deposition, nominal 1% Fe and 0.1% S residuals,
Lot. PD30L520-60805) were obtained from NanoLab (MA, USA).
2.2. Apparatus
Electrochemical measurements were conducted using
lAUTOLAB type III electrochemical analyser controlled by GEPES
4.9.007 software (Eco Chemie; Utrecht, Netherlands) installed on
a Pentium IV PC computer. Flow injection analysis set up included
a peristaltic pump (model ISM 834; ISMATEC, Germany) connected
to a four-way low-pressure valve Rheodyne type 5020 (Alltech,
Spain) with a 100-lL sample loop. The connections were made
with an interconnecting PTFE tube (0.8 mm i.d. and 1.6 mm o.d.).
The electrodes were inserted into a wall-jet cell (model EA-1096;
Metrohm; Herisau, Switzerland) containing a gold wire as counter
electrode and Ag/AgCl/3 M NaCl (Metrohm) as reference electrode.
Spectrophotometric measurements were carried out using a
double-beam spectrophotometer Hitachi U-2000 (Japan).
pH was monitored with a 654 pH-meter from Metrohm (Heri-
sau, Switzerland).
2.3. Working electrodes
Glassy carbon electrodes (GCEs) of 3-mm diameter were pur-
chased from CH Instruments (model CHI104; Austin, TX, USA).
They were polished with 0.3- and 0.05-lm alumina slurries (Bueh-
ler; Spain) on polishing cloths (Buehler; Spain) and subjected to
ultrasonic cleaning in water for 1 min before use.
GCEs modified with CNT dispersions were prepared by drop-
ping the CNT dispersion (15 lL) onto the clean GCE surface and
leaving it to dry for at least 2 h. The CNT dispersions were prepared
by dispersing 2.0 mg of the as-received CNTs in 4.0 mL of disper-
sant solution followed by ultrasonic treatment using an ultrasonic
probe Sonics Vibra Cell (model VCX130; Sonics & Materials, Inc.,
Newtown, CT, USA) for 60 min. Three different dispersants were
used: ethanol/water (1:1 v/v), 0.5% (w/v) Nafion in ethanol/water
(1:1 v/v) and 0.5% (w/v) polyethylenimine (PEI) in ethanol/water
(1:1 v/v). The corresponding GCE modified with these CNT disper-
sions were labelled as GC/(CNT/EtOH), GC/(CNT/Nafion) and GC/
(CNT/PEI), respectively.
2.4. Procedures
2.4.1. Electrochemical experiments
Cyclic voltammetry experiments were carried out using a stan-
dard three-electrode cell (10 mL) system with different working
electrodes, an Ag/AgCl/3 M KCl (BAS MF-2063) reference electrode
and a platinum wire (1-mm diameter) auxiliary electrode. The
potentials given in these experiments were referred to this elec-
trode system. All working electrodes were subjected to 20 scans
between 0.20 and +1.10 V at 0.050 V s 1 in supporting electrolyte
before each experiment.
1185
Electrochemical detection using the flow injection analysis set
up was performed in the amperometry mode by applying a fixed
potential (referred to the electrode system described in Sec-
tion 2.2). The detecting currents were allowed to reach a stable
baseline prior to the amperometric monitoring.
2.4.2. Spectrophotometric experiments
The Folin–Ciocalteu assay, FC, was carried out following the Sin-
gleton and Rossi (Singleton & Rossi, 1965) method using gallic acid
as reference standard. Wine samples (50 or 250 lL for red and
white wines, respectively) or appropriate volumes of gallic acid
standard were mixed with 0.50 mL of Folin–Ciocalteu reagent
and 5 mL of water. 3.0 mL of 1 M Na2CO3 were added 2 min later
and the mixture was made-up to 10.0 mL in a volumetric flask.
Solutions were kept in the dark for 2 h and the absorbance of the
blue complex formed was measured at 765 nm with 1.0-cm quartz
cuvettes.
The 280 nm absorbance index, I280, (Luque de Castro et al.,
2005) was obtained from the absorbance measurement at this
a
wavelength of wine samples diluted with water (1:20 and 1:100
dilution factor for white and red wines, respectively) in 1.0-cm
quartz cuvettes. The index values were calculated by multiplying
the absorbance by the dilution factor of each sample.
Colour intensity index, CI, (OIV, 2012) was estimated from the
absorbance at 420, 520 and 620 nm of undiluted wine samples,
using the following expression CI = A420 + A520 + A620. In this case,
1.0- and 0.2-cm quartz cuvettes were employed for white and
red wines, respectively. The absorbance values of red wines were
multiplied by 5 in order to normalise the optical path length before
the calculation of CI.
All experiments were carried out at room temperature (20–23°
C).
3. Results and discussion
3.1. Cyclic voltammetry experiments using CNT-modified electrodes
The first objective of this work was to examine the electro-
chemical performance of the electrodes modified with carbon
nanotubes towards polyphenolic compounds usually found in
wines in chemical conditions (pH and ionic strength) similar to
that found in such matrix (Jackson, 2008). Indeed, preliminary
studies were conducted using cyclic voltammetry in order to com-
pare the electrochemical performance of GCE modified with a CNT
layer obtained from the dispersions described in Section 2.3. For
this purpose, four representative phenolic acids were employed:
gallic acid, a benzoic acid derivative with three hydroxyl substitu-
ents in its aromatic ring, and caffeic, ferulic and p-coumaric acids,
three derivatives from cinnamic acid with a different number of
hydroxyl and methoxy groups in their aromatic rings. The cyclic
voltammograms of solutions of these compounds (0.50 mM each)
in 100 mM acetate buffer containing 100 mM NaCl with a pH 4.5
were obtained (between 0.20 and +1.10 V at 0.050 V s 1) with
the different working electrodes and the results obtained (mean
value from three different electrode surfaces) are detailed in the
Supplementary information Section, Table 2.
In general, those compounds with a catechol-like structure (caf-
feic and gallic acids) were electrochemically oxidised at lower
overpotentials than those with one hydroxyl group, with the high-
er peak potentials shown with p-coumaric acid. This trend is usu-
ally observed when using carbon-based electrodes (Makhotkina
and Kilmartin, 2010). The voltammograms of electrodes modified
with a CNT layer provided signals at lower overpotentials with
higher peak and capacitative (background) currents than those ob-
served at bare GCE as a result of the improved electrochemical
1186 A. Sánchez Arribas et al. / Food Chemistry 136 (2013) 1183–1192

