Analytica Chimica Acta 732 (2012) 100–104

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Analytica Chimica Acta

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Ultrasound assisted extraction of phenolic compounds from grapes

Ceferino Carrera, Ana Ruiz-Rodríguez, Miguel Palma ∗ , Carmelo G. Barroso
Centro Andaluz de Investigaciones Vitivinícolas, Campus de Excelencia Internacional Agroalimentario (ceiA3), Universidad de Cádiz,
C/República Saharaui s/n, 11510 Puerto Real, Cádiz, Spain

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

Article history: A new ultrasound-assisted extraction method was developed for the determination of phenolic com-
Received 1 September 2011 pounds present in grapes. Several extraction variables including extraction temperature (0–75 ◦ C), output
Received in revised form 20 October 2011 amplitude (20, 50 and 100%), duty cycle (0.2 s, 0.6 s and 1 s), the quantity of sample (0.5–2 g), and the total
Accepted 16 November 2011
extraction time (3–15 min) were evaluated. One of the most widely used extraction methods of polyphe-
Available online 23 November 2011
nol extraction has been used as reference method. Three parameters were compared: total amount of
phenolic compounds, total amount of anthocyanins and total amount of tannic components. The result-
Keywords:
ing method produced similar or higher recoveries for these three parameters; however a much shorter
Ultrasound-assisted extraction
Grapes
extraction time was needed: 6 min (ultrasound assisted extraction method) instead of 60 min (reference
Anthocyanins method).
Phenolic compounds Analytical properties for the new method were established, including limit of detection, limit of quan-
Tannins tification, repeatability and reproducibility.
The developed method was applied to two different types of grapes in different ripening degree.
© 2011 Elsevier B.V. All rights reserved.

1. Introduction interesting to have fast analytical methods of determining these

Phenolic compounds play a key role in the sensory properties
of red wines [1]. There are also very convenient health benefits
associated with their consumption [2]. The total contents of phe-
nolic compounds, as well as the ratio between the different types of
phenolic compounds in the red grape varieties are strongly related
to the quality of resulting wines. Therefore, the determination of
phenolic levels is very interesting information in setting the best
harvest date. Usually the concentration of the phenolic compounds
increases during berry ripening; however several factors can affect
its evolution, mainly related to the viticulture practices. Therefore
controlling the ripening of berries regarding phenolic levels is one
of the most critical stages in the production of red wines.
There are three parameters that are usually evaluated. First total
phenolic compounds, related generally to the organoleptic prop-
erties of wines and stability. In addition it is also interesting to
quantify the total anthocyanins, which are responsible for the red
color in red wines. Also interesting are the levels of total tannins,
because they are responsible for the astringency and body of wine,
as well as its relationship with the promotion of co-pigmentation
phenomena, binding to the anthocyanins and creating stable struc-
tures against the action of sulfur dioxide [3].
Changes for these parameters through the ripening period
are particularly rapid near the harvest date [4]. It is therefore
∗ Corresponding author. Tel.: +34 956016775; fax: +34 956016590.
E-mail address: miguel.palma@uca.es (M. Palma).
parameters, so that winemakers can have the information at appro-
priate times.
There are different extraction procedures, the most widely used
are based on the maceration of the berries under different condi-
tions of pH, alcoholic strength (ethanol concentration) and time,
followed by spectrophotometric determination of total phenolic
compounds, total anthocyanins and tannins in the extract obtained
[5]. Traditional extraction techniques typically require long macer-
ation. This time can range from 1 to 20 h [6,7]. These long extraction
times are because of most of the compounds of interest are located
in the skins of the grapes [8]. The extraction must have good repro-
ducibility and the solid–liquid soaking this parameter has higher
values using higher extraction times. There some papers related
to a faster determination of phenolic compounds in the extracts
[9], however research was not found related to the application of
fast/assisted extraction methods.
Ultrasonic-assisted extraction has been used for the extraction
of plant components in order to shorten the extraction time, lower
solvent consumption, increase extraction yields and improve the
quality of the extracts [10].
The technique of ultrasonic assisted extraction (UAE) is partic-
ularly attractive for its simplicity and low cost of equipment. It is
based on the use of energy derived from ultrasound (sound waves
with frequencies above 20 kHz) to facilitate the extraction of ana-
lytes from solid sample by the solvent, which is selected depending
on the nature of the solutes to be extracted [11]. This technique
has been used to extract various organic compounds from different
matrices, including phenolic compounds in cosmetic creams [12],

