Food Chemistry 173 (2015) 10591065

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

Analytical Methods

Rapid determination of total polyphenolic content in tea samples based

on caffeic acid voltammetric behaviour on a disposable graphite
electrode
Iulia Gabriela David a,, Ana-Maria Cristina Bizgan a, Dana Elena Popa a, Mihaela Buleandra a,
Zenovia Moldovan a, Irinel Adriana Badea a, Tuge Aya Tekiner b, Huveyda Basaga b, Anton A. Ciucu a
a
Department of Analytical Chemistry, Faculty of Chemistry, University of Bucharest, 4-12 Regina Elisabeta Av., 030018, District 3, Bucharest, Romania
b
Biological Sciences and Bioengineering Program, Sabanci University, 34457 Orhanl-Tuzla, Istanbul, Turkey

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

Article history: The present paper describes the voltammetric behaviour and the quantitative determination of caffeic acid
Received 8 May 2014 (CA) on a disposable pencil graphite electrode (PGE). The anodic peak current of CA recorded by differential
Received in revised form 23 October 2014 pulse voltammetry (DPV) varies linearly with CA concentration in the range 1  1073  103 M. The
Accepted 25 October 2014
detection and quantication limits were 8.83  108 M and 2.94  107 M caffeic acid, respectively. The
Available online 3 November 2014
mean recoveries of CA from Turkish green, white and black teas were 98.30%, 99.57% and 91.46%. For these
three tea types the corresponding total polyphenolic contents (TPCs) evaluated by DPV on PGE were 35.81,
Keywords:
34.59 and 31.21 mg caffeic acid equivalent/g tea, respectively. These TPC values were in good accordance
Caffeic acid
Pencil graphite electrode
with those obtained by the FolinCiocalteu method. The developed DPV on PGE method constitutes a
Total polyphenolic content simple and inexpensive tool for the rapid assessment of TPC of tea samples.
Tea 2014 Elsevier Ltd. All rights reserved.

1. Introduction investigation of polyphenols electrochemical behaviour and oxida-

Polyphenols are natural compounds with multiple benec
effects on human health as a result of their high antioxidant
activity generated by to the presence of phenolic groups in their
chemical structure. Based on the existence of a catechol group in
its molecule, caffeic acid (CA) (Fig. 1) is one of the most effective
polyphenolic antioxidants helping to prevent some diseases
caused by oxidative stress, like cancer and cardiovascular
diseases.
It was demonstrated that there is a correlation between the
antioxidant properties of polyphenols and their electrochemical
behaviour, i.e. a lower anodic peak potential indicates a stronger
antioxidant activity (Abou Samra, Chedea, Economou, Calokerinos,
& Kefalas, 2011; Simic, Manojlovic, Segan, & Todorovic, 2007)
whereas the electrochemical index, also called antioxidant
index, dened as the total phenolic content determined by electro-
chemistry, introduced by Blasco, Rogerio, Gonzalez, and Escarpa
(2005) was used for the determination of the antioxidants content
of agricultural products, herbs and herb extracts. Thus, electro-
chemical methods have proven to be powerful tools for the
Corresponding author. Tel.: +40 744884719; fax: +40 21 3159249.
E-mail address: gabrielaiulia.david@g.unibuc.ro (I.G. David).
tion mechanisms (Giacomelli, Ckless, Galato, Miranda, & Spinelli,
2002; Kallel Trabelsi, Belhadj Tahar, & Abdelhedi, 2004; Sochor
et al., 2013), for the determination of the total polyphenolic content
and of the antioxidant capacity of different matrices (Blasco et al.,
2005; Novak, eruga, & Komorsky-Lovric, 2010; Photinon,
Chalermchart, Khanongnuch, Wang, & Liu, 2010; Rebelo, Rego,
Ferreira, & Oliveira, 2013; Sen & Sarkar, 2013; Sousa, da Rocha,
Cardoso, Silva, & Zanoni, 2004; Tyszczuk, Skalska-Kaminska,
& Wozniak, 2011; Veljkovic et al., 2013; Ziyatdinova, Aytuganova,
Nizamova, & Budnikov, 2013; eruga, Novak, & Jakobek, 2011).
Voltammetric methods using different working electrodes are
often preferred for various types of analysis due to their high sen-
sitivity, quickness, simplicity, the possibility of performing analysis
in coloured or turbid solutions as well as the portability of the
instrumentation. Due to its inherent advantages (good electrode
material, disposable, cheap and commercial available) the pencil
graphite electrode (PGE) was used as working electrode for
the voltammetric analysis of various compounds (Aydinlik,
Ozkan-Ariksoysal, Kara, Sayiner, & Ozsoz, 2011; David, Badea, &
Radu, 2013; David, Matache, Radu, & Ciucu, 2013).
Polyphenols, including caffeic acid, are often found in herbs
(Tyszczuk et al., 2011), teas (Sen & Sarkar, 2013; Veljkovic et al.,
2013; Novak et al., 2010), coffee (Ziyatdinova et al., 2013), wine