characteristics of CNTs and the increased active surface area of the 3

layer (Henstridge, Dickinson, Aslanoglu, Batchelor-McAuley, & (A)

Current / µA
Compton, 2010). A closer inspection of these results reveals that 2
the charge of the dispersing polymer played an important role in
the changes on the voltammetric response observed when GCE
was modified using each type of CNT dispersion. The diminution 1
in the overpotentials, compared to bare GCE values, was similar
for all dispersions whereas the peak currents obtained with the 0
CNT/PEI layer were about twice and 10-fold higher than that regis- 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0
tered at CNT/EtOH and CNT/Nafion, respectively. Under these -1
Flow rate / mL min
experimental conditions, the phenolic acids studied are negatively
charged since their carboxylic acid group was deprotonated
(pKa < 4.5) in all cases (Kovachev, Canals, & Escarpa, 2010). The 3
(B)
electrostatic interactions amongst the phenolic acids and the pos-
itively charged PEI can explain the higher sensitivity observed
2
whereas the repulsion with the negatively charged Nafion impeded
an appropriate approach of the analytes towards the electrode

Current / µA
surface, thus notably lower peak currents were shown in this case. 1
Interestingly the catechol-like compounds showed a bigger
improvement in the voltammetric results obtained with CNT- b
0
modified electrodes, with a negative shifting of 0.10 V in the over-
2 0.0 0.2 0.4 0.6 0.8 1.0
potential of gallic acid and a decrease of 0.15 V in the peak-to-peak
difference of caffeic acid. Moderate decrease in the overpotentials
was shown in the cases of ferulic and p-coumaric acids, therefore 1
the electrochemical processes of these monophenols appeared to
be less prone to the beneficial effect of CNT in their voltammetric a
0
response.
It can be concluded from these experiments that electrodes 0.0 0.2 0.4 0.6 0.8 1.0
modified with the CNT dispersion in PEI offered the most promis- Potential / V
ing electrochemical performance for their possible application to
Fig. 1. Influence of the flow rate (A) and the detection potential (B) upon the
polyphenol analysis in wines, thus these electrodes were selected
amperometric signal of polyphenols at bare GCE (a) and GC/(CNT/PEI) (b). Injections
to be evaluated under flow conditions. of caffeic (squares), gallic (circles), ferulic (up triangle) and p-coumaric acid (down
triangle), 25 lM each. Carrier electrolyte, 100 (A) and 50 mM (B) acetate buffer of
pH 4.5 containing 100 mM NaCl. Sample loop, 100 lL. Detection potential in (A),
3.2. FIA of polyphenols with amperometric detection using CNT- +0.70 V. Flow rate in (B), 2.5 mL min 1. Each point is the mean value of 3 injections.