0003-2670/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.aca.2011.11.032
 C. Carrera et al. / Analytica Chimica Acta 732 (2012) 100–104
organic acids in grapes [13], phenolic compounds in strawberries
[14], soy isoflavones [15] and capsaicinoids in peppers [16]. Usually
the total extraction time is reduced between 3 and 10 times.
There are some previous applications of ultrasound assisted
extraction in the determination of phenolic compounds in spe-
cific parts of grapes. Ghassempour et al. [17] compared UAE and
microwave assisted extraction (MAE) in the recovery of antho-
cyanins from red grape skin. UAE showed slight lower recoveries
than MAE. Novak et al. [18] also applied UAE as extraction method
for the determination of flavonoids in red grape skins using an ultra-
sonic water bath. Fast extraction methods were obtained, between
15 and 30 min for different types of flavonoids.
Grape seeds have been also an interesting sample for UAE based
methods. Ghafoor et al. [19] developed and extraction method
using a sonication water bath in the recovery of total phenolic
compounds and anthocyanins from grape seed using 50 min as
extraction time.
Whole grapes are samples containing a high level of sugars
(>200 g L−1 ) and also lower levels for phenolics than grape seeds
and grape skins. Whole grapes allow for the determination of
the full content of phenolic in grapes including benzoic and cin-
namic derivatives found in the mass of grapes. Those compounds
are the first compounds released to the grape juice, so they are a
source for phenolics in grape must and wines, especially for white
wines.
This paper studies the feasibility of ultrasound-assisted extrac-
tion as an alternative to the classical maceration for extraction of
total phenolic compounds, condensed tannins and anthocyanins in
red whole grapes.
2. Materials and methods
2.1. Chemicals and solvents
Ethanol (Panreac, Barcelona, Spain) used was HPLC grade. Ultra
pure water was supplied by a Milli-Q water purifier system from
Millipore (Bedford, MA, USA).
2.2. Samples of grape
The red grape (var. Tempranillo) was employed for the devel-
opment of the ultrasound-assisted extraction method. They were
obtained from local vineyards from Bodegas Barbadillo (Sanlúcar de
Barrameda, Spain). The full berry (skin, pulp and seeds) was stud-
ied. The berries were triturated with a conventional beater, until
a homogeneous sample was obtained for the analysis. The tritu-
rated sample obtained was conserved in a freezer at −20 ◦ C until
its analysis.
2.3. Extraction procedure
The extraction of phenolic compounds originating from red
grapes by means of ultrasound was performed employing
water–ethanol mixture (50:50) which contains hydrochloric acid
(pH: 2.0) like solvent. Effects by the extraction temperature
(0–75 ◦ C), output amplitude of the nominal amplitude of the trans-
ducer (20, 50 and 100%), duty cycle (0.2 s, 0.6 s and 1 s), the quantity
of sample (0.5–2 g), and the extraction time (3–15 min) were stud-
ied.
Ultrasonic irradiation was applied by means of a UP200S sonifier
(200 W, 24 kHz) (Hielscher Ultrasonics, Teltow, Germany) which
was immersed in a water bath coupled to a temperature controller
(Frigiterm, J.P. Selecta, Barcelona, Spain).
101
2.4. Phenolic compounds determination
Determination of total phenolic compounds (PTOT ) and total