http://dx.doi.org/10.1016/j.foodchem.2014.10.139
0308-8146/ 2014 Elsevier Ltd. All rights reserved.
1060 I.G. David et al. / Food Chemistry 173 (2015) 10591065
(Mudnic et al., 2010; Photinon et al., 2010; Rebelo et al., 2013;
eruga et al., 2011), vegetables, fruits and fruit juice (Sousa et al.,
2004).
Nowadays, tea, which is one of the most consumed beverages of
the world, is considered to be one of the most important sources of
phenolic compounds in our diet. Turkey is recognised as having the
highest per capita consumption of tea and the Turkish tea as being
one of the healthiest. Green, white and black teas are all made from
the leaves of the same evergreen shrub Camellia sinensis but they
differ by their manufacturing process which leads to a given oxida-
tion degree of the polyphenols or other compounds existing in tea
leaves.
It is well known that catechins are the main polyphenols in
green tea (3050% of the solids and 90% of the total avonoids,
while () epigallocatechin gallate constitutes 50% of the polyphe-
nols) (Ahmad et al., 2014; Dias, Changarath, Damodaran, & Joshi,
2014; Lee, Kim, Kim, & Kim, 2014; Novak et al., 2010). During the
fermentation process these molecules are transformed into more
complex compounds (e.g. theaavins and thearubigins) which con-
stitute more than 20% of the solids and 7080% of avonoids in
black teas (Dias et al., 2014). It was also established that fresh
and processed tea leaves contain also other phenolic compounds
among them being also phenolic acids (Horzic et al., 2009). Pearson
correlation analysis emphasised that the antioxidant benets of
black tea depend strongly on the majority of tea active compounds
like catechins, theaavins, thearubigins and phenolic acids. It was
also established that the composition of teas originating from all
over the world depends on the geographical regions where the
plants were grown (Dias et al., 2014). The different levels of poly-
phenols (catechins, theaavins, thearubigins, phenolic acids) in
black and green teas cultivated in various parts of the globe can
be found in the database published by Obuchowicz, Engelhardt,
and Donnelly (2011).
Regardless of the exact tea composition, most studies on their
antioxidant activity are direct related to their total polyphenolic
content (TPC) without considering the contribution of the individ-
ual antioxidant component (Lee et al., 2014). Therefore for the
assessment of TPC of teas, in the present study we select caffeic
acid as reference compound based on the following considerations:
(a) according to Brand-Williams, Cuvelier, and Berset (1995) gallic
acid and caffeic acid have the strongest antiradical activity when
compared to other polyphenols whereas when compared to other
phenolic acids (e.g. ferulic or p-coumaric acids) found in teas, the
compounds bearing a catechol moiety (e.g. gallic acid, caffeic acid
and its derivative, chlorogenic acid) are more effective antioxidants
(Sousa et al., 2004); (b) studies carried out on leaves of yerba mate
emphasised that the caffeic acid content is less affected by indus-
trial processing, its content being similar in green and processed
leaves, indicating thus a higher stability compared to chlorogenic
acid (Turner, Cogoi, Isolabella, Filip, & Anesini, 2011); (c) voltam-
metry of caffeic acid is simpler: when compared with catechins
which exhibit two or even three irreversible oxidation peaks, the
CA presents only one reversible oxidation peak at about 500 mV,
situated in the same region with the rst oxidation peak of cate-
chins (Novak et al., 2010; Novak, eruga, & Komorsky-Lovric,
Fig. 1. Structure of caffeic acid (CA).
2009a,b) and (d) according to Photinon et al. (2010) CA has the
