Food Chemistry 134 (2012) 602–605

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

Analytical Methods

First approach on moisture determination in food products using alpha-pinene

as an alternative solvent for Dean–Stark distillation
Sadjia Bertouche a, Valérie Tomao b, Karine Ruiz b, Amina Hellal a, Chahrazed Boutekedjiret a,⇑,
Farid Chemat b
a
Laboratoire des Sciences et Techniques de l’Environnement (LSTE), École Nationale Polytechnique, BP 182, El Harrach, 16200 Alger, Algeria
b
Université d’Avignon et des Pays de Vaucluse, INRA, UMR 408, Sécurité et Qualité des Produits d’Origine Végétale, F-84000 Avignon, France

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

Article history: The conventional Dean–Stark (CDS) distillation is one of the most commonly analytical methods used to
Received 25 August 2011 determine water content in food products. Toluene is the recommended solvent for this method, but it is
Received in revised form 25 November 2011 also ranked on top of the list of the hazardous solvents despite its concern on safety, environmental and
Accepted 26 February 2012
health issues. The aim of this study is to use alpha-pinene instead of toluene (petroleum solvent) for
Available online 5 March 2012
moisture determination in food products using Dean–Stark distillation. The results obtained with the
two solvents, alpha-pinene or toluene, were comparable. Statistical tests showed no significant difference
Keywords:
between the two procedures used and assessed the efficiency of alpha-pinene Dean–Stark (PDS) method
Dean–Stark distillation
Alpha-pinene
and its reliability. Dean–Stark technique using alpha-pinene could be appropriate for the routine quality
Alternative solvent control analysis of moisture in food products, but also, easy to handle procedure for lab work students.
Green analytical chemistry Ó 2012 Elsevier Ltd. All rights reserved.

1. Introduction
In recent years, academic research in the area of green solvent is
focused on finding agro-solvents to reduce petroleum solvent re-
lated to environmental damage (Nelson, 2003). Several innovations
towards green solvents have been developed: solvent-free technol-
ogy (Huma, Abert-Vian, Maingonnat, & Chemat, 2009; Michel,
Destandau, & Elfakir, 2011), use of water as alternative solvent
(Kronholm, Hartonen, & Riekkola, 2007), use of ionic liquids that
have low vapor pressure and less emission of COV (Leveque & Crav-
otto, 2006; Scammells, Scott, & Singer, 2005). Bio- or agro-solvents
are an alternative which have less hazard risks, environmental and
safety impact and a real substitution to petroleum solvents. Terp-
enes, considered as renewable solvent, are a valuable alternative
to typical solvents. There are found in essential oils and oleoresins
of fruit and aromatic plants. The most commonly used terpene as
solvent is probably d-limonene which represents a major by-prod-
uct of the citrus fruits industry (Mira, Blasco, Berna, & Subirats,
1999; Njoroge, Koaze, Karanja, & Sawamura, 2005). Its physical
properties were compared with those of hexane in order to extract
fats and oil from oleaginous seeds (Virot, Tomao, Ginies, & Chemat,
2008b; Virot, Tomao, Ginies, Visinoni, & Chemat, 2008a) or oil from
rice bran (Liu & Mamidipally, 2005; Mamidipally & Liu, 2004). Lim-
onene was also compared with toluene in Dean–Stark procedure
based on the fact that its ability to form an azeotropic mixture with
water (Veillet, Tomao, Ruiz, & Chemat, 2010). In addition, d-limo-
nene was recently used as a green solvent for the extraction of
carotenoids especially lycopene as a substitute of dichloromethane
(Chemat-Djenni, Ferhat, Tomao, & Chemat, 2010).
Another monoterpene susceptible to be an interesting alterna-
tive solvent is alpha-pinene. It represents the major constituent
of the turpentine oils from most conifers and a component of the
wood and leaf oils obtained from leaves, bark, and wood of a wide
variety of other plants like rosemary, parsley, basil, yarrow, roses
and turpentine. Pinene was generally obtained by fractional distil-
lation of steam-distilled wood turpentine and commonly use in
fragrance and flavor industry.
The aim of the present study is the valorisation of the alpha-
pinene as green solvent in order to replace the toluene for Dean–
Stark distillation for moisture determination. In this way, we
developed a Dean–Stark procedure and compared the values ob-
tained with those of the reference method. The statistical tests
were run out in order to assess the efficiency and the reliability
of this new procedure.
2. Materials and methods
2.1. Plant material

⇑ Corresponding author. Onions, garlic, carrots, leeks, olives, caraway and coriander seeds,
E-mail address: c_boutekedjiret@yahoo.fr (C. Boutekedjiret). oregano and minced meat were commercial samples, purchased

