Food Chemistry 183 (2015) 169–172

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

Rapid Communication

A rapid method for simultaneously determining ethanol and methanol

content in wines by full evaporation headspace gas chromatography
Chun-Yun Zhang a, Neng-Biao Lin b, Xin-Sheng Chai a,⇑, Zhong-Li b, Donald G. Barnes c
a
State Key Laboratory of Pulp and Paper Engineering, South China University of Technology, Guangzhou, China
b
Inspection & Quarantine Technical Center of Zhuhai Entry-Exit Inspection & Quarantine Bureau of P.R.C, Zhuhai, China
c
School of Environment and Energy, South China University of Technology, Guangzhou, China

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

Article history: This work reports on a full evaporation headspace gas chromatographic (FE HS-GC) method for
Received 28 June 2014 simultaneously determining the ethanol (EtOH) and methanol (MeOH) content in wines. A small sample
Received in revised form 10 March 2015 (10 lL) was placed in a headspace sample vial, and a near-complete mass transfer of ethanol and metha-
Accepted 14 March 2015
nol from the liquid sample to the vapor phase was obtained within three minutes at a temperature of
Available online 20 March 2015
105 °C, which allowed the measurement of the EtOH and MeOH content in the sample by GC. The results
showed excellent precision and accuracy, as shown by the reproducibilities of 1.02% and 2.11% for EtOH
Keywords:
and MeOH, respectively, and recoveries that ranged from 96.1% to 104% for both alcohols. The method is
Ethanol
Methanol
efficient, accurate and suitable for the determination of EtOH and MeOH in wine production and quality
Wines control.
Full evaporation Ó 2015 Elsevier Ltd. All rights reserved.
Headspace GC

1. Introduction There are several methods available for quantifying alcohol spe-
cies in wines. Densimetric methods (European Commission, 2000;
Wine is a complex mixture obtained from the complete or par- OIV, 2009), based on pycnometry, and hydrostatic balance (or
tial fermentation of fresh grapes or grape must, containing water, hydrometry) have been used traditionally for the determination
alcohols, acids, sugars, mineral salts, pigments and aromatic com- of alcohols in wine, measured as the percentage by volume.
pounds (Cheynier, Schneider, Salmon, & Fulcrand, 2010). Ethanol However, these methods use distillation to separate the alcohols
(EtOH) is the second largest component (after water) in wine, with from other coexisting species (e.g., sugars) prior to the densitome-
concentrations ranging from 10% to 20% v/v (Pinney, 2012). The try. This distillation process is time-consuming, and the sensitivity
EtOH content is one of the most important parameters for process is poor, which can cause significant errors, especially in wines with
and quality control in wine industries (Collins, Miller, Altria, & low alcohol content. Several other analytical methods have been
Waterhouse, 1997). In addition to EtOH, there is a small amount developed to quantify specific alcohol components in wines and
of methanol (MeOH, also called ‘‘wood alcohol’’) in wines that is beverage products; e.g., titrimetric methods (AOAC, 1990), enzy-
produced as a by-product of the acetification involving the enzy- matic methods (Gulce, Gulce, Kavanoz, Coskun, & Yildiz, 2002;
mic hydrolysis of pectin methoxyl groups during fermentation Mizgunova, Zolotova, & Dolmanova, 1996), and colorimetric (spec-
(Ribereau-Gayon, Glories, Maujean, & Dubourdieu, 2000). Since trophotometric) methods (AOAC, 1990). The titrimetric and enzy-
MeOH is toxic to humans (Bindler, Voges, & Laugel, 1988; Robins, matic methods have low reproducibility and accuracy, while the
Angell, & Kumas, 1981), the MeOH content of wines is strictly regu- colorimetric methods are subject to interferences by colored sub-
lated by the International Office of Vine and Wine (OIV) at stances in wine. Because of the ability to separate chemical species
<400 mg/L for red wines and <250 mg/L for white or rose wines and high sensitivity of the detector, the combination of high per-
(OIV, 2011). Clearly, routine methods that can quantify the con- formance liquid chromatography (HPLC) (Chen et al., 1998; Kuo,
tents of these alcohols efficiently are desirable in both production Wen, Huang, Wu, & Wu, 2002) and gas chromatography (GC)
and quality control. (Liu, Liu, Zhang, & Zhang, 2001; Wilson, Ding, & Woods, 1991) is
regarded as the best method for the quantification of these alcohol
species. Unfortunately, HPLC or GC analysis requires sample pre-
⇑ Corresponding author. Tel./fax: +86 20 87113713. treatment to minimize the species (such as natural polymers, salts
E-mail address: [email protected] (X.-S. Chai).
and sugars) that might deteriorate or contaminate the instruments.