capability to give rise to the highest oxidation current.
Despite the fact that there are several electrochemical studies
related to caffeic acid (Sen & Sarkar, 2013; Abou Samra et al.,
2011; Blasco et al., 2005; Buratti, Scampicchio, Giovanelli, &
Mannino, 2008; Giacomelli et al., 2002; Kallel Trabelsi et al.,
2004; Photinon et al., 2010; Simic et al., 2007; Sochor
et al., 2013; Sousa et al., 2004; Tyszczuk et al., 2011; Veljkovic
et al., 2013), none of them have reported the use of bare or modi-
ed pencil graphite electrode as working electrode. To the best of
our knowledge there is only one paper related to the determination
of total phenolic content of teas using the PGE in amperometric
measurements in a ow system (Buratti et al., 2008) and one
regarding the study of the electrochemistry of eight avonoids
and their interaction with copper ions (Vestergaard, Kerman, &
Tamiya, 2005).
The present paper describes the voltammetric determination of
caffeic acid on a disposable pencil graphite electrode (PGE). The
developed differential pulse voltammetric (DPV) method was
applied to the assessment of total polyphenolic content (TPC) of
tea samples, expressed as mg of caffeic acid equivalent/g dry
weight tea (mg CAE/g tea). The results obtained for green, white
and black tea samples by DPV on PGE were compared with those
registered by the spectrophotometric FolinCiocalteu (FC) method.
2. Experimental
2.1. Reagents and solutions
Caffeic acid (97.0%), ethanol, methanol (P99.5%, ACS reagent),
H2SO4 (98.0%, ACS reagent), NaOH (pellets), Na2HPO42H2O and
KH2PO4 (p.a. ACS reagent), FolinCiocalteu reagent and Na2CO3
were purchased from SigmaAldrich (Munich, Germany).
A stock solution of 1  102 M CA was freshly prepared at the
beginning of each measurement set by dissolving the appropriate
weighted amount of analyte in 1 mL ethanol and diluting with
deionised water to the mark of a 10 mL dark volumetric ask.
The solution was stored in the refrigerator. More diluted solutions
with concentrations from 1  108 to 5  103 M CA used for the
voltammetric measurements were freshly prepared from the stock
solution by adding the appropriate supporting electrolyte and
diluting with deionised water to the mark of 5 mL dark volumetric
asks.
Three types of Turkish tea samples were used as follows: (i)
green tea aykur ORGANIC Zumrut Yesil Cay 125 g; (ii) black tea
aykur ORGANIC Rize Cay 400 g, both purchased from Istanbul
supermarkets and (iii) white tea bought from Istanbul free market.
The tea samples were analysed as infusions.
2.2. Instrumentation
Electrochemical measurements were carried out on a BAS CV-
500 CW Bioanalytical System Inc., USA galvanostat-potentiostat
coupled to a PC equipped with the CV-500 CW software for exper-
imental control and data acquisition. A 10 mL voltammetric cell
consisting of a pencil-graphite electrode (PGE), glassy carbon elec-
trode (GCE) or platinum (Pt) as working electrode, a Pt wire as aux-
iliary electrode and a Ag/AgCl (3 M KCl) reference electrode was
used for recording the voltammograms. For each measurement
the voltammetric cell was lled with 5 mL of the solution to be
analysed.
Before each measurement the solid electrodes (GCE and Pt)
were cleaned with alumina powder, rinsed with deionised water
and dried.
 I.G. David et al. / Food Chemistry 173 (2015) 10591065
For the preparation of the PGE, 6 cm long 0.5 mm HB pencil
leads were cut into the half. The cut edge was introduced into a
Rotring pencil acting as holder. Thus, the PGE consists of a 3 cm
long HB pencil lead xed in a Rotring pencil so that constantly
1.5 cm of the lead remained outside the pencil in order to be
exposed to the analyte solution. By soldering a metal wire to the