modified electrodes

The electroanalytical performance of GC/(CNT/PEI) coupled to a

FIA set up was evaluated through several experiments using caf-
feic, gallic, ferulic and p-coumaric acids as polyphenol probes and
acetate buffers as carrier electrolytes. The same experiments were
carried out using a bare GCE for comparison. These tests were fo-
cused in the optimisation of the experimental parameters in order
to establish a robust and reliable methodology for the amperomet-
ric detection and quantification of polyphenols, keeping in mind its
immediate application to wine samples.
The first parameter studied was the effect of varying the flow
rate, from 0.5 to 4.0 mL min 1, on the amperometric response of
25 lM caffeic and gallic acids at +0.70 V using 100 mM acetate buf-
fer of pH 4.5 with 100 mM NaCl as carrier electrolyte. In both cases,
the peak currents obtained at GC/(CNT/PEI) increased slightly with
the flow rate up to 2.0 mL min 1 levelling off after that (Fig. 1A). As
a consequence of the diffusive dispersion of analyte from the sam-
ple plug, broad peaks were obtained at flow rates below
2.0 mL min 1 whereas higher flow rates gave sharper signals. A
similar trend was observed when using the bare GCE but lower
currents (about three and two times lower at 2.0 mL min 1 for gal-
lic and caffeic acid, respectively) that stabilise at flow rate higher
than 3.0 mL min 1 were registered in the opposite. Regarding the
peak broadening observed, it was more important at electrodes
modified with the CNT dispersion and this effect could be related
to the CNT film porosity which increases the residence time of
the analyte in the electrode surface, therefore it takes more time
to restore the baseline signal after the sample injection. As a com-
promise between sensitivity and signal quality, a 2.5 mL min 1
flow rate was chosen for subsequent experiments.
Next, the attention was turned to the composition of the carrier
electrolyte, namely the pH, the buffer concentration and the ionic
strength. Therefore, the amperometric signal of 25 lM caffeic and
gallic acids at a detection potential of +0.70 V was employed to
optimise the carrier electrolyte composition. When 100 mM ace-
tate buffers covering the 3.5 to 5.5 pH range were tested, the peak
currents slightly increased up to a pH value of 4.5 and remained
constant after that for GC/(CNT/PEI) whereas almost constant cur-
rents were observed in the whole pH range for GCE (results not
shown). Consequently, the pH value of 4.5 was fixed for the follow-
ing evaluation of the effect of acetate concentration as well as the
ionic strength. In these cases, no variations were observed in the
amperometric currents obtained for acetate and NaCl concentra-
tions up to 200 mM (results not shown), thus a final carrier electro-
lyte composition consisting of 50 mM acetate buffer of pH 4.5
containing 100 mM NaCl was adopted hereafter. This composition
allowed a good buffering capacity combined with a high ionic
strength which can support the dilution of wine samples without
changing the pH or ionic strength of the carrier electrolyte.
Using these experimental conditions, the hydrodynamic vol-
tammograms of the four phenolic acids (25 lM each) were ob-
tained then for GC/(CNT/PEI) and bare GCE (Fig. 1B). In general,
the profile of the hydrodynamic waves obtained at bare GCE was
clearly more tailed than that provided by GC/(CNT/PEI), which only
needed about 0.15 V to attain the plateau in all cases whereas bare
GCE took about 0.30 V. This was accompanied with a negative shift
in the half-wave potential of ca 0.15 V for gallic and caffeic acids
and ca 0.10 V for ferulic and p-coumaric acids when comparing
 A. Sánchez Arribas et al. / Food Chemistry 136 (2013) 1183–1192 1187