anthocyanins (ATOT ): The analytical determination of phenolic com-
pounds and anthocyanins is done, as usual, from the absorbance at
280 and 520 nm respectively at pH = 1 [20].
Determination of total condensed tannins (TTOT ): The quantifica-
tion of condensed tannins was carried out using the method of
precipitation of condensed tannins with Methyl Cellulose (MCP)
[20,21]. The measure requires the preparation of a control sample
and a sample treatment. The amount of condensed tannins was
determined by subtracting the absorbance at 280 nm of the sample
treatment of A280 of control sample.
The measurement of absorbance of the samples was carried out
in a UV/vis Spectrophotometer V-530 (Jasco, Madrid, Spain)
3. Results and discussion
3.1. Extraction temperature
Usually the higher the temperature the higher the recovery
when working with solid–liquid extractions. On the other hand,
high temperatures promote the oxidation degradation reaction
of phenolic compounds. Therefore, high temperatures could pro-
vide higher recoveries and also higher degradation rates. These
two opposite effects make mandatory the evaluation of the full
temperature working range, starting at 0 ◦ C up to 75 ◦ C. Higher tem-
peratures were not checked because high loses of ethanol occurs
changing the solid/liquid ratio, then producing low repeatabilities.
The extractions were performed with a quantity of triturated red
grape of about 1 g of the sample, in 10 mL of solvent, at an output
amplitude of 100%, with duty cycle of 1.0 s for an extraction period
of 5 min. All the assays were performed in triplicate.
The amount of phenolic compounds extracted at the differ-
ent temperatures of the assay is presented in Fig. 1. In these
extraction conditions nonsignificant differences were found for
condensed tannins at assayed temperatures. However for total phe-
nolic compounds, also for anthocyanins, 10 ◦ C produced higher
recoveries with significant differences (p < 0.05). Recovery should
increase with increasing extraction temperature. However, most
likely degradation processes also increased due to oxygen and also
due to enzymatic activity at least at 30–40 ◦ C. Therefore, 10 ◦ C was
used as extraction temperature in the next experiments.
3.2. Output amplitude and duty cycle of the ultrasonic probe
Energy provided by ultrasound is needed to release the tar-
get compounds from the matrix; however it also can accelerate
degradation process for phenolic compounds. It has been found
that during ultrasound assisted extraction (for 30 min) degrada-
tion up to 75% can be produced [22] and all reactions are promoted
when high amplitudes are used, [23] including the formation of free
radicals. In those cases phenolic compounds can act as scavenging
compounds on reactive oxygen species, then suffering oxidation
reactions.
Therefore, for both amplitude and duty cycle different values
were evaluated starting at the lowest values allowed by the system.
Fig. 2 shows the results for total phenolic compounds, condensed
tannins and anthocyanins.
It can be noted that the higher the amplitude the higher the
recovery. In most cases there were not significant differences
between 100% amplitude and 50% amplitude. However recoveries
found using 100% amplitude were always significant higher than
using 20% amplitude.
102 C. Carrera et al. / Analytica Chimica Acta 732 (2012) 100–104