http://dx.doi.org/10.1016/j.foodchem.2015.03.048
0308-8146/Ó 2015 Elsevier Ltd. All rights reserved.
170 C.-Y. Zhang et al. / Food Chemistry 183 (2015) 169–172

The pre-treatment procedures, typically filtration and solvent sample loop temperature = 110 °C; transfer line
extraction, are not only complicated and time-consuming but also temperature = 115 °C; pressurization pressure = 2.00 bar; carrier
can easily introduce significant errors in the quantification. gas pressure = 1.50 bar; vial pressurization time = 15 s; sample
Conventional headspace gas chromatography (HS-GC), based on loop fill time = 10 s; and transfer time = 20 s.
vapor–liquid partition equilibrium of the analytes, has also been
used for the determination of EtOH (Wartts & McDonald, 1987)
and MeOH (Chai, Dhasmana, & Zhu, 1998) in complicated liquid 2.3. Procedures of sample preparation
matrices. The major advantage of HS-GC is that it can usually be
used without sample pre-treatment, because the analytes are 10 lL of the sample solution was placed in a headspace sample
released in a relatively pure form into the headspace. Thus, HS- vial (21.6 mL) by a micropipette. The sample vial was immediately
GC analysis is not only simpler but is also more efficient than the sealed with a PTFE/silicone septum and aluminum cap. The FE
techniques mentioned above. However, the HS-GC cannot simply equilibration was conducted at 105 °C for 3 min prior to HS-GC
be applied to quantify the EtOH content in wines, because the equi- measurement.
librium of EtOH between vapor and liquid phases does not follow
Henry’s Law (Teja, Gupta, Bullock, Chai, & Zhu, 2001) due to the
relatively high concentration of alcohol in wines. Although this
3. Results and discussions
problem can be solved by the internal standard calibration; i.e.,
spiking a known amount of analyte into the sample (Chai et al.,
3.1. Chromatogram of a wine sample measured by FE HS-GC
1998), this additional step makes the method less efficient, espe-
cially in the case of batch sample analysis.
Unlike the analysis in our previous work (Li et al., 2007, 2009),
Unlike the conventional HS-GC, the full evaporation (FE) tech-
in order to separate the EtOH and MeOH signals, generated by large
nique is based on a near-complete mass transfer, other than phase
concentration differences but similar retention times, the GC con-
equilibration, which means that the vapor analytes are indepen-
ditions had to be selected carefully. Fig. 1 shows a GC chro-
dent of the sample matrices (Kolb & Ettre, 2006). In FE conditions,
matogram of the FE HS-GC analysis of a wine sample under the
Henry’s Law is no longer a factor, which makes the calibration step
selected conditions. Fig. 1a is an enlargement of Fig. 1b and shows
much simpler. Also, compared with the conventional HS-GC meth-
that some minor volatile species (including MeOH) were well sepa-
ods, the time for headspace equilibration required in FE HS-GC is
rated from EtOH under these conditions. Clearly, EtOH is the domi-
much shorter, due to the very small samples used (Kolb & Ettre,
nant species in the vapor phase due to its high content in the
2006).
sample. Although MeOH is a minor component in wines, the GC
In previous studies, we developed two related FE HS-GC meth-
detector (FID) is sufficiently sensitive to quantify its content using
ods: one for the determination of MeOH in a pulping spent liquor
the FE HS-GC method.
(Li, Zhan, Fu, Liu, & Chai, 2007) and the other for determination
of EtOH in a fermentation process solution (Li, Chai, Deng, Zhan,
& Fu, 2009). In this work, we report on the development of a rapid
method to simultaneously quantify the EtOH and MeOH content in 3.2. Conditions for full evaporation
wines based on the FE HS-GC technique. The main focus is on the
optimization of the conditions during the analysis, with emphasis 3.2.1. Equilibration temperature
on the sample size and headspace equilibration time and tempera- In order to achieve a near-complete mass transfer of the volatile
ture. The reproducibility of the method and the recovery of spiked analytes from the liquid phase to vapor phase (i.e., a full evap-
analytes are also evaluated. The goal is to demonstrate a rapid oration), it is essential to equilibrate the sample at a high tempera-
method that can provide timely information during the wine mak- ture so that more volatile solutes can leave the liquid phase and
ing process and accurate data for quality control purposes. enter the headspace. However, if the temperature is too high, the
resulting high pressure increases the risk of the sample leaking
from or even bursting the vial.
2. Experimental In our previous studies (Li et al., 2007, 2009), we found that full
evaporation of aqueous samples at 105 °C (greater than water boil-
2.1. Chemicals and materials ing point) worked well with a sample size <100 lL. Therefore, we
chose 105 °C as the equilibration temperature in the present study.
All chemicals used in the experiments were analytical grade
and purchased from commercial sources without further purifica-
tion. A set of mixed standard solutions (EtOH concentrations of 0–
16.0% v/v and MeOH concentrations of 0–396 ppm (w/v)) were
prepared by adding different amounts of pure EtOH and MeOH
to distilled water
Wine samples used in the experiments were purchased from a
local commercial source.

