Total antioxidant capacity of some commercial fruit juices: Electrochemical and spectrophotometrical approaches

Article abstract of DOI:10.3390/molecules14010480

The aim of this paper was to assess the total antioxidant capacity of some commercial fruit juices (namely citrus), spectrophotometrically and by the biamperometric method, using the redox couple DPPH· (2,2-diphenyl-1- picrylhydrazyl)/DPPH (2,2-diphenyl-1

Full text of DOI:10.3390/molecules14010480

Molecules 2009, 14, 480-493; doi:10.3390/molecules14010480
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Total Antioxidant Capacity of Some Commercial Fruit Juices:
Electrochemical and Spectrophotometrical Approaches
1
2
2,
Aurelia Magdalena Pisoschi , Mihaela Carmen Cheregi and Andrei Florin Danet *
1
Chemistry and Biochemistry Department, Faculty of Veterinary Medicine, University of
Agronomic Sciences and Veterinary Medicine, bulevardul Marasti, no 59, Bucharest, Romania; E-
mail: apisoschi@yahoo.com (A-M. P.)
2
Analytical Chemistry Department, Faculty of Chemistry, University of Bucharest, Panduri Avenue
9
0-92, code 050657, Bucharest, Romania
*
Author to whom correspondence should be addressed; E-mail: andreidanet@yahoo.com.
Received: 26 November 2008; in revised form: 26 December 2008 / Accepted: 14 January 2009 /
Published: 20 January 2009
Abstract: The aim of this paper was to assess the total antioxidant capacity of some
commercial fruit juices (namely citrus), spectrophotometrically and by the biamperometric
method, using the redox couple DPPH· (2,2-diphenyl-1-picrylhydrazyl)/DPPH (2,2-
®
diphenyl-1-picrylhydrazine). Trolox was chosen as a standard antioxidant. In the case of
the spectrophometric method, the absorbance decrease of the DPPH· solution was
followed. For the biamperometric method, the influence of some parameters like the
®
potential diference, ΔE, DPPH· concentration, and Trolox concentration was investigated.
®
The calibration graph obtained for Trolox presents linearity between 5 and 30 µM, (y =
0
.059 x + 0.0564, where y represents the value of current intensity, expressed as μA and x
®
2
the value of Trolox concentration, expressed as μM; r = 0.9944). The R.S.D. value for
®
the biamperometric method was 1.29% (n = 10, c = 15 μM Trolox ). In the case of the
®
spectrophotometric method, the calibration graph obtained for Trolox presents linearity
between 0.01 and 0.125 mM (y = -9.5789 x+1.4533, where y represents the value of
®
2
absorbance and x, the value of Trolox concentration, expressed as mM; r = 0.9963). The
R.S.D. value for the spectrophotometric method was 2.05%. Both methods were applied to
total antioxidant activity determination in real samples (natural juices and soft drinks) and
the results were in good agreement.