GC/(CNT/PEI) with bare GCE. Under these conditions, the presence

of a CNT layer substantially improves the electrochemical perfor-
(A) 2

mance, reflected in clear plateau regions and lower overpotentials, 2 µA

when compared to bare GCE and this effect is more important for 3
1
compounds containing catechol-like groups, namely gallic and caf- 4
feic acids. According to that shown in the cyclic voltammetry
experiments reported in Section 3.1, the electrochemical oxidation
of these groups appears to be catalysed when using CNT, as previ-
b
ously suggested by Crevillén, Pumera, Gonzalez, and Escarpa
(2009), whereas ferulic or p-coumaric acids, which contains only 2
1
an hydroxyl substituent in their aromatic ring, displayed a moder- 3
ate decrease in their oxidation potential when compared to bare a 4
GCE. In our case, the combination of electrostatic interactions in-
duced by the dispersing polymer and the electrocatalytic proper- 0 10 20 30 40 50 60 70
ties of MWCNT can lead to a favoured orientation of analytes
resulting in such decrease in overpotentials. Another interesting (B) 2
feature that can be remarked from this experiment is that polyphe-
4
nols containing catechol-like groups can be detected at much low- 2 µA
er potential than all the other polyphenols, therefore these 1 3
compounds can be detected selectively and then quantified sepa-
rately. Therefore an electrochemical index of easily oxidizable
polyphenols (related to compounds with high antioxidant power)
can be estimated when the detection potential was set at +0.30 V b
whereas a complementary electrochemical index of total polyphe- 1 2
nols can be derived from measurements at +0.70 V. This conclusion 3
4
is in accordance to the findings that supported the methodology
proposed by Blasco et al. (2005), who employed a GCE in their a
work. The results obtained in our case, showing enhanced hydro- 0 10 20 30 40 50 60 70 80
dynamic profiles when using GC/(CNT/PEI), suggest a more robust Time / min
performance of CNT-modified GCE when compared to bare GCE.
The analytical characteristics of the amperometric detection of Fig. 2. Stability of the amperometric signal of bare GCE (a) and GC/(CNT/PEI) (b)
polyphenols using GC/(CNT/PEI) were evaluated using a series of after successive injection cycles of phenolic acids. Each cycle consisted of four
standard solutions of the four analytes with increasing concentra- successive injections of caffeic (1), gallic (2), ferulic (3) and p-coumaric (4) acids,
25 lM each. In (A), 10 cycles of injections were performed whereas in (B), one cycle
tions ranging from 1.0  10 7 to 1.0  10 4 M. The results obtained
of injections was followed by 20 successive injections of p-coumaric acid and then
are summarised in the Supplementary information Section, Table 3. six cycles were carried out. Detection potentials were +0.70 V in (A) and +0.80 V in
GC/(CNT/PEI) showed a remarkable enlargement on the concentra- (B). Other conditions as in Fig. 1B.
tion interval where the response was proportional to the analyte
concentration. Typically the response was linear within the whole
concentration range studied both at +0.30 or +0.70 V whereas the during the whole experiment. It is well-known that the electro-
linearity at bare GCE was limited up to 20 or 50 lM. Moreover, chemical oxidation of polyphenolic compounds (especially mon-
the correlation between the peak current and the concentration ophenols such as coumaric acid) in common carbon-based
of each analyte within the linear ranges was excellent at GC/ electrodes leads to the formation of polymeric derivatives that fi-
(CNT/PEI) and usually better than that observed at GCE. This lim- nally passivates the electrode surface and suppresses the electro-
ited linear range of GCE can be a result of a progressive surface chemical signal (Teófilo et al., 2008), as it has been demonstrated
fouling during the experiment which, on the opposite, is mini- in these experiments with bare GCE. Therefore it is clear that the
mised at GC/(CNT/PEI). The limits of detection (LOD) evaluated presence of the carbon nanotubes layer prevents the apparition
for the four analytes studied, using the two detection potentials, of any fouling symptom, even under severe conditions, and that
were below 0.1 lM when the GC/(CNT/PEI) were employed. Ten highly stable signals can be obtained, thus making these electrodes
fold higher LOD were calculated when using bare GCE at +0.30 V very attractive for analytical applications. This improvement in the
as well as for ferulic and p-coumaric acids at +0.70 V whereas more electrochemical signal is related to the presence of a high density
favourable values were obtained for caffeic and gallic acids. of edge-plane defects at the CNT surface (Ambrosi et al., 2010;
The stability and reproducibility of the amperometric signals Banks and Compton, 2005) which enhances the electron-transfer
were evaluated by performing several cycles of four successive kinetics and impedes fouling. Despite the controversy about the
injections of caffeic, gallic, ferulic and p-coumaric acids (25 lM influence of metallic and nanographite impurities in the electro-
for each analyte). The results obtained, displayed in Fig. 2A show chemical performance of CNT (Stuart & Pumera, 2011), it has been
that no signal variation was noticed when using GC/(CNT/PEI) after proved the importance of these edge-plane sites in the signal sta-
ten cycles of injections (40 injections) at a detection potential of bility shown by CNT towards the electrochemical oxidation of phe-
+0.70 V whereas an important decrease in the gallic, ferulic and nols (Arribas et al., 2007; Stuart and Pumera, 2011). The
p-coumaric acids signals (about the half of their initial signals) reproducibility of the signals of polyphenolic acids in these exper-
was suffered at bare GCE. This effect was more evident under more iments using the GC/(CNT/PEI), expressed as RSD (n = 10), was
severe experimental conditions, namely when a detection poten- 2.8%, 2.5%, 5.3% and 6.1% for caffeic, gallic, ferulic and p-coumaric
tial of +0.80 V was employed and 20 consecutive injections of acids, respectively. As a complement of these experiments, the in-
p-coumaric acid were carried out before six cycles of injections. ter-electrode reproducibility was estimated for GC/(CNT/PEI) by
Fig. 2B shows how the signals of phenolic acids provided by the replacing the electrode surface four times and using the ampero-
bare GCE decreased about 50% to 80% from their initial values metric signals of the four phenolic acids (3 injections for each com-
whereas the response of GC/(CNT/PEI) remained highly stable pound) at +0.70 V. Acceptable RSD values (10.8%, 11.0%, 10.2% and
1188 A. Sánchez Arribas et al. / Food Chemistry 136 (2013) 1183–1192