metallic part of the pencil one can assure the electrical contact
with the lead. The pencil was kept vertical so that 1 cm of the
graphite lead was introduced in the solution to be investigated. A
new graphite pencil lead was employed for each measurement.
Spectrophotometric measurements were performed in 1.00 cm
quartz cells by using a Jasco V 530 UVVIS spectrophotometer
running SpectraManager as software (Shimadzu, Japan).
All amounts of substances were weighted on a Shimadzu AX
200 (Japan) analytical balance. The pH-measurements were carried
out on a Consort P901 Scientic Instrument pH/mV/C metre
(Belgium) equipped with a combined pH-glass electrode.
2.3. Procedures
2.3.1. Preparation of tea infusion
Tea infusions were prepared by adapting procedures described
in the literature (Buratti et al., 2008; Veljkovic et al., 2013). Thus
about 1 g (green, white and black, respectively) tea leaves were
introduced in into a 50 mL Berzelius ask lled with 25 mL freshly
boiled water. The beaker was covered with a glass plate and left for
20 min to extract. The cooled extract was then ltered through
Blue Ribbon Quantitative Whatman lter paper without any other
pretreatment. The freshly obtained ltrate was immediately ana-
lysed in parallel on aliquots of the same infusion by differential
pulse voltammetry (DPV) and spectrophotometry (FolinCiocalteu
procedure) (FC) after a proper dilution.
2.3.2. Voltammetric measurements
Cyclic voltammetric (CV) measurements were performed in the
potential range 300 to 1000 mV at 100 mV/s scan rate if not sta-
ted otherwise.
Differential pulse voltammetric (DPV) recordings were carried
out in the potential range 300800 mV under following parame-
ters: scan rate (v) 50 mV/s, pulse period (PP) 200 ms, pulse width
(PW) 50 ms, sampling width (SW) 17 ms and pulse amplitude
(PA) 50 mV. All the experiments were done at room temperature
(25.0 0.2 C). In order to minimise an eventual adsorption of caf-
feic acid (or other polyphenols or matrix compounds from tea sam-
ples) onto the working electrode surface, the differential pulse
voltammograms were recorded immediately after insertion of
the PGE in the solution to be analysed.
The standard addition method was applied for the recovery
studies and for the assessment of the total polyphenolic content
(TPCDPV) of tea samples using DPV. An aliquot of 0.5 mL tea infu-
sion was diluted with 0.1 M H2SO4 solution to a nal volume of
5 mL. The measured pHs of the tea infusions were as follows: green
tea 5.21, white tea 5.22 and black tea 4.91. The analysed samples
pHs (after adding the H2SO4 to be a nal concentration of 0.1 M)
were 1.82, 1.82 and 1.81 for green, white and black tea, respec-
tively. DP voltammograms were recorded for the diluted tea sam-
ple solution before and after 3 successive additions of 0.1 mL of
1  103 M CA solution. The obtained peak currents were further
used to calculate the % recoveries and the TPCDPV is expressed as
mg CAE/g tea.
2.3.3. Spectrophotometric determination of total polyphenolic content
(TPCFC)
Total polyphenolic content (TPCFC) was determined by the mod-
ied FolinCiocalteu (FC) method using caffeic acid (CA) as stan-
dard (Singleton, Orthofer, & Lamuela-Raventos, 1999). TPC was
1061
expressed as mg CAE/g tea. Known volumes (0.10.5 mL) of
100 lg/mL caffeic acid stock solution (prepared in methanol) were
transferred into a series of 5 mL volumetric asks. A 2.5 mL volume
of FC reagent (freshly prepared by diluting 1:10 with deionised
water) was added to each ask. The solutions were set aside for
8 min. After that, 2 mL of sodium carbonate (7.8% w/v) was added
to each mixture, thoroughly mixed and made up to the mark with
deionised water. The volumetric asks were kept in the dark for