2.2. Apparatus and operations

HS-GC measurements were carried out with an automatic head-

space sampler (DANI HS 86.50, Italy) and a GC system (Agilent GC
7890A, US) equipped with a flame ionization detector and a DB-5
capillary column (30 m long, 0.35 mm ID), operating at a tempera-
ture of 30 °C for 2.8 min with nitrogen carrier gas (flow
rate = 3.8 mL/min). Headspace operating conditions were as fol-
lows: 3 min of strong shaking for sample equilibration at 105 °C; Fig. 1. GC chromatogram from FE HS-GC analysis for a wine sample.
 C.-Y. Zhang et al. / Food Chemistry 183 (2015) 169–172 171

3.2.2. Equilibration time

The equilibration time for the full evaporation of these two
alcohol species from liquid phase was investigated at 105 °C,
where 10 lL of wine sample was used. As shown in Fig. 2, the GC
signals for both EtOH and MeOH in the FE HS-GC testing reach at
the maximum values in a very short time (within two minutes).
Therefore, we chose three minutes as the equilibration time in
the following experiments.

3.3. Maximum sample size for the linear range

To obtain better detection sensitivity, a larger sample size (vol-

ume) in FE HS-GC measurement is desirable (Kolb & Ettre, 2006).
However, if the sample size is too large, part of the volatile solute
may remain in the condensed phase (Li et al., 2009) and thus the
full evaporation of the analytes in the sample would not be
achieved. Fig. 3 shows the effect of sample size on the full evap-
oration of these two alcohols from a wine sample in the headspace
vial. In Fig. 3a the linear range of the EtOH response extends to at
least 50 lL. In contrast, the GC signals for the MeOH are only lin-
early proportional over the volume range 0–30 lL, shown in
Fig. 3b. Therefore, unlike results observed in the previous work
(Li et al., 2007, 2009), greater amounts of EtOH in wine affected
the linear range of MeOH response due to the higher vapor pres-
sure in the headspace sample vial. Therefore, to ensure that the
GC responses for these alcohol species were located in the linear
range for both analytes, 30 lL was the maximum sample size used.