0308-8146/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2012.02.158
 S. Bertouche et al. / Food Chemistry 134 (2012) 602–605
from a local supermarket and stored at 4 °C in the dark prior to the
analysis.
2.2. Solvents
Toluene and alpha-pinene used for experiments were of analyt-
ical grade and were purchased from VWR international (Darms-
tadt, Germany).
2.3. Conventional Dean–Stark distillation (CDS)
Initially, moisture was determined according to the standard-
ized procedure AOCS Ja 2a-46 (1993). The apparatus is composed
of a flask of 500 ml with an electrical heater, a graduated Dean–
Stark receiver and a condenser. According to Harel and Talmi study
(1957), the weight of fresh sample used was fixed so as to obtain
an adequate amount of water in the receiver but not more than
10 ml which is its maximal countenance. Hundred milliliters of tol-
uene was used as distillation solvent.
At the beginning of the experiment, the electrical heating was
maximized until collection of the first droplets in the Dean–Stark
receiver. Then the heating was adapted to a distillation rate of
100 drops min1. When the greater part of the water has distilled,
heating was increased to a distillation rate of 200 drops min1 and
continued until the water level in the receiver did not increase
more than 0.1 ml in 30 min, then the distillation is stopped. In or-
der to recover the water droplets having adhered to the wall of the
condenser, 30 ml of toluene is added in top of this one. The Dean–
Stark collector was then allowed to stand at room temperature for
few minutes until the toluene layer was clear. The amount of water
contained in the Dean–Stark receiver was read and the moisture
content of the sample was calculated as follows:
Volume of water ðmlÞ  99:7
Moisture ð%Þ ¼
weight of fresh sample ðgÞ
were 99.7 is the density of water at 20 °C.
2.4. Alpha-pinene Dean–Stark distillation (PDS)
Dean–Stark distillation using alpha-pinene was performed
according to the above standardized method.
2.5. Gas chromatography and gas chromatography–mass
spectrometry analysis
To check if alpha-pinene is degraded during Dean–Stark distilla-
tion, the sample of recovered alpha-pinene after Dean–Stark distil-
lation was analyzed by gas chromatography (GC) and gas
chromatography coupled to mass spectrometry (GC–MS). A Hew-
lett–Packard computerized system comprising a 6890 gas chro-
matograph coupled to a 5973A mass spectrometer was used with
two fused-silica-capillary columns with different stationary
phases. The non-polar column was HP5MS (30 m  0.25 mm,
0.25 lm film thicknesses) and the polar one was a Stabilwax con-
sisting of Carbowax-PEG (60 m  0.2 mm, 0.25 mm film thickness).
GC–MS spectra were obtained using the following conditions: car-
rier gas He; flow rate 0.3 ml min1; split-less mode; injection vol-
ume 1 ll; injection temperature 250 °C; the oven temperature
program was 60 °C for 8 min increased at 2 °C min1 to 250 °C
and held at 250 °C for 15 min; the ionization mode used was elec-
tronic impact at 70 eV. The relative percentage of the components
was calculated from gas chromatography with flame ionization
detection (GC-FID). Most constituents were tentatively identified
by comparison of their GC Kovats retention indices (I), determined
with reference to an homologous series of C5–C28 n-alkanes and
603
with those of authentic standards available in the authors’ labora-
tory. Identification was confirmed when possible by comparison of
their mass spectral fragmentation patterns with those stored in the
MS database (National Institute of Standards and Technology and
Wiley libraries) and with mass spectra literature data (Adams,
1995; AFNOR, 2000; Arctander, 1994).
2.6. Reproducibility and statistical study
In order to study the reproducibility of the new PDS method,
two series of 10 essays were carried out on the onions under the
same conditions using both solvents: toluene and alpha-pinene.
Statistical study was performed using the Fisher-Snedecor’s test
to compare the variability of the results, and then the Student test in
order to compare the mean values obtained by the two different dis-
tillation procedures. Those two tests were performed with a = 0.05.
2.7. Generalization
With an aim of evaluating the effectiveness of the proposed
method, the essays were performed on several matrices and the re-
sults were compared to those obtained by conventional Dean–
Stark distillation (CDS). The matrices chosen are: two bulbs (onions
and garlic), two seeds (caraway and coriander), olives, carrots,
leeks, oregano and minced meat. All experiments were performed
in triplicate, both using CDS and PDS.
3. Results and discussion
3.1. Azeotropic distillation kinetics
The Dean–Stark procedure is an azeotropic distillation of a mix-
ture of immiscible solvents: water with toluene or alpha-pinene.
ð1Þ
Due to the difference in boiling point (Table 1), it is expected that
these solvents would have different kinetics of distillation and
recovery of water. Therefore we followed the distillation kinetics
for both toluene and pinene for moisture determination of corian-
der seeds. Fig. 1 show that kinetics for toluene CDS and PDS were
similar, only small variations could be observed. The boiling point
of alpha-pinene (Bp = 154 °C) is higher than toluene (111 °C), as
the meantime boiling point of azeotropic pinene/water (Bp = 97–
98 °C) is also higher than azeotropic toluene/water mixture
(84 °C). For this reason beginning of the water recovery is delayed
of 4 min. However, when the distillation started the water recovery
was faster when using alpha-pinene. Indeed 30 min provides the
amount of water comparable to those obtained after 105 min with
toluene. This reduction in the processing time represents a profit of
more than 70% in term of time and thus in consumption of energy.
The power consumption was determined with a Wattmeter at the
electrical heater power supply.
3.2. Alpha-pinene analysis
GC and GC/MS analysis of alpha-pinene recovered after
Dean–Stark distillation show the presence of alpha-phelandrene,
Table 1
Characteristics of toluene and alpha-pinene.
Characteristics Toluene Alpha-pinene
Boiling temperature (°C) 111 154
Density (g/cm3) 0.867 0.858
Environmental impact High Low
Renewable No Yes
Toxicity Yes Low
604 S. Bertouche et al. / Food Chemistry 134 (2012) 602–605