60 min. The absorbencies were measured at kmax = 760 nm against
a reagent blank sample. The calibration equation was found to be
Y = 0.1235X  0.0029 (R2 = 0.9988) where X = concentration of CA,
in lg/mL and Y = absorbance.
For samples measurement, an aliquot of 0.5 mL of tea infusion
was diluted with deionised water in a 5 mL volumetric ask. A
0.1 mL volume of this solution was transferred in another 5 mL cal-
ibrated ask and the general procedure applied. The TPCFC was cal-
culated using the calibration curve. The obtained results represent
the average of three measurements for each sample.
3. Results and discussions
3.1. Selection of the working electrode
The inuence of the electrode material on the voltammetric
response of CA was investigated by recording the cyclic voltammo-
grams of the polyphenol using various working electrodes: a Pt
electrode, a GCE and a PGE, respectively. As it can be observed from
the recorded cyclic voltammograms (Fig. 2) the most intense peaks
were obtained on PGE thus enabling a more sensitive determina-
tions using PGE in comparison to GCE and Pt electrode, respec-
tively. This fact was also emphasised by the slopes of the
equations describing the dependence of the anodic peak currents
(Ipa) on the CA concentrations in the range 2.5  1043 
103 M obtained at the three electrodes in phosphate buffer
solution (PBS) pH = 7.00, namely for:
Pt: Ipa = 0.0045  CCA  4  107 (R2 = 0.9963);
GCE: Ipa = 0.0104  CCA + 2  106 (R2 = 0.9960) and
PGE: Ipa = 0.0398  CCA + 5  107(R2 = 1.0000).
The contamination of the electrode surface during the voltam-
metric measurements due to the adsorption of different com-
pounds existing in the matrix or produced during the
electrochemical process is an undesired effect because it perturbs
the subsequent measurements. From the literature it is well known
that during their electrooxidation, phenolic compounds may lead
to the electrode surface fouling by covering it with a non-conduct-
ing polymeric lm (de Carvalho, Kubota, & Rath, 2003). Therefore,
Fig. 2. Cyclic voltammograms recorded on (1) Pt; (2) GCE and (3) PGE for a
1  103 M CA in PBS pH = 7.00 solution; v = 100 mV/s.
1062 I.G. David et al. / Food Chemistry 173 (2015) 10591065
in order to ensure the determinations reproducibility, a time con-
suming electrode surface cleaning step is necessary before each
voltammetric measurement. Thus the use of a disposable, easy to
replace (renew) electrode, like the PGE, is worth.
Considering the above mentioned aspects as well as the proper-
ties of the pencil graphite electrode (PGE) as a good electrode
material (e.g. chemical inertness, low background current and
large potential window), with its economic advantages of being
cheap and easy commercially available, in this study the PGE was
used as working electrode for further voltammetric measurements
of CA.
3.2. Selection of the supporting electrolyte
In order to select the optimum supporting electrolyte, cyclic
voltammograms were recorded at PGE for a 3  103 M CA solution
using 0.1 M H2SO4, PBS pH = 7.00 and 0.1 M NaOH as supporting
electrolyte. In 0.1 M NaOH CA did not present any voltammetric
peak whereas the best dened cyclic voltammograms (with sharp
peaks) were obtained using 0.1 M H2SO4 as supporting electrolyte
(Fig. 3). It can also be observed that in 0.1 M H2SO4 the potentials of
both anodic and cathodic peaks of CA (Fig. 3) are shifted towards
more positive potentials when comparing with the CV obtained
in PBS pH = 7.00 (Fig. 2). This observation is consistent with the
conclusions reported by Giacomelli et al. (2002) related to the
inuence of the solution pH on CA electrooxidation. According to
these authors at pH 6 2 (i.e. in 0.1 M H2SO4 in our case) the solu-
