Food Anal.

Methods (2014) 7:1043–1046

DOI 10.1007/s12161-013-9711-8

A Novel Method for the Determination of the Ethanol Content

in Soy Sauce by Full Evaporation Headspace
Gas Chromatography
Mengru Liu & Hailong Li & Huaiyu Zhan

Received: 3 May 2013 / Accepted: 30 August 2013 / Published online: 11 September 2013
# Springer Science+Business Media New York 2013

Abstract A full evaporation headspace gas chromatographic fermentation process. It is produced when the grain ferments,
(HSGC) method is described for rapid determination of etha- while tamari, which is usually made without grain, contains
nol in soy sauce. The results demonstrated that a near- little or no alcohol. Ethanol is added after the tamari has been
complete mass transfer of ethanol in the soy sauce sample to aged to prevent molds and fungi developing (Kataoka 2005).
the vapor phase (headspace) was achieved within 2 min at The ethanol in soy sauce is usually a small quantity, but even
105 °C when a very small (≤40 μL) sample was added to a 20- small amounts of ethanol affect the taste of soy sauce
mL headspace sample vial. The ethanol in the vapor phase (Steinhaus and Schieberle 2007). The amount of ethanol in
was then determined by HSGC using a flame ionization soy sauce is one of the key parameters for soy sauce quality.
detector. The results showed that the method has an excellent Therefore, the ethanol determination in soy sauce is critically
measurement precision (relative standard deviation <0.41 %) important as not only does it influence the taste and aroma but
and accuracy (recovery=99.85±1.85 %) for ethanol quantifi- also the certification of soy souce.
cation in soy sauce samples. The method requires no sample Traditional analysis methods, such as colorimetry (LAU
pretreatment, so it is a simple, rapid, and sensitive method for and LUK 1994; Zanon et al. 2007), dichromate oxidation
the accurate quantification of ethanol content in soy sauce and (Caputi et al. 1968), enzymatry (Mccloskey and Replogle
suitable for applications in food industry. 1974; Gonchar et al. 2001; Pavlishko et al. 2005) as well as
the advance techniques such as gas chromatography (Stackler
Keywords Ethanol . Soy sauce . Headspace . Gas and Christensen 1974; McLachlan et al. 1999; Naviglio et al.
chromatography . Full evaporation 2001; Wang et al. 2003) and high performance liquid chro-
matography (Kupina 1984; Lefebvre et al. 2002; Yarita et al.
2002), are widely used for quantification of ethanol in the food
Introduction industry. Nevertheless, the makeup of soy sauce is very com-
plex, as they contain not only a significant amount of nonvol-
Ethanol is a volatile, colorless liquid that has a slight odor. It is atile species and color substances but also suspended solids.
widely used as a carrier in many food flavors. In cooking, For that reason, it is usually necessary to pretreat the sample,
ethanol is often used to remove any undesirable fishy or using procedures such as chemical reaction, distillation, and
gamey odors. Many fermented foods, such as soy sauce, solvent or solid-phase extraction, in order to minimize the
contain an amount of ethanol (Luh 1995; Blandino et al. impact of these interfering species in subsequent ethanol
2003). Soy sauce contains ethanol that is a by-product of the analysis. This pretreatment makes the traditional procedures
more complicated, time-consuming, and subject to errors.
The full evaporation (FE) technique was one of the early
M. Liu
Industrial Technology Research Institute, South China University of headspace gas chromatographic (HSGC) techniques (Zhu and
Technology, Guangzhou 510640, China Chai 2005). It was initially developed by Markelov and
Guzowski (1993). The FE-HSGC technique is based on a
H. Li (*) : H. Zhan
near-complete evaporation or transfer of the analytes from a
State Key Laboratory of Pulp and Paper Engineering, South China
University of Technology, Guangzhou 510640, China condensed matrix into a vapor phase in the headspace of the
e-mail: [email protected] sample vial. Therefore, the sample matrix effect could be
1044 Food Anal. Methods (2014) 7:1043–1046

completely eliminated. In FE-HSGC technique, a very small

size (such as microliter level) of liquid sample is usually used
in order to achieve a full evaporation in a very short period of
time; consequently, vapor–liquid equilibrium and sample pre-
treatments are not required.
In the present work, we have demonstrated an FE-HSGC
technique for rapid determination of ethanol in the sample of
soy sauce. The effects of FE-HSGC conditions (e.g., equili-
bration temperature, equilibration time, and sample size) on
experimental results were also probed.

Materials and Methods

Samples

Fig. 1 FE-HSGC chromatogram from ethanol in a soy sauce sample

The samples were purchased from local markets. Samples
analysis
were stored in the dark at room temperature.