Molecules 2009, 14
481
Keywords: Antioxidant activity; 2,2-Diphenyl-1-picrylhydrazyl; 2,2-Diphenyl-1-
picrylhydrazine; Biamperometry; Pt electrode; Citrus juices.
1
. Introduction
Oxidation is one of the most important free radical-producing processes in food, chemicals and
even in living systems. Free radicals play an important role in food and chemical material degradation,
contributing also to more than one hundred disorders in humans [1-6]. Highly reactive free radicals
and oxygen species present in biological systems can oxidize nucleic acids, proteins and lipids,
initiating degenerative diseases [7, 8]. Antioxidants significantly delay or prevent the oxidation of
easily oxidable substrates.
Plants contain high concentrations of numerous redox-active antioxidants, such as polyphenols,
carotenoids, tocopherols, glutathione, ascorbic acid and enzymes with antioxidant activity, which fight
against hazardous oxidative damage of plant cell components. In animal cells, antioxidant production
is much more limited and oxidative damage is involved in the pathogenesis of most chronic
degenerative diseases (including cancer and heart diseases) and aging [9-11]. Therefore, plant-sourced
food antioxidants like vitamin C, vitamin E, carotenes, phenolic acids, phytates and phytoestrogenes
have been recognized as having the potential to reduce disease risk.
The intake of food rich in α-tocopherols, β-carotene and ascorbic acid has been associated with
reduced oxidative-stress related diseases. Phenolic acids, polyphenols and flavonoids scavenge free
radicals such as peroxide, hydroperoxide or lipid peroxyl, thus inhibiting the oxidative mechanism that
lead to degenerative diseases [12-15].
Many analytical methods have been used for antioxidant monitoring, for instance phenolics in fruit
have been monitored by HPLC [16, 17] or colorimetrically using the Folin Ciocalteu reagent [18]. The
total antioxidant capacity of foods and plant extracts has been assessed by using spectrophotometric
+
methods with DPPH· (2,2-diphenyl-1-picrylhydrazyl) [19-21], ABTS · (2,2’-azinobis(3-ethylbenzo-
3
+
thiazoline-6-sulfonic acid)) [19, 20] or Fe -TPTZ (2,4,6-tripyridyl-s-triazine) [22].
The antioxidant content and total antioxidant activity were also evaluated electrochemically, by
means of voltammetric or amperometric methods. Phenolics were determined by HPLC and FIA with
amperometric detection [23], amperometric biosensors [24] and voltammetric techniques [25].
Biamperometric determination of the total antioxidant capacity uses redox couples like DPPH·/DPPH
+
[
26] or ABTS· /ABTS, where the ABTS radical cation is bienzymatically produced by glucose oxidase
and peroxidase, immobilized in a flow-through reactor [27]. Cyclic voltammetry results of antioxidant
capacity determination in buckwheat products showed good correlation with the data obtained by
spectrophotometry with DPPH· [28].
The aim of this work is to perform a comparative study regarding the determination of total
antioxidant capacity of some fruit juices, by using the spectrophotometric method with DPPH· and
biamperometry with a DPPH·/DPPH redox couple.
Spectrophotometry–principle of the method: the spectrophotometric method for assessing the total
antioxidant activity is based on the absorbance decrease monitoring of the DPPH· radical (2,2-

Molecules 2009, 14
482
diphenyl-1-picrylhydrazyl) in the presence of antioxidants. DPPH· is characterized as a stable free
radical due to the delocalization of the spare electron over the molecule. Thus, the molecule cannot
dimerise, as would happen with other free radicals. The delocalization gives rise to a deep violet
colour characterized by an absorption band at about 520 nm. When a DPPH· solution is mixed with a
substance which can donate a hydrogen atom (see Figure 1), the reduced form is generated,
accompanied by the loss of the violet colour [19, 21, 29].
Figure 1. Reaction of DPPH· free radical with an antioxidant.
Biamperometry–principle of the method: the biamperometric method is based on the measurement of
the current flowing between two identical Pt working electrodes polarized at a small potential
difference and immersed in a solution containing a reversible redox couple. Indirect biamperometric
measurement relies on reaction of the analyte with the indicating redox couple, its selectivity
depending on the specificity of the reaction involving the oxidized or reduced form of the redox pair
3
+
2+
-
3-
4-
and the analyte. Fe /Fe , I
2
/I , Fe(CN)
6
/Fe(CN)
6
are redox couples commonly used in
biamperometric measurements. The redox pair chosen in this study was DPPH·/DPPH. Antioxidants
react with DPPH· (radical form) generating DPPH (reduced form), like in the reaction presented in
Figure 1, the intensity of the resulted current being proportional to the residual concentration of DPPH·
after its reaction with the analyte (antioxidant).
In the present study, we used two identical Pt electrodes where the reduction of the DPPH· radical
and oxidation of the reduced form (DPPH) take place as follows:
-
-
Electrode 1: DPPH· + e → DPPH
Electrode 2: DPPH → DPPH· + e
The reduction of DPPH· at electrode 1 gives rise to a cathodic current, while the oxidation of DPPH
at electrode 2 generates an anodic current. The biamperometric detector response is linear for that
constituent of the redox couple which is present in lower concentration. Working conditions were
chosen for a DPPH· concentration smaller than DPPH concentration. Each antioxidant addition in a
solution containing the couple DPPH·/DPPH decreases the concentration of the oxidized (radical) form
and increases the concentration of the reduced form, thus generating a current proportional to the
concentration of antioxidant. In the case of the proposed method, cathodic current is limited by the
lower concentration of DPPH· radical in the indicating mixture [26].