tion contains predominantly the undissociated caffeic acid
(H3CA) whereas at pH = 7.00 (i.e. PBS in our situation) the ionic
species H2CA is preponderant. Despite the fact that both species
involve 2 electrons for their oxidation, the different magnitude of
Ipa (Ipa ; H2 SO4 8:5  10 5 A and Ipa,PBS = 4  105 A) obtained for
103 M CA by CV in the two supporting electrolytes with different
pH values was attributed to the fact that the two species have dif-
ferent diffusion coefcients in 0.1 M H2SO4 and PBS pH = 7.00,
respectively. The same authors (Giacomelli et al., 2002) indicated
that the diffusion coefcient decreases with increasing pH of the
analysed solution. Having in mind that in 0.1 M H2SO4 CA presents
a sharper and more intense anodic peak than in PBS pH = 7 for fur-
ther studies 0.1 M H2SO4 was employed as supporting electrolyte.
3.3. Voltammetric behaviour of CA
The cyclic voltammograms recorded for CA at different scan
rates (v) from 10 to 1000 mV/s (Fig. 3) present both anodic and
cathodic peaks with peak potentials shifting with the potential
scan rate. For all scan rates the value of the peak potential separa-
tion, DEp = Epa  Epc, is higher than the theoretical value of 59/n mV
(where n is the number of exchanged electrons) and increases with
Fig. 3. Cyclic voltammograms recorded on PGE at different potential scan rates (a)
10; (b) 25; (c) 50; (d) 100; (e) 250; (f) 500 and (g) 1000 mV/s for a 3  103 M CA in
0.1 M H2SO4 solution.
v. These observations are correlated with the fact that the ratio Ipa/
Ipc is higher than 1 which indicates a quasi-reversible behaviour of
CA on the PGE in 0.1 M H2SO4. For a further evaluation of the elec-
trochemical behaviour of CA the apparent heterogeneous electron
transfer rate constant (ks) was calculated according to the equation
(Laviron, 1979):
RT a1  anF DEp
log ks a log1  a 1  a log a  log  ;
nF v 2:3RT
where a is the charge transfer coefcient estimated from the slope
of the plot of the anodic and respectively cathodic peak potential
versus decimal logarithm of scan rate, n is the number of electrons
involved in the reaction and the other symbols have their usual
meanings (R = 8.314 J/mol  K, T = 298 K, F = 96,493 C/mol). Accord-
ing to this equation, for the obtained a value of 0.56 (theoretical
value is 0.53), ks was 0.123 0.02 s1 indicating that in 0.1 M
H2SO4 CA the electron transfer process on the PGE surface is
promoted.
Both the anodic (Ipa) and cathodic (Ipc) peak currents varied lin-
early with the square root of the potential scan rate (v) increasing
(Ipa = 2  105  v1/2 + 4  105; R2 = 0.9985 and Ipc = 2  105
 v1/2 + 6  106, R2 = 0.9998), indicating a diffusion controlled
electrode process of CA. The regression equations of the log Ip =
f(log v) plots for both the anodic and cathodic peak are:
Ipa = 0.3854  log v  4.4478; R2 = 0.9877 and Ipc = 0.4868  log
v  4.7655; R2 = 0.9999, respectively. The slope values of these
plots near to 0.5 conrmed also the observation that the electro-
chemical oxidation of CA on PGE is diffusion controlled (Brett &
Oliveira-Brett, 1993, chap. 9).
3.4. Linear range and detection limit
In CV the maximum current intensities of both anodic and
cathodic peaks depend linearly on CA concentration in the
range 1  1045  103 M obeying the equations Ipa = 0.0911 
CCA  6  106 (R2 = 0.9996) and Ipc = 0.0644  CCA + 2  106 (R2 =
0.9952), respectively.
Fig. 4. Differential pulse voltammograms recorded in 0.1 M H2SO4 on PGE for
different concentrations of CA: (a) 1  107 M; (b) 1  106 M; (c) 5  106 M; (d)
1  105 M; (e) 5  105 M; (f) 1  104 M; (g) 2.5  104 M; (h) 5  104 M; (i)
1  103 M and (j) 3  103 M.
 I.G. David et al. / Food Chemistry 173 (2015) 10591065 1063