13.5% for caffeic, gallic, ferulic and p-coumaric acids, respectively)
of peak currents were obtained in all cases in this comparison,
reflecting the appropriate reproducibility in the CNT-casting pro-
cess. The main evidence of the last experiments is the remarkable
improvement in the signal stability conferred by the presence of
the CNT layer that supports the reliability of long-term analysis
of polyphenol samples without electrode conditioning, thus
enhancing the reproducibility as well as saving time.
3.3. Determination of total polyphenols in wine samples
For a practical application of the GC(CNT/PEI) electrode to pol-
yphenol analysis in wines, its performance should be robust and
reliable during long periods of time in order to analyse the higher
number of wine samples, independently of their type or polyphe-
nol content. In this sense, several studies were carried out to eval-
uate and demonstrate the utility of the GC/(CNT/PEI) for the
analysis of real wine samples using the FIA conditions proposed
in the previous section.
Initially, the stability of the amperometric signals at +0.30 and
+0.70 V was examined for repetitive injections of diluted white
and red wine samples (1:10 and 1:100 dilution factors, respec-
tively). As can be seen in the Fig. 3, the signals obtained using
the GC/(CNT/PEI) were stable for more than 30 min (15 injections)
at +0.70 V for both red and white wine. No fouling symptoms were
observed during these experiments both at +0.70 V or +0.30 V (re-
sults not shown) and the reproducibility of the analytical signals
(expressed as RSD) was lower than 2.7% (n = 15) in all cases. On
the opposite, an important signal decreasing was noticed when
the experiments were carried out with a bare GCE. For example,
only the 15% of the initial signal at +0.70 V remained after 10 injec-
tions of diluted red wine samples. Supplementing these experi-
ments, the reproducibility of the electrode modification was
estimated using the signals at +0.70 V of 5 different GC/(CNT/PEI)
(A)
2 µA
b samples spiked with Na2SO3 in a common concentration (0.1 g L 1)
a
0 5 10 15 20 25 30
(B)
2 µA
b flow of carrier electrolyte and cannot interact with sulphite before
a nals from typical substances found in the wine matrix. Finally,
0 5 10 15 20 25 30
Time / min
Fig. 3. Successive injections of ‘‘Alectum’’ white wine (A) and ‘‘Alcorta’’ red wine (B)
samples obtained using bare GCE (a) and GC/(CNT/PEI) (b). White and red wines
were subjected to 10- and 100-fold dilution, respectively, in carrier electrolyte.
Detection potential, +0.70 V. Other conditions as in Fig. 2.
after 20 injections of red and white wines and the calculated RSD
were 9.1% and 6.8% (n = 5), respectively. These results demonstrate
once again the improved resistance to passivation of the active
sites on the electrode surface in the presence of the CNT layer, thus
superior signal stability was obtained. Similarly to that mentioned
in the previous section, the electrochemical oxidation of polyphe-
nols, as well as other fouling substances present in wine matrix
such as sugars or organic acids, led to the formation of products,
typically polymers, that adsorbs onto the electrode surface and
hinders the subsequent electron transfer, therefore the electro-
chemical signal decreases as observed at bare GCE. The presence
of multiple ‘‘edge-plane’’ sites in the CNT guaranteed fast electron
transfer for remarkable extended time whereas the influence of
passivating products was minimised (Ambrosi et al., 2010; Banks
and Compton, 2005). Further long-term stability experiments
showed that one GC/(CNT/PEI) provided stable signals at +0.70 V
for 4 days (40 wine injections per day) before a decrease of 30%
from the initial signal was noticed.
The reliability of the GC/(CNT/PEI) response towards wine sam-
ples was also evaluated by conducting several experiments. The ef-
fect of the dilution rate of wine sample was checked in the 1:50 to
1:5 and 1:250 to 1:50 ranges for white and red wines, respectively.
The results obtained showed that the amperometric signals ob-
tained at +0.30 and +0.70 V were proportional to the dilution factor
in all cases (regressions of 0.997 and 0.996, n = 5, were obtained for
white and red wines, respectively). This resulted in an extended
range of useful dilution factors when compared to that obtained
by Kilmartin et al. (2001) using cyclic voltammetry at GCE. Since
the amperometric currents of white and red wines at dilution fac-
tors of 1:10 and 1:100, respectively, were high enough and were
within the linear range of gallic acid response (Supplementary
information, Table 3), these sample treatments were kept for sub-
sequent studies. The effect of other substances present in the wine
matrix upon the amperometric signal was studied next by record-
ing the response of injections of model wine samples diluted 10
times (Fig. 4A). In this case no signal variations were observed both
at +0.30 (not shown) or +0.70 V, therefore the contribution of eth-
anol, organic acids or sugars to the amperometric current of the
real samples can be considered negligible and the electrochemical
oxidation of polyphenols was the responsible of these signals un-
der these conditions. The effect of sulphite was also evaluated
since it is an additive usually employed as antioxidant in wine
making, especially in white wines (Jackson, 2008). Thus white wine
were employed for this purpose (injections ‘‘c’’ in Fig. 4A) and an
increase of only 4.4% compared to the initial signal was registered
at +0.70 V whereas no signal variation was observed at +0.30 V
(not shown). Makhotkina and Kilmartin, (2010) reported that the
main interference from sulphite arise from its rapid interaction
35
with quinone derivatives generated electrochemically, resulting
in the regeneration of the former polyphenol or the formation of
an electroactive sulphonic acid derivative. Thus an increase in
the voltammetric anodic signal of polyphenols is observed. In our
case, polyphenols are oxidised at the electrode surface and subse-
quently the oxidation products are rapidly flushed away by the
leaving the electrode surface. Therefore the proposed experimental
conditions allow to minimise the development of interfering sig-
white and red wine samples were spiked with gallic acid in order
35
to evaluate the recovery and some of the results obtained at
+0.70 V are displayed in Fig. 4B. It can be noticed that red and
white wines provided similar results and recovery values higher
than 92% and 94% were obtained at detection potentials of +0.30
and +0.70 V, respectively. The main conclusion extracted from
these studies is that the wine matrix does not interfere in the
 A. Sánchez Arribas et al. / Food Chemistry 136 (2013) 1183–1192 1189