Molecules 2009, 14
483
2
. Results and Discussion
2
.1. Spectrophotometry: the study of DPPH· absorbance decrease in the presence of antioxidants
Following the explained working procedure (see Experimental), the value of the absorbance
®
diminishes as the antioxidant concentration increases because more DPPH· is quenched by Trolox . It
®
can be noticed that the absorbance signal for blank reached the steady state two minutes after Trolox
addition and therefore the following readings were done at this time point.
®
The calibration graph (Figure 2) is linear in the range 0.01 to 0.125 mM for Trolox , with an
equation of y = -9.5789 x + 1.4533 (where y represents the value of absorbance and x, the value of
®
2
Trolox concentration, expressed as mM) and a correlation coefficient of r = 0.9963. The flattening of
®
the graph which begins at 0.150 mM Trolox concentration is a consequence of DPPH· almost
®
complete quenching by Trolox . The value of inhibition (Q%, defined as: 100x(A -A )/A , where A
0
c
0
0
®
represents the absorbance for blank and A
c
, the absorbance at different values of Trolox
®
concentration) calculated for 0.150, 0.175 and 0.200 mM Trolox on the final solution were 89.00,
9
5.86% and 96.66%, respectively.
®
Figure 2. Absorbance variation for DPPH· 0.12 mM, in the presence of different Trolox
concentrations.
2
.2. Electrochemical method
2
.2.1. Study of the reversibility of the DPPH·/DPPH redox couple
To verify the reversibility of the redox DPPH·/DPPH couple, cyclic voltammetry experiments were
2
performed with a three electrode electrochemical cell equipped with two Pt strip electrodes (30 mm
surface) and a saturated calomel electrode as reference. The potential was swept from -100 to 600 mV.
The registered voltammogram (Figure 3) shows the characteristics of a reversible redox couple, that is
a 59 mV difference between the anodic peak corresponding to DPPH oxidation and cathodic peak
corresponding to DPPH· reduction.

Molecules 2009, 14
484
Figure 3. Cyclic voltammogram obtained for the DPPH·/DPPH redox couple; conditions:
DPPH· concentration = 0.5 mM; DPPH concentration = 0.5 mM; potential sweep speed =
0 mV/s; (for details see Experimental).
5
2
.2.2. Study of the influence of the potential difference, ΔE, applied between the two electrodes on the
analytical signal
The influence of the potential difference, ΔE, applied between the two electrodes, on the analytical
signal was studied and a series of chronoamperograms were registered in the two-electrode
electrochemical cell (see Figure 4). The value of the current intensity obtained at different values of
ΔE and read after one minute, was plotted against DPPH· concentration (Figure 5). By analysing the
data plotted in Figures 4 and 5, we notice that the current value on the plateau due to DPPH oxidation
and DPPH· reduction, increases with the potential difference, ΔE.
The results presented in Figure 5 prove that the analytical signal is the greatest at 200 mV potential
difference. Moreover, a proportionality can be noticed between current intensity and DPPH·
concentration. Therefore, this value of the potential difference was chosen for investigating the
®
influence of the Trolox concentration and real sample analysis. Greater values of the potential
difference were not taken into account, because of electrode reactions which can occur at the electrode
surface, involving other compounds present in the analysed sample, thus giving rise to interferences.

Molecules 2009, 14
485
Figure 4. Chronobiamperograms illustrating the influence of the potential difference, ΔE:
1) 50 mV, (2) 100 mV, (3) 150 mV, (4) 200 mV; conditions: DPPH· concentration = 100
(
µM, DPPH concentration = 110 µM; (for details see Experimental).
Figure 5. The analytical response plotted against DPPH· concentration at different values
of ΔE: (1) 50 mV, (2) 100 mV, (3) 150 mM, (4) 200 mV; DPPH concentration = 110 µM.
(
for details see Experimental).
®
2
.2.3. Study of the influence of the Trolox concentration on the analytical signal
®
Increasing concentrations of Trolox , from 5 to 30 µM, were added to the DPPH·/DPPH mixture
solution and the obtained chronoamperograms are presented in Figure 6.

