ANNEX  
The Annex to Regulation (EC) No 2870/2000 is amended as follows:  
(1) in the table of contents ,  the following point  is added : 
‘XI. Determination of ¹⁴C content in ethanol’ ; 
(2) in point  5 (Reagents and Materials) of part III.2 (Gas chromatographic determination of 
volatile congeners: aldehydes, higher alcohols, ethyl acetate and methanol) of Chapter 
III, the following p oints  are inserted :  
‘5.13a For ethyl alcohol of agricultural origin only, ethanol absolute (CAS 64 -17-5).’; 
‘5.14.1a For ethyl alcohol of agricultural origin only, standard solution A sh all be 
prepared pipetting the reagents with reduced volumes of higher alcohols with the purpose 
of having standard solutions with concentr ations close to the legal limits for ethyl alcohol 
of agricultural origin.’ ; 
‘5.14.2 a For ethyl alcohol of agricultural origin only, standard solution B sh all be 
prepared pipetting a suitable internal standard with the reduced volumes with the purpose 
of having standard solutions with concentrations close to the legal limits for ethyl alcohol 
of agricultural origin.’ ; 
(3) the following Chapter XI is added : 
‘XI.  DETERMINATION OF ¹⁴C CONTENT IN ETHANOL  
1.  Introduction  
Determination of the ¹⁴C content in ethanol permits a distinc tion to be made 
between alcohol from fossil  fuels (synthesis alcohol) and alcohol from recent 
raw materials (fermentation alcohol).  
2.  Definition  
The ¹⁴C content of ethanol shall be considered as  the ¹⁴C content determined 
using the  method described here  or the method described in standard EN 
16640 Method C . 
The natural ¹⁴C content in the atmosphere (the referen ce value), which is 
absorbed by living vegetation by  assimilation, is not a constant value. The 
reference value is therefore determined on ethanol from raw materials of the most recent vegetation period. This annual reference value is determined 
according to standard EN 16640 . However, another reference value can be 
accepted where it is certified by an accredited body.  
3.  Principle  
The ¹⁴C content of samples containing alcohol with at least 85 % mass ethanol 
is determined directly by liquid  scintillation c ount.  
4.  Reagents  
4.1.  Toluene scintillator  
5,0 g 2,5 -diphenyloxazole (PPO)   
0,5 g p -bis-[4-methyl -5-phenyloxazolyl(2)] -benzene (dimethyl -POPOP) in 1 
litre analytical grade toluene.  
Commercial, ready -to-use toluene scintillators of this composition may also be  
used.  
4.2.  ¹⁴C standard  
n-Hexadecane ¹⁴C with an activity of about 1 × 10 ⁶ dpm/g (approximately 1,67 
× 106 cBq/g) and a guaranteed  accuracy of determined activity of ± 2 % rel.  
4.3.  ¹⁴C-free ethanol  
Synthesis alcohol from raw materials of fossil  origin with at least 85 % mass 
ethanol, to determine the background . 
4.4.  Alcohol from recent raw materials of the most recent vegetation period with at 
least 85 % mass ethanol as  reference material.  
5.  Apparatus  
5.1.  Multi -channel liquid scintillation  spectrometer with processor and automatic 
external standardisation and  display of the external standard/channel r atio 
(usual design: three meter channels and two external standard  channels).  5.2.  Low-potassium counter tubes suitable for the spectrometer,  with dark screw -
tops containing a polyethylene  insert.  
5.3.  Volumetric pipettes, 10 ml.  
5.4.  Automatic dosing device 10 ml.  
5.5.  250 ml round -bottom flask with ground -glass stopper.  
5.6.  Alcohol distillation apparatus with heating mantle, e.g. type Mi cko. 
5.7.  Microliter syringe 50 μl.  
5.8.  Pycnometer funnel, pycnometers, 25 ml and 50 ml.  In alternative, equivalent 
equipment, such as electronic densimetry, should be allowed.  
5.9.  Thermostat with a temperature stability of ± 0,01 °C.  
6.  Procedure  
6.1.  Adjusting the equipment  
The equipment sh all be adjusted according to the manufacturer's instructions. 
Measuring conditions are  optimal when the value E ₂/B, the quality index, is at 
its maximum.  
E = efficiency  
B = background  
Only two meter channels are optimised. The third is left fully open for control 
purposes.  
6.2.  Selection of counter tubes  
A larger number of counter tubes than will later be needed are each filled with 
10 ml of ¹⁴C-free synthesis  ethanol and 10 ml toluene scintillator. Each is 
measured for at least × 100 minutes. Tubes whose backgrounds vary by more 
than ± 1 % rel. from the mean are  discarded. Only tubes new from the factory 
and from the same batch may be used.  6.3.  Determination of the external standard/channel ratio (ESCR) . 
During the process of setting the channels ( point 6.1) the  ESCR is determined 
using the  appropriate computer  program when the efficiency is determined. 