Table 1
Comparison of previously published electrochemical methods applied for CA determination.

Technique Electrode Linear range (lM) LoD (lM) Sample References

LSV Pt rotating disk 10100 2.13 Tincture Reyes-Salas, Rangel-Ordoez, Manzanilla-Cano,
Barcel-Quintal, and Dosal-Gmez (2002)
SWV Pb-lm-GCE 0.010.5 0.004 Herbs Tyszczuk et al. (2011)
SWV Guanine-GCE 2.224.44 0.33 Beverages Barroso, Delerue-Matos, and Oliveira (2012)
Adenine-GCE 0.552.78 0.388
FI amperometry PGE 5.55111 Tea infusion Buratti et al. (2008)
Amperometry GCE at +0.5 V 2.22333 0.22 Blasco et al., 2005
GCE at +0.8 V 0.16
Amperometry GCE and Pt 5.551387.65 Tea, beverages Sen & Sarkar, 2013
CV GCE 1001000 Orange juice Sousa et al. (2004)
DPV MWNT-GCE 0.00935030 0.0025 Coffee Ziyatdinova et al. (2013)
DPV PGE 0.103000 0.0883 Tea infusions Our

Table 2
Recovery (expressed as CA concentration) and TPCDPV values from green, white and black tea samples (3 sample replicates), and comparison with TPCFC.

Tea CCA, 105 M CCA SDa, 105 M R SDb, % TPCDPV SDc, mg CAE/g tea; TPCFC SDe, mg CAE/g tea; Relative error F-valueg
sample added found RSDd, % RSD, % er%f

Green tea 0 7.95 0.61

1.96 9.66 1.01 97.24 4.59 35.81 2.74 37.23 2.42
3.85 11.55 0.96 97.76 3.24 7.65 6.57 3.82 1.28
5.66 13.59 0.67 99.85 0.77
White tea 0 7.68 0.12
1.96 9.51 0.41 98.70 5.55 34.59 0.53 36.83 0.79
3.85 11.01 0.15 95.52 1.50 1.54 2.44 6.07 2.22
5.66 13.94 0.22 104.48 0.70
Black tea 0 6.93 0.41
1.96 8.12 0.47 91.31 1.08 31.21 1.84 32.57 1.34
3.85 9.93 0.69 92.02 2.95 5.90 4.18 4.18 1.89
5.66 11.47 0.68 91.03 2.41
1.96 8.12 0.47 91.31 1.08
a
Average concentration and standard deviation for 3 sample replicates,
b
Average recovery and standard deviation,
c
Average total polyphenolic content by differential pulse voltammetry and standard deviation,
d
Relative standard deviation,
e
Average total polyphenolic content by FolinCiocalteu method and standard deviation,
f
er% = ((TPCFC  TPCDPV)/TPCFC)  100.
g
Tabled F-value for m1 = 9, m2 = 2 at 95% condence level, two-sided is 4.26.

