EN 14103 Fat and oil derivatives - Fatty Acid Methyl Esters (FAME) - Determination of ester and linolenic acid methyl ester contents
6 Procedure
6.1 Operating conditions
The chromatographic analysis conditions will be chosen taking into account the characteristics of the column being used and the type of carrier gas (hydrogen or helium).
By way of indication, an example of analysis conditions is described below:
- column temperature: 60 °C hold for 2 min, programmed at 10 °C.min-1 up to 200 °C, programmed at 5 °C.min-1 up to 240 °C, final temperature hold for 7 min;
- injector temperature & detector temperature: 250 °C;
- carrier gas flow rate 1-2 ml.min-1, a minimum flow rate of 1 ml.min-1 shall be warranted when operating at the maximum temperature;
- injected volume: 1 µl;
- hydrogen pressure = 70 KPa;
- split flow = 100 ml.min-1.
6.2 Internal standard purity determination
Prepare a solution of nonadecanoic acid methyl ester (3.2) in toluene (3.1) approximately at 10 mg.ml-1. Analyse 1 µl of this solution by gas chromatography according to the conditions described in (6.1), but the final temperature should be held for 20 min at 240 °C, instead of 7 min. Calculate the purity of the nonadecanoic acid methyl ester taking into account all the peak eluted in the chromatogram, excepted the solvent peak. The purity of nonadecanoic acid methyl ester should be at least 99.5 % (m/m). If the purity is lower than 99.5 % (m/m) do not use this standard for this determination.
NOTE No correction of the purity of the internal standard is performed for the calculation of the FAME content.
6.3 FAME sample preparation and analysis
Accurately weigh approximately 100 mg (accuracy +/- 0.1 mg) of homogenized sample in a 10 ml tube (4.3), and approximately 100 mg (accuracy +/- 0.1 mg) of nonadecanoic acid methyl ester (3.2), and dilute with 10 ml of toluene (3.1).
NOTE Standard and samples should be let at ambient temperature, in their container closed, at least 3 h prior being weighed, in order to limit the water absorption during weighting.
Analyse 1 µl of this solution by gas chromatography according to the conditions described in (6.1).
For each sample, two test portions are prepared and give rise, each one, to two chromatographic analyses.
6.4 Identification
The chromatographic conditions (injected quantity, oven temperature, carrier gas pressure and split flow rate) shall be adjusted so as to correctly visualize the methyl ester peaks of the lignoceric (C24:0) and nervonic (C24:1) acids.
The integration shall be carried out as from the hexanoic acid methyl ester (C6:0) peak up to that of the nervonic acid methyl ester (C24:1) taking all the peaks identified as fatty acid methyl esters into consideration. In order to identify properly the fatty acid methyl esters, some commercial solutions may be used.
NOTE If some unknown thin peaks are found (others than saturated and mono-unsaturated FAME) between the linolenic acid methyl ester (C18:3) and the nervonic acid methyl ester (C24:1), presence of fish oil in the sample can be suspected.
As a general rule, the separation is done according to carbon atom chain length, unsaturated FAME are eluted after the corresponding saturated ones.