Fig. 1. Kinetics of water distillation of coriander seeds depending on the solvent.

Table 2
Reproducibility and statistical comparison test.

Experimentsa Mean SD RSD (%)

1 2 3 4 5 6 7 8 9 10
Reproducibility study
CDS 85.4 90.6 86.4 88.8 88.3 87.7 87.9 87.0 88.5 87.4 87.8 1.41 1.6
PDS 89.7 85.0 88.1 89.6 87.2 90.1 89.8 84.5 87.1 86.7 87.8 2.03 2.31
Statistical comparison test S2CDS S2PDS FCALb FTABc TCALd TTABe
2.01 4.12 2.07 3.18 0.67 2.10
a
Water in onion expressed as % fresh weight.
b
F value calculated using Fisher-Snedecor’s test.
c
F value tabulated for a = 0.05 and 9 and 9 degrees of freedom.
d
t value calculated using student’s test.
e
t value tabulated for a = 0.05 and 18 degrees of freedom.

camphene, beta-pinene, alpha-pinene oxide but with contents

Table 3
lower than 0.1%. The content of alpha-pinene remains higher than Water determination of different food matrices depending on the solvent of
98%, then, recovered alpha-pinene could be suitable for be reuse as distillation.
a solvent in subsequent distillation.
Food product (% water)

3.3. Reproducibility and statistical study Toluene Pinene Limonenea

Coriander seeds 9.5 ± 0.2 9.6 ± 0.1
The results of the reproducibility and reliability of the new PDS Caraway seeds 10.3 ± 0.2 10.3 ± 0.1
Onion 87.8 ± 1.4 87.3 ± 2.4 89.4 ± 1.4
method obtained for the water determination of onions were sum-
Garlic 70.8 ± 0.6 70.3 ± 0.8 68.6 ± 1.9
marized in Table 2. The mean value obtained with alpha-pinene Carrot 90.5 ± 2.0 90.5 ± 3.0 89.5 ± 0.7
was comparable with that obtained using toluene (87.8 ± 2.03 Leek 89.0 ± 2.1 86.3 ± 0.6 88.0 ± 1.0
and 87.8 ± 1.41, respectively). The relative standard deviations Olive 47.3 ± 0.8 49.1 ± 0.9
Oregano 14.3 ± 0.2 14.3 ± 0.1
were of 1.6% and 2.31% for toluene and alpha-pinene which means
Minced meat 59.3 ± 2.0 57.1 ± 0.7 64.0 ± 0.3
that the results were very little dispersed around the mean value.
a
This was confirmed using the Fisher-Snedecor’s test that gave no Values cited in the literature: (Veillet et al., 2010).
significant difference in the dispersion of the results. The tabular
value obtained by the Student test table for a = 0.05 was 2.10
and the calculated value was 0.67 which mean that there was no tained for the water contents found with the two methods (CDS and
significant difference from the statistical point of view. The two PDS) are comparable. All the results given by the PDS were not sta-
statistical tests validated the new PDS method as a good alterna- tistically different from those obtained by CDS, which make this
tive to the conventional one for the water determination. new method a green alternative to the toluene distillation for the
moisture determination in food matrices. If we compare these re-
3.4. Generalisation sults with those cited in the literature for limonene (Veillet et al.,
2010), we can see that the values obtained are comparable (Table
The results of the generalization of the PDS method to other 3) from moisture content point of view, reproducibility of the re-
products were given in Table 3. We can observe that the values ob- sults and kinetics of distillation. Thus, alpha-pinene is as effective
 S. Bertouche et al. / Food Chemistry 134 (2012) 602–605
as limonene and can be used like green solvent for the determina-
tion of the water content of the food products to replace toluene.
4. Conclusion
In this study, we use for the first time alpha-pinene as solvent
for water determination of food products using the Dean–Stark
procedure. The results obtained by this new PDS method are com-
parable in term of moisture contents and statistical tests with
those obtained by conventional procedure (CDS) using toluene as
distillation solvent. PDS method allows a substantial savings in
term of extraction time (30 min vs 105 min) and energy consump-
tion. Dean–Stark technique using alpha-pinene is a laboratory
exercise which easily teaches fundamental ‘‘green’’ food analytical
chemical lessons and successfully incorporates green ‘‘food analyt-
ical chemistry’’ into the teaching and the research laboratory to
both developed and developing nations.
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