APPENDIXThe Determination of Ethano

APPENDIX
The Determination of Ethanol in Beer by Gas
Chromatography
Warning and Safety Precautions
For the recommended practices for the safe storage
and handling of compressed gases, refer to the BOC
Users' Guide. Both ethanol and n-butanol are flam
mable and must be kept away from sources of ignition.
n-Butanol is harmful by inhalation and also by adsorp
tion through skin.
SCOPE
The determination of ethanol in beer by gas liquid
chromatography using direct injection.
FIELD OF APPLICAT1ON
2.1 The method can be used to determine the etha
nol content of all beers and partly-fermented worts,
but not non-alcoholic beers, i.e. with ethanol contents
below 0.5% V/V. The method can be used to deter
mine the alcohol content of shandy. The method is
applicable to products with an ethanol concentration
between 0.5 and 8% V.V.
PRINCIPLE
3.1 The beer sample is de-gassed by filtration and
attemperated to 20°C.
3.2 The sample is diluted with known quantity
of n-butanol (as an internal standard).
3.3 The diluted liquid sample is injected into gas
chromatographic column, previously calibrated using
standard ethanol solutions.
3.4 The percentage of ethanol V/V in the sample
is calculated from the peak areas of the ethanol and nbutanol
peaks, using previously calculated calibration
factor.
REAGENTS
4.1 Unless otherwise stated, use only reagents of
recognized analytical grade and distilled water, or
water of equivalent purity.
4.2 Ethanol, absolute. Lower strengths may be
used provided the concentration is known.
4.3 n-Butanol, new batches must be checked chromatographically
to ensure that there are no inter
ferences with the ethanol peak.
4.4 n-Butanol (0.5% V/V), internal standard. Pip
ette 10 ml of n-butanol into a 2 litre volumetric flask
and dilute to litres with distilled water.
APPARATUS
5.1 Gas chromatograph, fitted with flame ionization
detector and an oven capable of operating isothermally
at 115°C.
5.2 Auto-sampler for the gas chromatograph—to
inject 0.5 or fi of liquid sample, OR a 10 (il gastight
positive displacement syringe.
5.3 Auto-diluter, capable of diluting to
10% 0.1%, OR ml grade bulb pipette, 20 ml
grade bulb pipette.
5.4 Computing integrator, OR Chart recorder and
integrator.

Results (English) 1:

Care must be taken when interpreting computed signals from integrators; they should not be assumed tobe accurate. If possible, the peak areas given shouldbe checked against manual calculation made by measuring peak height and width. For this purpose, traceof peaks should always be produced in addition to theprinted value for peak area.5.5 Water bath, controlled to 20°C 0.5°C.5.6 Thermometer—accurate to 0.1°C.5.7 Chromatography column 2 metres mm,glass or stainless steel packed with 15% Carbowax 20Mon Chromosorb WAW DMCS, 100/100 mesh (e.g.Phase Separations).5.8 Filter funnels.5.9 Filter paper—Watman No. 1.5.10 100 ml conical flask.5.11 Ultrasonic bath.5.12 Pipettes—Grade bulk ml, ml, ml and20 ml.5.13 Volumetric flasks—Grade A, 250 ml and100 ml.SAMPLE PREPARATION6.1 Adjust the temperature of the sample to 20°Cand filter 50 ml into conical flask. Discard the first25 ml of filtrate and cover the filter funnel with clockglass to prevent loss of ethanol.6.2 Place the flask in the ultrasonic bath and sonicate to remove any remaining carbon dioxide. Seal theflask until required for analysis.PROCEDURE7.1 Preparation of Standards7.1.1Ethanol standard 1% VYV, accurately pipette1.0 ml of ethanol into 100 ml volumetric flask. Makeup to volume with distilled water at 20.0°C 0.1°C.7.1.2 Ethanol standard 5% V.V, prepare as in 7.1.1using 5.0 ml of ethanol.7.1.3 Ethanol standard 8% V/V, accurately pipette20.0 ml of ethanol into 250 ml volumetric flask. Makeup to volume with water at 20.0°C 0.1CC.7.1.4 Ensure that pipettes, syringes, ethanol, waterand resulting diluted solutions are all at20.0°C±0.1°C.7.1.5 Check the ethanol content of the standard solutions by measuring the specific gravities either bygravity bottle or meter.7.2 Calibration7.2.1 Attemperate the n-butanol internal standardand the ethanol calibration standards to 20°C 0.1°Cin the water bath.7.2.2 Prepare the gas chromatograph according tothe manufacturer's instructions.Typical conditions:Oven temperature 115°CInjector temperature 150°CDetector temperature 200°CCarrier gas NitrogenCarrier gas flow rate 45 ml/min7.2.3 Dilute ml of each ethanol standard with 20 mlof n-butanol internal standard using an auto-diluter orby pipetting, into clean, dry 50 ml conical flask.Note: Ensure that both solutions are at 20°C 0.1°Cbefore diluting. Temperature accuracy at this stage iscritical to the accuracy of the method. Mix thoroughly.7.2.4 Inject 0.5 or (i of the first diluted standardethanol solution from 7.2.3 into the gas chromatograph.7.2.5 Determine the areas of the ethanol and n-butanol internal standard peaks.7.2.6 Repeat steps 7.2.4 and 7.2.5 for each standardethanol solution in duplicate.7.3 Analysis7.3.1 Attemperate the filtered, degassed beer sampleto 20°C 0.1°C.7.3.2 Dilute ml of the beer sample with 20 ml ofn-butanol internal standard using the same equipmentas for the ethanol standards, into clean, dry 50 mlconical flask. Mix thoroughly.7.3.3 Inject 0.5 or /ul of the dilute beer from 7.3.2into the gas chromatograph.7.3.4 Calculate the area of the peaks due to theethanol and n-butanol internal standard.CALCULATION8.1 Plot graph ofArea of ethanol peakArea of internal standard peakagainst ethanol concentration V/V (after correcting forpurity) from the results obtained for each of the calibration standards. The graph should be linear andshould pass through the origin.8.2 Fit either visually or by using linear regression,the best straight line to the graph. Calculate the coefficient of correlation (r) and provided 0.99, calculate the gradient to give the factor F.Ethanol concentrationArea ethanol peak/Area internal standard peak8.3 Once the linearity of the calibration range isestablished, subsequent calibrations to determine thefactor can be performed by carrying out triplicatedeterminations of the standard nearest to the expectedconcentration of ethanol in the sample.