Fig. 1. Effect of extraction temperature on the recovery of phenolic compounds.

Fig. 2. Effect of the ultrasonic amplitude and duty cycle on the recovery of phenolic compounds.

The same effect was found for the duty cycle, the higher the
cycle the higher the recovery. No significant differences were found
between results obtained using either 0.5 or 1.0 s; however 1.0 was
selected for later experiments as total recovery was higher.
3.3. Quantity of sample
In general, by reducing the quantity of sample while holding
the volume constant, the recovery is increased, since the ratio of
mass/volume of solvent is diminished. The disadvantage of this
practice is the decrease of the signal in the subsequent detection
system, because of a lower total amount of extracted compounds
in the same volume.
In this study sample quantities of 0.5, 1.0 and 2 g of grape have
been employed while maintaining the solvent volume constant at
10 mL of water–ethanol mixture. In Fig. 3 it can be observed that
the extraction of phenolic compounds was lower using 2 g of grapes
instead of 1 g. On the other hand, there were no significant differ-
ences between the results from the experiments using either 0.5 or
1 g. Using 1 g a higher signal is going to be obtained in the final mea-
surement method, then it was decided to follow the optimization
using 1 g of solid material.

Fig. 3. Effect of the solid/liquid ratio on the recovery of phenolic compounds.

 C. Carrera et al. / Analytica Chimica Acta 732 (2012) 100–104 103

Fig. 4. Effect of the extraction time on the recovery of phenolic compounds.

3.4. Extraction time
When the rest of the variables are optimized extractions times
of 3, 6, 9, 12 and 15 min were evaluated to determine the kinetic of
the extraction process. Fig. 4 shows the results. It can be observed
that, at extraction times longer than 6 min, there were no sig-
nificant differences in the quantities of phenolic compounds and
condensed tannins extracted. For anthocyanins, the longest time
assayed (15 min) showed a significant lower recovery than the
results obtained using 6 min, i.e. degradation clearly occurs after
15 min of extraction for anthocyanins. Anthocyanins are a part of
total phenolic, therefore this degradation should be also observed
for total phenolic compounds; however it was not recorded due
to anthocyanins are a low percentage of total phenolic compounds
(<10%) and also the degradation products for anthocyanins could be
other phenolic compounds counting for total phenolic compounds.
3.5. Comparison of the proposed extraction with the classical
method
study, and 3 more extractions on each of the two consecutive days.
Repeatability results (RSD) ranged from 2.5% for anthocyanins to
3.8% for condensed tannins. Reproducibility results ranged from
1.6% for anthocyanins to 4.3 for condensed tannins.
The limits of detection and quantification were established after
running the extraction of a blank six times. The LOD values (n = 6)
and LOQ values (n = 6) were 0.06 and 0.19 mg g−1 respectively for
total phenolic compounds, 0.01 and 0.04 mg g−1 respectively for
anthocyanins and 0.12 and 0.40 mg g−1 respectively for condensed
tannins.
3.7. Application to real samples
The suitability of this method for real samples during ripening
period was evaluated by monitoring the ripening process for two
cultivars (irrigate and non-irrigated) for the same grape variety.
Table 1 shows as grapes produced in the irrigated vineyard section

The proposed method is compared with the classical method

of extraction of phenolic compounds [20,21]; in this method the
extraction step is carried out by stirring the solid sample and sol-
vent for 1 h.
Fig. 5 shows the results for an extraction carried out by both
the classical method and the optimized UAE conditions. Compared
to the reference method, ultrasound-assisted extraction recoveries
obtained were significantly higher for total phenolic compounds
and not significantly different for both anthocyanins and condensed
tannins.

3.6. Analytical properties

The repeatability and reproducibility of the method developed

have been studied. A total of 15 extractions were performed, dis-
tributed as follows: 9 extractions performed on the first day of the Fig. 5. Comparison between classical and ultrasound assisted extraction method.

Table 1
Monitoring of ripening of Tempranillo grapes (irrigated and not irrigated) with the ultrasound assisted extraction method.

Sampling date Non-irrigated Irrigated

Total phenolic R.S.D. Anthocyanins R.S.D. Condensed R.S.D. Total phenolic R.S.D. Anthocyanins R.S.D. Condensed R.S.D.
compounds tannins compounds tannins