The external standard used is ¹³⁷caesium, which is already built -in by the 
manufacturer.  
6.4.  Preparation  of sample  
Samples having an ethanol content of at least 85 % mass and free from 
impurities, which absorb at wavelengths below 450 nm may be measured . The 
low residue of esters and a ldehydes  has no disruptive effect . The alcohol 
content of the sample is pr eviously determined with an approximation of 0 ,1 
%. 
7.  Measurement of samples using external standard  
7.1.  Slightly extinguished samples such as those described in point 6.4 with an 
ESCR of around 1 ,8 may be measured  via the ESCR, which provides a 
measure of the efficiency.  
7.2.  Measurement  
10 ml each of the samples prepared according to point 6.4 is pipettes into a 
selected counter  tube checked for  background and 10 ml of toluene scintil lator 
is added via an automatic dosing device. The samples in  the tubes are 
homogenised by suitable rotary movements;  the liquid shall  not be allowed to 
wet the polyethylene  insert in the screw -top. A tube containing ¹⁴C-free fossil 
ethanol is prepared in the same way to measure the  background. Check the 
relevant an nual ¹⁴C value a duplicate of recent ethanol from the latest 
vegetation  period is prepared, a tube being mixed with internal standard, see 
point 8. 
The control and background samples are placed at the beginning of the 
measurement series, which sh all contain no more than 10 samples for analysis. 
Total measuring time per sample is at least 2 × 100 minutes,  with the 
individual samples being measured in part stages of 100 minutes so that any equipment drift or  other defect can be detected. (One cycle ther efore 
corresponds to a measuring interval of  100 minutes per  sample).  
Background and control samples shall  be freshly prepared every four weeks.   
In the case of slightly extinguished samples (ESCR circa 1,8) the efficiency is 
only negligibly affected by th e change in this value. If the change is within ± 5 
% rel. the same efficiency can be expected. For more greatly  extinguished 
samples, such as denatured alcohols, the efficiency may be established via the 
extinction correction graph. If an appropriate comp uter program is not 
available the internal standard shall  be used,  and this  gives an unambiguous 
result.  
8.  Measuring samples using internal standard hexadecane¹⁴C  
8.1.  Procedure  
Control and background samples (recent and fossil ethanol) and the unknown 
material  are each measured as  duplicates. One sample of the duplicate is 
prepared in a non -selected tube and an accurately dosed quantity  (30 μl) of 
hexadecane ¹⁴C is added as internal standard (added activity around 26 269 
dpm/gC approximately  43 782 cBq/gC). For the sample preparation and 
measuring time of the other samples see  point 7.2, but  the measuring time for 
the samples with the internal standard may be reduced to  about five minutes by 
presetting  at 10 ⁵ pulses. One duplicate each of back ground and control samples 
is used per measuring series; these are  placed at the beginning of the 
measuring series.  
8.2.  Handling the internal standard and counter tubes  
To prevent contamination when measuring with the internal standard these 
shall  be stored and handled well  away from the area where the samples for 
analysis are prepared  and measured. After measurement the tubes  checked for 
background may be re -used. The screw -tops and tubes containing the internal 
standard shall be  disposed of.  
9.  Express ion of the results  9.1.  The unit of activity of a radio -active substance is the becquerel; 1 Bq = 1 
decay/sec.  
Indication of specific radio -activity is expressed as becquerels relative to one 
gram  carbon = Bq/gC.  
To obtain more practical results , these shall be express ed in centi -bequerels = 
cBq/gC.  
The descriptions and formulae used in the literature, based on dpm, may also 
be used . To obtain corresponding figures in cBq  merely multiply the dpm 
figure by 100/60.  
9.2.  Expression of results with external standard  
(cpm ₚᵣ – cpm NE) · 1,918  · 100 
cBq/g C =   ________________________  
V · F · Z · 60  
9.3.  Expression of results with internal standard  
(cpmₚ ᵣ – cpm NE) · dpm IS · 1,918  · 100 
cBq/g C =   ______________________________  
          (cpm IS – cpmₚᵣ) · V · F · 60 
9.4.  Abbreviations  
cpmₚ ᵣ = the mean sample count rate over the total measuring time.  
cpm NE = the mean background pulse rate calculated in the same way.  
cpm IS = the amount of added internal standard added (calibration radioactivity 
dpm).  
dpm IS = the quantity of internal standard added (calibration radioactivity dpm).  
V = the volume of the samples used in ml.  
F = the content in grammes pure alcohol per ml corresponding to its 
concentration.  
Z = the efficiency corresponding to the ESCR value.  
1,918 = the number of grammes alcohol per gramme carbon.  10.  Reliability of the method  
10.1.  Repeatability (r)  
r = 0,632 cBq/g C; S ₍ᵣ₎ = ± 0,223  cBq/g C  
10.2.  Reproducibility  (R) 
R = 0,821 cBq/g C; S ₍R₎ = ± 0,290 cBq/g C. ’