Chemicals eliminate the interference from the coexisting volatile and

nonvolatile species found in soy sauce samples.
All chemicals used in the experiments were from commercial
sources. A standard ethanol solution (800 mg/L) was prepared Conditions for FE Headspace Analysis
by adding 100 μL pure solvent in 100 mL water.
In FE-HSGC analysis, the key to success is to achieve a near-
Apparatus and Operations complete mass transfer of analyte from the liquid phase to vapor
phase as quickly as possible (Chai et al. 2004). Therefore, the
All measurements were carried out using an HSS 80.65 effects of major experimental parameters in headspace sampler
Automatic Headspace Sampler (DANI, Italy) and Model operation on the full evaporation should be discussed.
HP-7890 capillary gas chromatograph (Agilent, USA).
Headspace sampler operating conditions were as follows: Equilibration Temperature
2 min strong shaking for the sample equilibrium at a temper-
ature of 105 °C, vial pressurization time of 0.2 min, sample The vapor–liquid equilibrium (VLE) partition coefficient is a
loop fill time of 1.0 min, and loop equilibration time of function of temperature; it decreases with increasing temper-
0.05 min. GC conditions were as follows: HP-5 capillary ature and then begins to increase with temperature after a
column at 40 °C, high-purity nitrogen carrier gas flow rate
of 3.5 mL/min. A flame ionization detector was employed
with hydrogen and air with flow rates of 40 and 400 mL/min,
respectively.
The sample preparation and measurement steps were as
follows: inject microliter level (or weigh the equivalent
amount) of sample solution into a closed sample vial by micro
syringe and place it in the headspace sample tray for HSGC
measurements.

Results and Discussion

Headspace Analysis of Ethanol from Soy Sauce

Figure 1 shows that the ethanol of soy sauce sample can be well
separated from the other volatile species under the given GC
conditions. Therefore, the FE-HSGC method can effectively Fig. 2 Effect of the equilibration temperatures
Food Anal. Methods (2014) 7:1043–1046 1045

Table 1 Repeatability
testing Sample Ethanol content (mg/L)

1 16.45
2 16.36
3 16.40
4 16.32
5 16.49
average 16.40
RSD (%) 0.41

Thus, the vapor ethanol content can be calculated as

below,
ClV l
Cg ¼ ð1Þ
Vt
Fig. 3 Effect of the equilibration time on the amount of ethanol detected where V l and V t are the volumes of the liquid sample and the
in the vapor phase
headspace sample vial, respectively.
According to Eq. (1), the vapor ethanol content (C g) is
point. As shown in Fig. 2, the two-phase equilibrium is increased with the sample size introduced in a given vial vol-
achieved at a temperature above 105 °C after 2 min for a sample ume. However, a larger sample size requires a longer sample
size of 20 μL. Therefore, we chose a temperature of 105 °C for transfer time or high temperature to achieve near-complete mass
the rest of the study. transfer. Figure 4 shows the effect of the sample size on the
ethanol full evaporation of the soy sauce at the given conditions.
Equilibration Time It can be seen that the linear responses can be obtained if the
sample size is less than 40 μL. Consequently, 40 μL is the
For an efficient analysis, a short VLE time is desired. The maximum sample size that could be used for accurate determi-
effect of equilibration time is shown in Fig. 3. It can be seen nation of ethanol in the present FE-HSGC method.
that the VLE can be achieved within 2 min at the given
temperature (105 °C). Completeness of Ethanol Interphase Transfer

Sample Size For quantification analysis, it is essential to achieve a near-

complete mass transfer of analyte from the liquid phase to
In FE headspace technique, it assumes that the ethanol vapor phase. To verify a near-complete interphase transfer
in the liquor is near-completely released to vapor phase. under these conditions, we placed 20 μL of soy sauce sample
into a small metal dish that was, in turn, placed in a sample
vial. After the first measurement by HSGC at 105 °C (head-
space sampler oven temperature) with an equilibration time of
2 min, the dish was transferred to another sample vial for
second measurement at same conditions. The result showed
that the ratio of the GC signal peaks (P) of these two

Table 2 Method validation

Sample Ethanol content (mg/L) Recovery (%)

Added Measured

1 4.0 3.92 98.0

2 8.0 8.12 101.5
3 16.0 16.27 101.7
4 24.0 23.92 99.7
5 32.0 31.72 99.1
Fig. 4 Effect of sample size on the amount of ethanol detected in the
vapor phase Twenty microliters of samples was used
1046 Food Anal. Methods (2014) 7:1043–1046

measurements (i.e., P2/P1) was less than 0.72 %, which ex- Conflict of Interest Mengru Liu declares that he has no conflict of interest.
perimentally proved that a near-complete full evaporation was Hailong Li declares that he has no conflict of interest.
Huaiyu Zhan declares that he has no conflict of interest.
achieved within 2 min at 105 °C.