Molecules 2009, 14
486
®
Figure 6. Chronobiamperograms obtained for different Trolox concentrations: (1) 0 µM,
2) 5 µM, (3) 10 µM, (4)-15 µM, (5) 20 µM, (6) 25 µM, (7) 30 µM; conditions:110 µM
DPPH/100 µM DPPH·, potential difference, ΔE = 200 mV (for details see Experimental).
(
The analytical signals obtained for different concentrations of DPPH· and a concentration of 110
®
μM DPPH, as well as for different Trolox concentrations, in the presence of 100 μM DPPH· and 110
μM DPPH were represented in Figure 7. The biamperometric detector responses were read at 60
®
seconds. In both cases, the value of the intensity registered for 0 μM DPPH· or 0 μM Trolox
concentration (blank) was substracted from the analytical signal obtained at different concentrations of
®
®
DPPH or Trolox , respectively. The obtained ΔI values were plotted against DPPH· and Trolox
concentrations (see Figure 7).
Figure 7. Analytical response of the biamperometric detector plotted against DPPH·
®
concentrations (1) for 110 μM DPPH and ΔE = 200 mV and Trolox concentrations; (2)
for 100 μM DPPH· and 110 μM DPPH, ΔE = 200 mV (for details see Experimental).

Molecules 2009, 14
487
The slope difference between the two graphs presented in Figure 7 can be explained in terms of the
®
stoichiometry of the reaction DPPH·/Trolox and the way the solvent affects DPPH· concentration, as
reported previously [26, 29, 30]. Molyneux [21] also observed a difference between DPPH· theoretical
(
stoichiometrical) concentration and the one reacting with ascorbic acid.
According to the experimental data plotted in Figure 7, 100 µM DPPH· is equivalent to
®
approximately 20 µM Trolox , which corresponds to a approximate 5/1 ratio. Considering 2/1
®
stoichiometry for the reaction between DPPH· and Trolox [29], that implies a difference between
calculated DPPH· and the one determined in the experiment. The result is a 60 µM difference between
®
prepared DPPH· concentration (100 µM) and the one reacting with Trolox (40 µM). This discrepancy
®
is confirmed by the results found by other authors for the reaction DPPH·/Trolox and it can be
explained by the fact that the solvent (ethanol) affected active DPPH· concentration [26, 30].
Milardovic et al. [30] found a 40 µM difference between prepared DPPH··and the one determined in
®
the biamperometric experiment. The spectroscopic calibration curve for Trolox , obtained using 200
µM DPPH· in ethanol-water solution shows the same equivalent concentration range as established by
®
the calibration curve for Trolox , determined amperometrically [30]. The same equivalent
concentration of DPPH in the radical form, determined spectrometrically and electrochemically
implies that the active DPPH· concentration was affected by the solvent, which agrees with other
literature data [21]. In addition to that, in the case of ethanol, solvent properties, such as the ability to
form hydrogen bonds with the antioxidant, influence the level of the relative activity towards
DPPH·[31].
®
The accuracy of biamperometric measurements of Trolox was excellent, RSD% = 1.29% (c = 15
®
µM Trolox , n = 10). The detection limit (calculated as 3 x the standard deviation of the blank signal)
was 0.0768 µM, whereas the limit of quantification (calculated as 10 x the standard deviation of the
®
blank signal) was 0.256 µM. The Trolox calibration curve [Figure 7 (2)] was employed for the
analysis of real samples (commercial, as well as juices obtained by fruit squeezing).
2
.3. Analysis of real samples
Natural juices obtained by squeezing fruit, which contained fruit pulp, needed previous
centrifugation and 10-fold dilution with distilled water, while clear soft drinks did not necessitate any
special sample preparation before analysis.
Spectrophotometry: For soft drinks, the dilution degree varied between 1/2 and 1/50, while for
juices obtained by pressing fruit greater dilution degrees are required, between 1/100 and 1/150. The
®
working procedure used for standard antioxidant solutions (Trolox ) was also applied to fruit juices.
The biamperometric method was also applied for total antioxidant capacity determination in juice
real samples. For soft drinks, the dilution degree was comprised between 1/5 and 1/400, whereas for
juices obtained by pressing fruit, the dilution degree ranged between 1/500 and 1/750. The
experimental conditions and working procedure were the same as those used for the standard
®
antioxidant, Trolox . The obtained results are presented in Table 1. The highest values of TEAC
(
Trolox Equivalent Antioxidant Capacity) were registered for natural juices obtained by squeezing
fruit.

