Iliade-143 Ethanol Gc-Fid

ILIADe 143:2019 | CLEN Method
Determination of Ethanol in Alcoholic Products by GC-FID
Version 14 October 2019

Determination of Ethanol in Alcoholic Products by GC-FID
(Gas Chromatography - Flame Ionisation Detection)
1. Scope
The method is suitable to determine ethanol in Completely Denatured Alcohol (CDA)
required for calculation of the content of denaturing substances as specified in
Regulation (EC) 3199/93 of 22 November 1993 and its amendments, concerning the mutual
recognition procedures for the complete denaturing of alcohol (CDA) for the purpose of
exemption from excise duty.
In addition, this method is also suitable for some alcoholic beverages such as vodka, rum,
whisky, brandy, fruit distillates as well as fraudulent distillates containing denaturants. The
method can be used also for determination of ethyl alcohol in commercial mixtures such as
antifreeze and screen-wash preparations.
2. Principle
Ethanol is separated from other volatile substances (i.e. methanol, ethyl acetate or
denaturants such as IPA and MEK) using capillary column gas chromatography and is
detected by FID. The ethanol concentration is determined against a calibration curve using
an internal standard.
3. Reagents and materials

3.1. Chemicals used and their associated safety phrases
− Ethanol (ethyl alcohol) ; CAS 64-17-1; purity 99.8 %.
− Methanol (methyl alcohol);CAS 67-56-1; purity 99.8 %.
− 1,4-dioxane; CAS 123-91-1; p.a. grade;
1,4-dioxane is used as internal standard. Alternatively, any other substance
chromatographically sufficiently separated from all other components and not
present in the sample can be used as an internal standard.
3.2. Gases and other consumables
− Gases: helium purity min. 5.0; hydrogen purity min. 4.6; synthetic air free of
hydrocarbons
− Automatic pipette: 0 - 10 ml.
− Chromatographic capillary column DB624 (J&W) length 30 m, inner diameter 0.32
mm and thickness of stationary phase 1.8 µm. Alternative GC capillary columns to
the recommended DB624 (30 m x 0.32 mm x 1.8 µm) can be used if a good
separation of ethyl alcohol from possible interferences can be achieved. The
alternative columns include e.g. RXI-624 SILMS, TRB 624, RX-1, DB-1, HP-5 and other
capillaries based on non-polar stationary phases.
− Chromatographic syringe 10 µl, 500 µl, 1000 µl
− Glass vials 2 ml and 20 ml with cap and septum
− Tips for automatic pipette
− Tool for closing and opening of glass vials 2 ml and 20 ml
The chemicals, gases, and consumables mentioned here may be replaced by those
having equivalent specifications.
4. Apparatus
4.1. Analytical balance with precision of 0.1 mg.
4.2. An electronic densimeter.
4.3. A gas chromatograph (GC) with split/split less injector, flame ionization detector, auto
sampler and PC for control of the GC and data processing.
5. Procedure
5.1. Preparation of calibration solutions
Solutions for calibration points 1-5 are prepared as follows: an empty 20 ml glass vial
(including septum and cap) is weighed and its weight (ms) is recorded. The balance is then
tarred. 15 ml of methanol is added to the 20 ml glass vial. The vial is closed by cap and
weighed (the weight of methanol mm is recorded). The balance is tarred. Then 0.5 ml of
internal standard (e.g. 1,4-dioxane) is added through a septum using a syringe, the content
of the vial is mixed and weighed (the weight of internal standard mi is recorded). The
balance is tarred. Finally 10, 100, 300, 700 or 1000 µl of ethyl alcohol (for calibration
standards 1 to 5, respectively) are added to the vial through the septum using a syringe. The
content of the vial is mixed and weighed (the weight of ethyl alcohol ma is recorded). The
balance is tarred and the vial is removed from the balance. Final weight reading mf is
recorded (as positive value). The maximum acceptable difference between mf and the sum
of all the weights is 0.5 mg (see Equation 1). When the difference is higher, the preparation
of that particular calibration solution must be repeated. Each calibration solution is mixed,
then opened and 2 ml of the calibration solution is transferred to a 2 ml glass vial. The 2 ml
glass vial is closed and stored in the refrigerator until the GC analysis is completed.
Note: Depending on final ethanol concentration in the sample the calibration range can be
modified providing the quality control conditions described below are met.
5.2. Sample preparation
Solid impurities in sample must be filtrated.
Sample preparation is carried out exactly as described above, however, instead of ethanol,
1000 µl of the sample is added and the weight mvz is recorded. Every sample is prepared in
duplicate.
5.3. Chromatographic conditions
With regard to the operation of the GC, the analytical sequence is programmed in the GC
software (e.g. sample number, sample position in auto sampler, analytical method, amount
of the internal standard, dilution factor).
The chromatographic conditions to be used with the GC capillary column DB624 (30 x 0.32
mm x 1.8 µm) are as follows: inject volume: 1 µl, split approx. 120 ml/min., injector
temperature 230 °C, detector FID temperature 230 °C, temperature gradient: 45 °C (4 min) /
10 °C/min / 90 °C (0 min) / 20 °C/min / 200 °C (2 min), carrier gas helium velocity (32 cm/s).
If a different type of GC column is used or if the ethyl alcohol peak co-elutes with the peak of
another substance (i.e. separation of ethanol peak is less than 0.75 %) or if the shape of the
ethyl alcohol peak is not obvious (broad peak, tailing peak, etc.), the GC temperature
programme may be modified.
Between each sample injection, the auto-sampler must be programmed to thoroughly rinse
the injection syringe using methanol to prevent any possible cross-contamination.
The chromatographic conditions can be modified for other columns used to achieve a good
separation of ethyl alcohol and internal standard from possible interferences.
5.4. Quality control