(A) (B) f
1 µA c
b 3 µA

g j
e

i
d
c
a b
a
0 5 10 15 0 10 20 30

Time / min
Fig. 4. Interference (A) and recovery (B) studies of wine samples at GC/(CNT/PEI). Injections in (A) were model wine (a), ‘‘Alectum’’ white wine (b) and ‘‘Alectum’’ white wine
spiked with 0.10 g L 1 of Na2SO3 (c). Each sample was 10-fold diluted in carrier electrolyte. Injections in (B) were 2.0 (a), 5.0 (b), 10.0 (c), 20.0 (d), 50.0 (e) and 100 lM (f) gallic
acid standards, ‘‘Viñas del Vero’’ white wine (g) and ‘‘Viñas del Vero’’ white wine spiked with 20 lM gallic acid (h) and ‘‘Pueblo Viejo’’ red wine (i) and ‘‘Pueblo Viejo’’ red wine
spiked with 20 lM gallic acid (j). White and red wine samples were subjected to 10- and 100-fold dilution in carrier electrolyte, respectively. The diluted samples were spiked
with gallic acid. Detection potential, +0.70 V. Other conditions as in Fig. 2.

Table 1
Total polyphenols in white and red wines by electrochemical (EI 0.30 V and EI 0.70 V) and spectrophotometric (Folin–Ciocalteu assay, FC; 280 nm absorbance index, I280; colour
intensity index, CI) assays (three replicates each, n = 3).

Samples Year EI 0.30 Va EI 0.70 Va EI 0.7–0.3a,b EI 0.3/0.7c FCa I280 CI

White wine Marqués de Riscal 1 2009 9.7 49.2 39.5 0.246 104 7.6 0.13
Marqués de Riscal 2 2009 10.2 55.7 45.5 0.224 88.5 7.7 0.17
Martín Códax 1 2009 14.6 64.7 50.1 0.291 107 8.8 0.15
Martín Códax 2 2009 15.6 65.3 49.7 0.314 112 8.8 0.16
Martín Códax 3 2009 18.1 76.4 58.3 0.310 111 8.6 0.11
Alectum Albariño 2009 15.3 73.7 58.4 0.262 146 9.7 0.18
Viña Esmeralda 2009 12.3 53.1 40.8 0.295 80.0 5.1 0.08
Viñas del Vero 2009 11.6 61.6 50.0 0.232 128 10.0 0.24
Red wine Izadi 1 2006 203 590 387 0.524 914 50.6 10.7
Izadi 2 2006 182 573 391 0.465 953 52.4 9.55
Izadi 3 2006 164 550 386 0.425 942 53.0 8.47
Izadi 4 2006 159 585 426 0.379 951 49.9 8.29
Alcorta 1 2007 117 463 346 0.338 840 44.6 8.11
Alcorta 2 2007 126 490 364 0.346 859 46.2 6.80
Alcorta 3 2007 110 447 337 0.326 827 46.1 8.46
Alcorta 4 2007 122 558 436 0.280 923 48.1 7.90
Heredad 1 2006 104 439 335 0.310 969 49.2 9.70
Heredad 2 2006 100 451 351 0.285 972 47.6 9.63
Heredad 3 2006 108 491 383 0.282 968 48.4 9.66
Pueblo Viejo 1 2007 90.0 386 296 0.304 734 41.0 6.14
Pueblo Viejo 2 2007 116 492 376 0.309 793 43.5 6.11
a
Gallic acid equivalents (mg L 1).
b
Polyphenols with high oxidation potential, calculated as the difference amongst polyphenols detected at +0.70 V and +0.30 V.
c
Ratio amongst polyphenols with low oxidation potential (detected at +0.30 V) and high oxidation potential (EI 0.7–0.3 V).