Molecules 2009, 14
488
The previously mentioned results also prove that the greatest TEAC values are obtained for natural
products: juices obtained by pressing fruit and fruit extracts.
Table 1. Results, expressed as TEAC obtained for total antioxidant activity determination
in fruit juices by spectrophotometry and biamperometry and comparison with literature
data.
Spectrophotometry
Biamperometry
Literature Data
ΔI value
TEAC value
TEAC value
(biamperometry)
mM Trolox
Absorbance
value
(biampero
metry)
µA
TEAC values (mmoles
Product
(spectrophotometry)
mM Trolox
Trolox/kg or mM Trolox)
Orange juice
9.25
0.89
9.07
0.64
8.74 mmoles/kg
fresh weight orange by
+
.
ABTS method [13]
.88 mM
sweet oranges extract by
8
*
**
DMPD /FeCl method [32]
3
Lemon juice
6.5
0.86
6.25
0.68
7.0 mmoles/ kg
***
fruit (limes) by FRAP [7]
.3 mmoles Kg fruit (limes)
7
by FRAP [33]
Fanta orange
Cappy
0.71
1.03
1.15
0.70
0.69
0.67
-
-
0.064
0.0615
grapefruit
Frutti fresh
Tutti Frutti
Fanta lemon
4.0
0.732
0.870
4.32
1.45
0.51
0.74
-
1.54
2.20 mM
lemon soft beverage by
+
.
ABTS method [13]
Prigat orange
Prigat peach
2.40
1.24
0.790
0.795
2.46
1.20
0.67
0.63
3.02 mM
orange soft beverage
+
.
by ABTS method [13]
2.51 mM
peach soft beverage by
+
.
ABTS method [13]
*
The value represents the hydrophilic antioxidant activity (HAA) [32], determined for the aqueous
citrus extract; **N,N-dimethyl-p-phenylenediamine; ***ferric reducing antioxidant power assay.
®
For verifying the degree of recovery, known Trolox amounts (from a more concentrated, 1 M
solution, with the exception of Cappy grapefruit, for which a 100 mM Trolox solution was used) were
added to the respective juices, which were subsequently analysed by using the experimental conditions
®
described at the working procedure. Due the small volume required from the concentrated Trolox
solution (comprised between 6 and 94 µl) no correction of volume was taken into account for

Molecules 2009, 14
489
calculating the degree of recovery (see Table 2). As can be seen in Table 1, the results obtained in this
study are in good agreement with the previously published data.
Table 2. Determination of the degree of recovery of known concentrations of Trolox
introduced in the analysed samples, by using the biamperometric method.
Measured TEAC
Recovery Measured TEAC value
Recovery
value (mM
Trolox) after 1st
addition
1.73
st
nd
Product
% after 1
addition
(mM Trolox) after 2nd % after 2
addition
addition
Prigat peach
Prigat orange
Fanta lemon
Fanta orange
Cappy grapefruit
Frutti freshTutti Frutti
Lemon juice
101.76
101.15
102.56
102.10
101.15
97.47
2.16
4.45
2.48
1.21
0.11
8.14
11.2
16.80
98.18
99.77
101.22
100.83
98.65
97.83
99.55
101.39
3.50
2.0
0.97
0.0875
6.16
8.7
13.05
99.42
101.80
Orange juice
3
. Conclusions
A sensitive and rapid biamperometric method was designed for monitoring the total antioxidant
content in fruit juices. The method has proved its accuracy by the degrees of the recovery obtained for
®
known quantities of Trolox , added to the analysed samples ranging between 97.47 and 102.56%, as
well as the results comparable to those of the spectrophotometric method. The precision of the method
®
was proved by the R.S.D. value of 1.29% (n = 10, c = 15 μM Trolox ).
Although the dynamic and linear ranges are more extended in the case of the spectrophotometric
method (0.01-0.125 mM linearity for the spectrophotometric method and 5-30 μM for the
biamperometric method), the biamperometric method has turned out to be more sensitive, allowing the
®
detection of concentrations as low as 5 µM Trolox .
The results obtained in this study are in good agreement with the ones previously published in
literature, for similar products [7, 13, 32], as shown by the comparison presented in Table 1. Thus,
biamperometry can be successfully used in food industry for assessing the antioxidant content of
natural fruit juices and soft drinks, being an accurate, rapid and relatively cheap method.
4
. Experimental
4
.1. Reagents and solutions
®
2
,2-Diphenyl-1-picrylhydrazyl (DPPH·), 2,2-diphenyl-1-picrylhydrazine (DPPH) and Trolox
(
Sigma Aldrich). Potassium dihydrogen phosphate and sodium monohydrogen phosphate (Riedel de
®
Haën). Potassium chloride (Sigma Aldrich). Methanol and ethanol (Sigma Aldrich). Trolox was
chosen as water-soluble antioxidant for both methods and a 1mM stock solution was prepared by
dissolving the antioxidant in distilled water which was previously boiled and chilled until it reached
room temperature. For spectrophotometric experiments, DPPH· was dissolved in methanol as to obtain