Method Calibration, Precision, and Valuation References

The reproducibility testing of the present method was inves- Blandino A, Al-Aseeri M, Pandiella S, Cantero D, Webb C (2003)
tigated. The results show that the relative standard deviations Cereal-based fermented foods and beverages. Food research inter-
national 36:527–543
(RSD) in five measurements is <0.41 % (as shown in Table 1),
Caputi A, Ueda M, Brown T (1968) Spectrophotometric determination of
in which the random errors are associated with the uncer- ethanol in wine. Am J Enol Vitic 19:160–165
tainties in both sampling and HSGC detection. Chai X-S, Hou Q, Schork F (2004) Determination of residual monomer in
In this work, a simple external standard calibration can be polymer latex by full evaporation headspace gas chromatography.
Journal of Chromatography A 1040:163–167
employed. The calibration was performed based on adding
Gonchar MV, Maidan MM, Pavlishko HM, Sibirny A (2001) A new
different volumes (0∼40 μL) of ethanol standard solution into oxidase-peroxidase kit for ethanol assays in alcoholic beverages.
a set of headspace sample vials, and then the samples were Food Technology and Biotechnology 39:37–42
tested by FE-HSGC. Using the data from the GC measure- Kataoka S (2005) Functional effects of Japanese style fermented soy
sauce (shoyu) and its components. Journal of bioscience and bioen-
ment on these samples, a standard calibration curve was
gineering 100:227–234
obtained, that is Kupina S (1984) Simultaneous quantitation of glycerol, acetic acid and

ethanol in grape juice by high performance liquid chromatography.
A ¼ 24:33ð14:27Þ þ 52:02ð0:81Þ  C n ¼ 8; R2 ¼ 0:999 Am J Enol Vitic 35:59–62
ð2Þ LAU OW, LUK SF (1994) Spectrophotometric method for the determination
where A and C represent the GC signal peak are of ethanol in of ethanol in beverages and beer samples using cerium (IV) as reagent.
International journal of food science & technology 29:469–472
the vapor phase and its absolute amount (in microgram) added
Lefebvre D, Gabriel V, Vayssier Y, Fontagne-Faucher C (2002) Simultaneous
in the headspace sample vial, respectively. The limit of quan- HPLC determination of sugars, organic acids and ethanol in sourdough
titation (LOQ) in this method is 2.09 μg. process. LWT-Food Science and Technology 35:407–414
To validate the present method, we prepared a set of sample Luh B (1995) Industrial production of soy sauce. Journal of Industrial
Microbiology & Biotechnology 14:467–471
solutions by accurately spiking different volumes (0–250 μL)
Markelov M, Guzowski JP (1993) Matrix independent headspace gas
of ethanol standard solution into 2.0 mL of a soy sauce chromatographic analysis. This full evaporation technique.
sample. The original sample (i.e., without added ethanol) Analytica chimica acta 276:235–245
was measured as a reference. Thus, the net contribution from Mccloskey LP, Replogle LL (1974) Evaluation of an enzymatic method
for estimating ethanol in wines using an" enzyme kit". Am J Enol
the added ethanol in the FE-HSGC measurement for these
Vitic 25:194–197
spiked samples can be calculated by Eq. (2). As shown in McLachlan DG, Wheeler PD, Sims GG (1999) Automated gas chromato-
Table 2, the recoveries achieved in the present method are in graphic method for the determination of ethanol in canned salmon.
the range of 98.0–101.7 %. The good ethanol recoveries Journal of agricultural and food chemistry 47:217–220
Naviglio D, Romano R, and Attanasio G (2001) Rapid determination of
indicate that the present method is justifiable for the determi-
ethanol content in spirits and in beer by high resolution gas chro-
nation of ethanol content in soy sauce samples. matography. Industrie delle Bevande 30
Pavlishko N, Ryabinina O, Zhilyakova T, Sakharov IY, Gerzhikova V,
Gonchar M (2005) Oxidase-peroxidase method of ethanol assay in
fermented musts and wine products. Appl Biochem Microbiol 41:
Conclusions 604–609
Stackler B, Christensen E (1974) Quantitative determination of ethanol in
We successfully demonstrated a full evaporation headspace wine by gas chromatography. Am J Enol Vitic 25:202–207
Steinhaus P, Schieberle P (2007) Characterization of the key aroma
gas chromatographic technique for determination of ethanol in
compounds in soy sauce using approaches of molecular sensory
soy sauce. By choosing a very small sample size (≤40 μL) and science. Journal of agricultural and food chemistry 55:6262–6269
a high temperature (105 °C), a near-completion of ethanol Wang M, Choong Y-M, Su N, Lee M (2003) A rapid method for determi-
mass transfer from soy sauce to vapor phase can be achieved nation of ethanol in alcoholic beverages using capillary gas chromatog-
raphy. J Food Drug Anal 11:133–140
within 2 min. The present method is simple, rapid, and accu-
Yarita T, Nakajima R, Otsuka S, Ihara T, Takatsu A, Shibukawa M (2002)
rate and suitable for applications in the food industry. Determination of ethanol in alcoholic beverages by high-performance
liquid chromatography–flame ionization detection using pure water as
Acknowledgment The authors acknowledge the Natural Science Foun- mobile phase. Journal of Chromatography A 976:387–391
dation of China (nos. 31100438 and 21006034) and the State Key Laboratory Zanon JP, Peres MF, Gattás EA (2007) Colorimetric assay of ethanol
of Pulp and Paper Engineering Program (201154) for sponsoring the research. using alcohol dehydrogenase from dry baker's yeast. Enzyme and
microbial technology 40:466–470
Compliance with Ethics Requirements This article does not contain Zhu J, Chai X-S (2005) Some recent developments in headspace gas
any studies with human or animal subjects. chromatography. Curr Anal Chem 1:79–83