electrochemical signal which can be assigned certainly to the
polyphenol content in the sample.
The polyphenol content of the nine wine brands (21 bottles ana-
lysed, 3 sample replicates were analysed in each assay) was mea-
sured using the FIA methodology and the results obtained,
expressed as gallic acid equivalents, are summarised in Table 1.
The polyphenol content in red wines was significantly higher than
that estimated in white wines, independently from the detection
potential. The comparison of the values obtained in this work with
those found in the literature using electrochemical detection is dif-
ficult, as described recently by Arribas, Martinez-Fernández, and
Chicharro (2012), because the scarce FIA examples and the more
frequent voltammetric ones have been carried out using bare
GCE and usually catechin as reference compound. Regarding to
FIA schemes, Mannino et al. (1998) obtained polyphenol contents
ranging 225–1050 mg L 1 and 40–145 mg L 1 (as catechin) for 16
red wines and 6 white wines, respectively, using a GCE and
+0.40 V as detection potential whereas Blasco et al. (2005) reported
945 (at +0.50 V) and 1050 mg L 1 (at +0.80 V) and 175 (at +0.50 V)
and 245 mg L 1 (at +0.80 V) when they analysed only one red wine
and one white wine, respectively. These authors employed the val-
ues obtained at low detection potentials as an antioxidant power
marker and the polyphenol contents reported are higher than that
shown in Table 1 (EI 0.30 V). This difference can be understood in
1190 A. Sánchez Arribas et al. / Food Chemistry 136 (2013) 1183–1192
the basis of the higher detection potentials and the reference com-
pound employed. In our case we detected more selectively poly-
phenols with low oxidation potential at +0.30 V (as shown in
Fig. 1B) whereas Mannino et al. (1998) and Blasco et al. (2005) de-
tected a wider range of polyphenols, probably because their goal
was to report a general antioxidant power index. Supplementing
this comparison, the works of Kilmartin, Zou, and Waterhouse
(2002) and Seruga et al. (2011) using voltammetric methods for
the estimation of total polyphenolic content are the only closer
comparative references available for this work. Kilmartin et al.
(2002) reported 40–125 mg L 1 and 1800–2400 mg L 1 of gallic
acid equivalents for five white wines and six red wines analysed,
respectively. In addition, Seruga et al. (2011) analysed 11 red wines
and found total polyphenol contents of 850–2500 mg L 1 as cate-
chin equivalents. The values obtained for white wines analysed
in this work (Table 1, EI 0.70 V) are similar to that reported by
Kilmartin et al. (2002) whereas for red wine are quite lower. This
difference can be explained taking into account that red wines ana-
lysed in this work are aged wines and during the ageing process
usually a diminution of the content of monomeric polyphenols
occurs. These former polyphenols were oxidised and polymerised
to more complex forms that have lower contribution to total
polyphenol indexes (Jackson, 2008; Roginsky et al., 2006).
Another important feature shown in Table 1 is the estimation of
the content of polyphenols with high oxidation potential (EI 0.7-
0.3; assigned to low antioxidant power) in the analysed wines. It
can be seen that the proportion of polyphenols detected at
+0.30 V, assigned to higher antioxidant power, was higher in the
red wine samples (especially ‘‘Izadi’’, ‘‘Alcorta’’ and ‘‘Pueblo Viejo’’
brands) whereas the presence of polyphenols with high oxidation
potential was notably noticed in white wines samples (‘‘Marqués
de Riscal’’, ‘‘Viñas del Vero’’ and ‘‘Alectum’’ brands). For example,
Aznar, Checa, Oliver, Hernández-Cassou, and Saurina (2011) and
Carrascosa et al. (2012) have reported the presence of significant
content of tyrosol and coumaric acid derivatives (compounds with
high oxidation potential) in Spanish red and white wines, respec-
tively. However other authors (Makris, Psarra, Kallithrakac, &
Kefalas, 2003; Paixao, Pereira, Marques, & Camara, 2008) have
shown variable contents of this type of polyphenols. Such a large
differences in the phenolic content between wines from different
regions and/or countries are likely to be result of grape varieties,
vineyard location, climate, soil type, different wine processing
techniques and ageing. These proportions (Table 1, EI 0.3/0.7)
might be employed as another qualitative parameter of wines
since these values are related to the particular distribution of
600
500
-1
EI / GAE mg L
400
300
200
100
0
0 250 500
FC / GAE mg L
Fig. 5. Correlation graphs of the electrochemical indexes (EI) at +0.30 V (squares) and +0.70 V (circles) with spectrophotometric assays: Folin–Ciocalteu (FC), 280 nm
absorbance index (I280) and colour intensity index (CI).
polyphenols thus they are characteristic for each sample. Therefore
they have an interesting potential for wine classification purposes.
It is also remarkable that the results obtained for different bottles
from the same brand and supplied from different markets were
reasonably similar, therefore the possible difference in the storage
time and/or conditions between such bottles did not affect to their