Molecules 2009, 14
490
®
a stock solution of 0.24 mM concentration. For the working antioxidant solutions, Trolox
concentrations varied between 0.01 and 0.20 mM and were prepared by diluting the stock solution
with methanol.
For all chronoamperometric experiments, the DPPH·/DPPH mixture solution was prepared by
DPPH· and DPPH dissolution in an ethanolic phosphate buffer solution, so as to reach concentrations
of 150 µM DPPH· and 165 µM DPPH, respectively. For the cyclic voltammetry experiments, the
DPPH·/DPPH mixture solution of 0.5 mM (each component) was prepared by dissolution of the
necessary amount of DPPH·/DPPH in the ethanolic phosphate buffer. The ethanolic phosphate buffer
solution was prepared by mixing 0.055 M potassium dihydrogen phosphate with 0.055 M sodium
monohydrogen phosphate in an 1/4 ratio in order to reach a pH = 7.40, adding absolute ethanol, up to
4
0% (v/v) concentration in the final solution, and then solid KCl as supporting electrolyte, up to 0.33
M concentration in the final solution.
4
.2. Instrumentation
Spectrophotometer Spekol 11 Carl Zeiss Jena; potentiostat-galvanostat coupled to computer
(
laboratory-made by Prof. dr. Slawomir Kalinowski, Warmia and Mazury University, Olsztyn,
Poland); magnetic stirrer (Metrohm AG); centrifuge (Qualitron, Korea); two identical Pt strip
2
electrodes (Radelkis, 30 mm surface, mounted in a 25 mL electrochemical cell) for biamperometric
determinations. In the case of cyclic voltammetry experiments, these Pt strip electrodes were used as
working and auxiliary. A saturated calomel electrode (Radelkis) was introduced as reference. The
scheme of the biamperometric system is presented in Figure 8.
Figure 8. Scheme of the biamperometric system with two identical Pt electrodes (1 and 2)
used for measuring the total antioxidant activity; the potentiostat enables measuring of the
current intensity (I), at a fixed (applied) voltage (V); electrode 1 is connected to the
WORKING ELECTRODE output, while electrode 2 is connected to the REFERENCE and
AUXILIARY outputs of the potentiostat.
Working procedure: The spectrophotometric method: the value of the absorbance was monitored at
5
15 nm using a 1 cm spectrophotometric cell. The blank solution (0.12 mM DPPH·) was prepared by

Molecules 2009, 14
491
diluting the 0.24 mM solution with methanol in 1/1 ratio). The analysed samples were measured versus
the blank sample. 10 mL volumetric flasks were used, in which 0.24 mM DPPH· solution (5 mL) and
®
different volumes of 1 mM Trolox solution (or a volume comprised between 0.12 and 5 mL of the
analysed sample) were added, then methanol was added up to 10 mL, so that Trolox5 mL of solution
concentrations varied within the range 0.01 mM - 0.20 mM in the final solution. The decrease of
DPPH· absorbance was monitored.
In the case of the electrochemical method, 25 mL volumetric flasks were used, to which the
indicating mixture solution (16.7 mL) containing 150 µM DPPH· and 165 µM DPPH were first added,
so as to have 100 µM and 110 µM concentrations, respectively, in all final solutions. Then, different
®
volumes of 1 mM Trolox solution were added. The ethanolic phosphate buffer solution was made up
®
to 25 mL volume, so that Trolox concentrations varied within the range 5 - 30 µM in the final
solutions. Before each determination, both Pt electrodes were cleaned electrochemically in 1.25 M
H
2
SO
ultrasonically in a water bath for two minutes. All experiments were performed under stirring using a
5 mL electrochemical cell. For the biamperometric experiments we employed a ΔE of 200 mV
potential difference between the two electrodes.
4
solution by applying four potential pulses of -1.5 V (versus SCE) for three seconds and
2
5
. Acknowledgements
This work was performed with the support from the Romanian National Agency for Research, in
the frame of the Research Projects no. 183, CNCSIS code TD-387 and PN II 71-098/2007.
6
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Sample Availability: Samples are available from the authors.
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2009 by the authors; licensee Molecular Diversity Preservation International, Basel, Switzerland.
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