17/08/10 11.22 1.20 1.32 0.16 5.52 0.57 9.77 0.24 0.98 0.02 4.52 1.33
26/08/10 11.29 0.14 1.39 0.02 5.87 0.27 10.92 0.27 1.14 0.04 4.76 0.08
31/08/10 12.51 0.48 1.46 0.07 6.72 1.13 9.46 0.30 0.94 0.03 3.75 0.65
22/09/10 12.28 0.49 1.37 0.05 6.16 0.81 10.58 0.48 1.07 0.05 3.95 0.66
31/10/10 12.87 0.43 1.51 0.05 6.59 0.22 10.78 0.20 1.23 0.03 4.93 1.69
104 C. Carrera et al. / Analytica Chimica Acta 732 (2012) 100–104
contain lower values for all analyzed phenolic compounds. Regard-
ing the extraction method, it can be seen that analyzing different
kind of samples, i.e. different degrees of ripening, very similar RSD
were found for all of them. Therefore, no influence by the degree of
ripening near the harvest time on the suitability of the extraction
method was observed.
4. Conclusions
Ultrasound-assisted extraction, by means of the method devel-
oped, allows the quantitative and reproducible extraction of the
phenolic compounds (total phenolic, total anthocyanins and con-
densed tannins) present in grape, in a short time (6 min), employing
ethanol–water as extracting solvent. Given its low instrumen-
tal requirement, its simplicity and its analytical capabilities, the
method developed is suitable for the routine analysis of phenolic
compounds in grapes during ripening.
Compared with usual method, ultrasound-assisted extraction
produced similar or even higher recoveries.
Acknowledgements
This work was carried out with the funding received for the
project RTA2009-00022-C02 funded by INIA.
References
[1] R. Gawel, Aust. J. Grape Wine Res. 4 (1998) 74–95.
[2] C. Santos-Buelga, A. Scalbert, J. Sci. Food Agric. 80 (2000) 1094–1117.
[3] B.W. Zoecklein, K.C. Fugelsang, B.H. Gump, Wine Analysis and Production,
Aspen Publishers, Philadelphia, USA, 1995.
[4] R.L. Hanlin, M.O. Downey, Am. J. Enol. Vitic. 60 (2009) 13–23.
[5] S. Fragoso, M. Mestres, O. Busto, J. Guasch, J. Agric. Food Chem. 58 (2008)
4071–4076.
[6] J.A. Kennedy, C. Saucier, Y. Glories, Am. J. Enol. Vitic. 57 (2006) 239–248.
[7] K.A. Joutei, Y. Glories, Rev. Fr. Oenol. 153 (1995) 28–31.
[8] Y. Cadot, M. Chevalier, G. Barbeau, J. Sci. Food Agric. 91 (2011) 1963–1976.
[9] S. Fragoso, L. Acena, J. Guasch, O. Busto, M. Mestres, J. Agric. Food Chem. 59
(2011) 2175–2183.
[10] W. Lijun, L.W. Curtis, Trends Food Sci Technol. 17 (2006) 300–312.
[11] M. Soni, K. Patidar, D. Jain, S. Jain, J. Pharm. Res. 3 (2010) 636–638.
[12] M. Padilla, M. Palma, C.G. Barroso, J. Chromatogr. A 1091 (2005) 83–88.
[13] M. Palma, C.G. Barroso, Anal. Chim. Acta 458 (2002) 119–130.
[14] M.C. Herrera, M.D. Luque de Castro, J. Chromatogr. A 1100 (2005) 1–7.
[15] M.A. Rostagno, M. Palma, C.G. Barroso, J. Chromatogr. A 1012 (2003) 119–128.
[16] G.F. Barbero, A. Liazid, M. Palma, C.G. Barroso, Talanta 75 (2008) 1332–1337.
[17] A. Ghassempour, R. Heydari, Z. Talebpour, A.R. Fakhari, A. Rassouli, N. Davies,
H. Aboul-Enein, J. Liq. Chromatogr. Relat. Technol. 31 (2008) 2686–2703.
[18] I. Novak, P. Janeiro, M. Seruga, A.M. Oliveira-Brett, Anal. Chim. Acta 630 (2008)
107–115.
[19] K. Ghafoor, Y.H. Choi, J.Y. Jeon, I.H. Jo, J. Agric. Food Chem. 57 (2009) 4988–4994.
[20] C.J. Sarneckis, R.G. Dambergs, P. Jones, M. Mercurio, M.J. Herderich, P.A. Smith,
Aust. J. Grape Wine Res. 12 (2006) 39–49.
[21] M.D. Mercurio, P.A. Smith, J. Agric. Food Chem. 56 (2008) 5528–5537.
[22] M.J. Biesaga, Chromatogr. A 1218 (2011) 2505–2512.
[23] M.D. Luque de Castro, F. Priego-Capote, Anal. Chim. Acta 583 (2007) 2–9.