polyphenol content.
These results were compared to those obtained using the spec-
trophotometric assays, namely the Folin–Ciocalteu assay (FC), the
280 nm absorbance index (I280) and the colour intensity index
(CI). The polyphenol content estimated by the FC method was
notably higher than that obtained electrochemically but the corre-
lation of spectrophotometric and electroanalytical signals was
good (Fig. 5 and Table 4 in the Supplementary information Sec-
tion). Similar observations have been reported elsewhere (Blasco
et al., 2005; Kilmartin et al., 2002; Mannino et al., 1998; Seruga
et al. 2011). The FC assay is based in the absorbance of the blue
complex formed by the reduction of Mo(VI) species by reductant
substances present in the sample. It is well known that not only
polyphenols are able to reduce Mo(VI) but also other compounds
in the wine matrix (sulphite or reducing sugars among others)
can contribute to the spectrophotometric signal, therefore an over-
estimation of total polyphenols by this method is usually found
(Blasco et al., 2005; Escarpa and González, 2001; Everette et al.,
2010). Indeed, the polyphenol content values estimated by the
electrochemical method are expected to be more realistic but it
is also expected the good correlation of signals obtained using both
methods since they are based in the reducing ability of the com-
pounds responsible of signal generation. It should be noted that
better correlation was obtained using the electrochemical signal
at +0.70 V (less selective) than that at +0.30 V, related to the poly-
phenol fraction with higher antioxidant power. A similar trend in
the correlation results (Fig. 5 and Table 3 in the Supplementary
information Section) was observed when compared with I280, re-
lated to the presence of aromatic rings in the sample, and CI, re-
lated to the presence of coloured anthocyanins and condensed
tannins. The electrochemical results showed the higher correlation
values with the I280 index. It is notable the high correlation of the
results obtained from signals at +0.30 V (easily oxidizable polyphe-
nols) and it might be related to the presence of catechins in the
samples analysed, since these compounds possess easily oxidizable
orto-diphenolic groups (catechol-like structure) as well as they
strongly absorb at 280 nm (Makhotkina and Kilmartin, 2010). On
the contrary, CI presented lower correlation values when com-
pared to the electrochemical signals both at +0.30 or +0.70 V. This
750 1000 0 15 30 45 0 4 8 12
-1
I280 CI
 A. Sánchez Arribas et al. / Food Chemistry 136 (2013) 1183–1192
is probably a consequence of the smaller contribution of anthocy-
anins and the polymeric forms of condensed tannins to the electro-
chemical signal because these compounds usually contain more
mono-phenolic groups which are not able to be electrochemically
oxidised at moderate overpotentials (Aguirre et al., 2010;
Makhotkina and Kilmartin, 2010).
The high correlation degree observed in the analysis of these
wine samples, even when the polyphenol content was very differ-
ent among them, demonstrates the practical application of the GC/
(CNT/PEI) electrode to the determination of different relevant frac-
tions of polyphenols and can be considered an appropriate alterna-
tive to the spectrophotometric assays.
4. Conclusion
This work demonstrates that the GC/(CNT/PEI) showed excel-
lent electrochemical performance for the detection of caffeic, gallic,
ferulic and p-coumaric acids using a FIA set up, with good sensitiv-
ity, wide linear range and excellent stability. These valuable prop-
erties were also demonstrated for the amperometric detection of
total polyphenols in wines. The presence of the CNT layer con-
ferred a highly stable and robust electrochemical response of these
GC/(CNT/PEI) during the analysis of different wine samples for ex-
tended periods of time. The results obtained for the different sam-
ples assayed showed a high correlation with those found using the
spectrophotometric methods.
The highly stable response of the electrochemical transducers
integrating the CNT nanostructures, as evidenced in this work,
proves the actual possibilities of these devices for real applications
in routine analysis where stable and robust responses are required
during long periods of time. Other important feature of the pro-
posed protocol is the possibility to estimate the content of poly-
phenols with low oxidation potentials (high antioxidant power)
as well as the total polyphenol amount in a fast, simple and direct
way just by changing the detection potential. Moreover, the excel-
lent electroanalytical properties of these GC/(CNT/PEI) anticipate
that useful methodologies for the polyphenol mapping and the
characterisation of wine types can be readily developed in connec-
tion to HPLC or capillary electrophoresis.
Acknowledgements
The authors wish to thank the Ministerio de Ciencia e Innova-
ción (Grant CTQ2009-09791) for financial support and M. M.-F. fel-
lowship (BES-2010-037292).
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.foodchem.2012.
09.027.
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