Data for calibration curve construction are obtained by the measurement of 5 calibration
solutions. The peak of the internal standard (this peak is defined as reference) and the ethyl
alcohol peak are identified. A first order curve (line) is constructed from the points obtained
by combination of the area of ethyl alcohol peak and its corresponding concentration. The
calibration curve does not include the point [0; 0]. The calibration curve must have a
correlation coefficient higher than 0.999. If not, the calibration must be repeated. The
maximum acceptable intercept of the calibration curve (line) is |0.01| (absolute value). In
case of a higher intercept, it is necessary to find the source of this problem and to repeat the
calibration.
A quality control (QC-80) solution of approx. 80 % ethyl alcohol is prepared. The correctness
of the calibration is checked by analysing the QC-80 solution immediately after the
calibration. If the result for the QC-80 standard is outside the limits given for this standard
(±0.4 % mass) the calibration must be repeated or at least re-evaluated. The method is
checked by controlling the QC-80 standard at the beginning and at the end of a sample
sequence (first and last vials contain QC-80). If the sequence contains more than 10 vials,
this control standard is placed into the sequence after each 10 vials. The sample results are
accepted only if the results for the QC-80 standard are within the limits given for this
standard. If the result for the QC-80 standard is outside the limits given for this standard, it is
not possible to accept the results for the samples placed before and after this standard.
These samples must be prepared and measured again.
Note: Depending on samples different quality control solutions can be used having ethanol
concentration close to the sample analysed e.g. QC-35 for distillates or QC-90 for denaturized
alcohol.
Each sample is prepared in duplicate: if the difference between the first and the second
sample result is higher than the repeatability limit, the sample must be prepared and
measured in duplicate again.
6. Calculations
The concentrations of ethyl alcohol as well as the internal standard are calculated for each
calibration solution using the Equations 2 and 3. The ethanol content in the sample is
calculated using the GC software and is obtained from the calibration curve (evaluation is
done using the area of the peaks) and multiplied by sample dilution factor (using Equations 4
and 5).
Equation 1 𝑚𝑚𝑓𝑓 − (𝑚𝑚𝑠𝑠 + 𝑚𝑚𝑚𝑚 + 𝑚𝑚𝑖𝑖 + 𝑚𝑚𝑒𝑒 ) ≤ 0.5 [mg]

0.78924 ma
Equation 2 C EtOH = wv × × [% mass]
ρ EtOH mm + mi + ma
mi
Equation 3 Ci = 100 × [% mass]
mm + mi + ma
mm + mi + mVZ
Equation 4 R= [-]
mVZ
Equation 5 wEtOH = R × w´ EtOH [% mass]
wEtOH
Equation 6 wv = ρVZ × [% vol.]
0.78924
ρEtOH Density of calibration standard by 20 °C in g.cm-3
ρVZ Density of the sample by 20 °C in g.cm-3
0.78924 Density of ethyl alcohol by 20 °C in g.cm-3
Concentration of the internal standard in calibration solution in %
CI
mass
CEtOH Concentration of ethyl alcohol in calibration solution in % mass
ma Weight of the calibration standard
mi Weight of the internal standard in g
mm Weight of methanol in g
Weight of full 20 ml vial (with cap, septum, methanol, internal
mf
standard and calibration standard/sample) in g
ms Weight of empty 20 ml vial with cap and septum in g
mvz Weight of the sample in g
R Dilution factor
Concentration of ethyl alcohol in the sample solution in % mass
w’EtOH
(reading from the calibration curve)
wEtOH Concentration of ethyl alcohol in sample in % mass
wv Concentration of ethyl alcohol in % volume
Duplicate measurements are rounded to 0.01 % mass.
The final result is the arithmetic mean of duplicate measurements for each sample, rounded
to 0.1 % mass.
It is possible to recalculate the result from % mass to % vol. using the Equation 6.
7. Expression of results
Result is given with maximum 3 significant digits, with one decimal.
8. Precision
Precision data obtained from the 1st CLEN proficiency test on completely denatured alcohol,
performed in 2019 (final report issued 4 September 2019) by 41 laboratories on 3 samples.
Ethanol Matrices
Completely
denatured Burning alcohol Screen wash
alcohol (CDA)
Ethanol content
94.6 89.6 33.9
(robust mean), % vol.
Repeatability (SD), % vol. 0.2 0.2 0.1
Repeatability, % 0.2 0.2 0.3
Reproducibility (SD), % vol. 0.7 0.7 0.2
Reproducibility, % 0.7 0.7 0.7
Annex – examples of chromatograms
Example of the typical chromatogram of a sample containing ethanol:
Typical chromatogram of a formulation including the internal standard (1,4-dioxane) and a series of
other potential denaturants.

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ILIADe 143:2019 | CLEN Method

Determination of Ethanol in Alcoholic Products by GC-FID

Version 14 October 2019

3. Reagents and materials

5.4. Quality control

Equation 1 𝑚𝑚𝑓𝑓 − (𝑚𝑚𝑠𝑠 + 𝑚𝑚𝑚𝑚 + 𝑚𝑚𝑖𝑖 + 𝑚𝑚𝑒𝑒 ) ≤ 0.5 [mg]

Example of the typical chromatogram of a sample containing ethanol:

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