Due to its limited sensitivity CV is mainly used to investigate the The value obtained for 10 solutions containing 1  106 M CA
voltammetric behaviour of the CA and has less applicability for under the optimised conditions was 6.40%.
quantitative assays. Therefore, for the quantitative determination
of CA the more sensitive differential pulse voltammetry (DPV)
3.6. Recovery studies and analytical applications
was further used. The inuence of CA concentrations on the anodic
peak current recorded by DPV in 0.1 M H2SO4 on a PGE (Fig. 4) was
DP voltammograms recorded at PGE for green, white and black
investigated in the concentration range 1  1083  103 M CA.
tea samples present well-dened oxidation peaks at about 500 mV.
The obtained linear range was 1  1073  103 M CA and it is
This peak is due to the oxidation of the sum of the different poly-
described by the equation Ipa = 0.0930  CCA + 4  106 (R2 =
phenols contained in the analysed tea sample and therefore its cur-
0.9954). It is worth mentioning that the obtained linear range of 4
rent intensity is proportional to the sample total polyphenolic
orders of magnitude is one of the largest reported in the literature
content (TPCDPV). The current intensity of this peak increases line-
(see Table 1).
arly by three successive additions (each of 0.1 mL) of 1  103 M
There are several calculation methods for the detection limit
CA solution. This observation indicates that the total polyphenolic
(LoD) and the quantication limit (LoQ) determination. In this case
content (TPCDPV) can be estimated from the current intensity of the
the data from the linearity study were used: LoD 3scmin =b and
tea sample peak by applying the standard addition method, but
LoQ 10scmin =b, where scmin is the standard deviation of the small-
without determination of the caffeic acid content. The measure-
est concentration from the calibration curve and b is the calibration
ments were done in triplicate and the results were expressed as
curve slope (Gonzlez & Herrador, 2007). The values obtained for
mg caffeic acid equivalents per gram of dry weight tea (mg CAE/
the detection and quantication limits were: 8.83  108 M and
g tea). Recoveries of caffeic acid added to the analysed tea samples
2.94  107 M CA, respectively. For comparison, Table 1 presents
(3 additions to each sample) were calculated and the overall aver-
some previously published examples of electrochemical determi-
age values were 91.45 2.03% (for black tea), 98.30 3.07% (for
nations of caffeic acid.
green tea) and 99.57 4.89% (for white tea) (Table 2).
For comparison, the total polyphenolic contents of the three
3.5. Repeatability types of teas (green, black and white, respectively), expressed as
mg CAE/g tea, were also assessed by the spectrophotometric
The precision of the method was evaluated on the repeatability FolinCiocalteu method (TPCFC). Despite some disadvantages and
level, expressed as percentage relative standard deviation (RSD%). lack of selectivity, the FolinCiocalteu method, which is an electron
1064 I.G. David et al. / Food Chemistry 173 (2015) 10591065
transfer one, is unanimously considered suitable as a measure of
total phenolics in natural products (Prior, Wu, & Schaich, 2005)
and some modications of the initial procedure have been made
in time and gallic acid used rst as reference was replaced by other
compounds, caffeic acid being one of them (Maranz, Wiesman, &
Garti, 2003). Therefore, we consider that the use of caffeic acid as
reference in both methods (FC and this new one) can ensure a quite
reliable comparison of the results obtained. Thus, in order to
evaluate the results of DPV method, the data obtained by the
FolinCiocalteu method are considered as reference to calculate
the relative error values (*** JCGM 100: 2008, chap. 4). The
comparative results and the relative error (er %) of the total poly-
phenolic content estimated for the three tea types by the two
methods (DPV on PGE and FolinCiocalteu) are summarised in
Table 2. The relative error values were ranged between 3.82%
and 6.07% and proved the good agreement of the results.
The results of the proposed method were statistically compared
with those of the FolinCiocalteu method applying the F test
(Hartmann et al., 1995; Skoog, West, & Holler, 1996, chap. 4). For
all of the three tea samples the calculated F values at 95% con-
dence levels are smaller than the theoretical ones (Table 2). Thus,
one can conclude that there are no signicant differences between
the performances of the two methods (they present close
precisions).
4. Conclusions
The present paper describes for the rst time a study regarding
the voltammetric behaviour of caffeic acid at a cheap, disposable
pencil graphite electrode, eliminating thus the time consuming
step of cleaning the surface of solid electrodes. This working elec-
trode was used for the rst time for the determination of total
polyphenolic content in Turkish green, black and white tea sam-
ples using differential pulse voltammetry. The developed DPV
method at the PGE is a sensitive (LoD = 8.83  108 M), rapid and
inexpensive tool for the evaluation of TPC in tea, expressed as
mg CAE/g tea. The average recoveries of CA were 98.30%, 99.57%
and 91.46% for green, white and black tea, respectively. The statis-
tical analysis and the relative errors of 3.82%, 6.07% and 4.18% for
green, white and black tea, respectively, indicated that the results
of TPC estimated by DPV on PGE were in good accordance with
those obtained by the well-known, more time consuming spectro-
photometric FolinCiocalteu method which needs about one hour
for reaction in order to develop a stable colour to be measured (see
Section 2.3.3. Spectrophotometric determination of total polyphe-
nolic content).
Acknowledgements
Authors thanks for nancial support to Romania Turkey Joint
Project 604/2013 nanced by UEFSCDI and Tubitak 112 O 420.
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