3.4. Evaluation of the method

3.4.1. Calibration and the limit of quantification Fig. 3. (a) Effect of sample size on the full evaporation of EtOH. (b) Effect of sample
A simple external standard calibration was employed in the size on the full evaporation of MeOH.
present FE HS-GC method. The calibration was based on placing
10 lL of the standard solutions containing EtOH (concentrations The limit of quantitation (LOQ) was calculated from the follow-
from 0% to 16.0% v/v) and MeOH (concentrations from 0 to ing equation (MacDougall & Crummett, 1980).
396 ppm) into a set of headspace sample vials and analyzing the
vapor by FE HS-GC. From these data we obtained the following a þ 10jDaj
LOQ ¼ ð3Þ
calibration equations for EtOH and MeOH; i.e., s

AE ¼ 126ð103Þ þ 941ð11ÞC E ðn ¼ 7; R2 ¼ 0:999Þ ð1Þ where a, s and Da represent the intercept, the slope, and the uncer-
tainty of the intercept in Eqs. (1) and (2), respectively. Eq. (3) gives
an LOQ of 1.23% v/v for EtOH and an LOQ of 13.0 mg/L for MeOH.
AM ¼ 0:455ð0:073Þ þ 0:0911ð0:0016ÞC M ðn ¼ 7; R2
¼ 0:999Þ ð2Þ 3.4.2. Method precision and accuracy
The reproducibility of the present method was investigated by a
where AE and AM represent, respectively, the GC peak area for EtOH
sextuplicate determination of EtOH and MeOH content in the same
and MeOH in the calibration test, and C E (% v/v) and C M (mg/L)
wine sample under the optimized conditions and procedures. The
represent the corresponding concentrations of EtOH and MeOH.
relative standard deviations (RSD) for EtOH and MeOH determina-
tion were less than 1.02% and 2.11%, respectively.
To evaluate the recovery of the method, we prepared a set of
sample solutions by accurately spiking 1 mL of wine sample with
different volumes (10–70 lL) of pure EtOH and a MeOH standard
solution (4752 ppm). The unspiked wine sample, which contained
12.3% v/v of EtOH and 257 mg/L of MeOH, served as a reference.
The net contribution from the added EtOH and MeOH in these
make-up samples was determined. The results in Table 1 show that

Table 1
Method evaluation.

Sample Ethanol, % v/v Methanol, lg/mL Recovery, %

Added Measured Added Measured Ethanol Methanol
1 3.00 3.12 142 148 104 104
2 4.00 4.17 190 193 104 101
3 5.00 4.99 238 233 99.8 97.9
4 6.00 5.78 285 274 96.3 96.1
Fig. 2. Effect of equilibration time on the full evaporation of alcohols from a wine 5 7.00 7.23 332 324 103 97.6
sample.
172 C.-Y. Zhang et al. / Food Chemistry 183 (2015) 169–172

the recoveries for both EtOH and MeOH were in the range of 96.1– Collins, T. S., Miller, C. A., Altria, K. D., & Waterhouse, A. L. (1997). Development of a
rapid method for the analysis of ethanol in wines using capillary
104%, which indicates that the present method is sufficiently accu-
electrophoresis. American Journal of Enology and Viticulture, 48, 280–284.
rate and robust for the determination of these alcohols in wine Commission Regulation (EC) No 2870/2000 laying down Community reference
samples. methods for the analysis of spirits drinks, (2000). Official Journal of the European
Communities, 333, 20–46.
Gulce, H., Gulce, A., Kavanoz, M., Coskun, H., & Yildiz, A. (2002). A new
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Kolb, B., & Ettre, L. S. (2006). Static headspace-gas chromatography-theory and
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