BS EN 14105 Fat and oil derivatives - Fatty Acid Methyl Esters (FAME) - Determination of free and total glycerol and mono-, di-, triglyceride contents
7 Procedure
7.1 Operating conditions
The chromatographic analysis conditions shall be chosen taking into account the characteristics of the column being used and the type of carrier gas (hydrogen or helium). It is however recommended to observe an analysis time of about 30 min to 35 min to ensure triglycerides elution.

EXAMPLE By way of indication, an example of analysis conditions is described below:
column temperature: 50 °C hold for 1 min, programmed at 15 °C/min up to 180 °C, programmed at 7 °C/min up to 230 °C, programmed at 10 °C/min up to 370 °C, final temperature hold for 15 min;
detector temperature: 380 °C;
carrier gas pressure (hydrogen): 80 kPa;
volume injected: 1 µl.

7.2 Analysis of the calibration solutions
Using a microsyringe (4.10), add 150 µl of MSTFA (3.1) to each of the four calibration solutions (5.5), close hermetically the vials and shake vigorously. Store 15 min at room temperature, then add 8 ml of n-heptane (3.4) using a graduated cylinder (4.11).

Analyse 1 µl of each reaction mixture by gas chromatography under the conditions defined under 7.1, using only the first part of temperature programme, stopping the analysis when the temperature of 230 °C has been reached. Each reaction mixture gives rise to two chromatographic analyses. Samples are stable for some hours after derivatisation.

NOTE The silylated standard solutions are only stable one day.

7.3 Analysis of the commercial mixture of monoglycerides
Using microsyringes (4.10), transfer 200 µl of commercial mixture of monoglycerides dissolved in pyridine (3.10) and 150 µl of MSTFA (3.1) into a 10 ml vial (4.6). Avoid contact with humidity.

Hermetically close the vial and shake vigorously.

Store 15 min at room temperature, and then add 8 ml of n-heptane (3.4).

Analyse 1 µl of the reaction mixture by gas chromatography according to the conditions described under 7.1.

7.4 Preparation and analysis of the samples
Accurately weigh approximately 100 mg (accuracy +/- 0.1 mg) of homogenized sample in a 10 ml vial (4.6).

Using precision microsyringes (4.8 and 4.9), add 80 µl of 1,2,4-butanetriol stock solution (5.1), 200 µl of standard glycerides stock solution (5.3), 200 µl of pyridine (3.10) and 200 µl of MSTFA (3.1). Avoid contact with humidity.

Hermetically close the vial and shake vigorously. Store 15 min at room temperature, and then add 8 ml of n-heptane (3.4). Analyse 1 µl of the reaction mixture by gas chromatography according to the conditions described under 7.1.

Carry out the determination in duplicate, by preparing two independent samples.

7.5 Identification
The analysis of the calibration solutions under the same operating conditions as those used for the analysis of the sample allows the identification of the peaks by comparison of the retention times. Due to the overlapping of the elution zones of the methyl esters and of the monoglycerides, it is therefore advised, in order to identify the monoglyceride peaks, to inject the commercial mixture composed of monopalmitin, monosterarin and monoolein (5.4), the latter having been previously submitted to the derivatisation reaction.

A chromatogram of a rapeseed oil methyl ester sample, obtained under the operating conditions and preparation described under 7.1 is presented in Annex A. Internal glyceride standards may be analysed under the above mentioned chromatographic conditions, after silyl derivatisation.

7.6 Calibration
For glycerol only, the study of the variation of weight ratio versus area ratio makes it possible to verify the linearity of the response and to work out a calibration function.

For mono-, di- and triglycerides it is assumed that, within the considered concentration range the detector response is regarded as linear.

7.7 Column performance control
For each analysis, evaluate the relative response factor for glyceryl dinonadecanoate (Di C38) versus glyceryl trinonadecanoate (Tri C57), by using the following equation:
RRF = (ADiC38/MDiC38)/(ATriC57/MTriC57)
where
ADiC38 is the peak area of internal standard Di C38;
MDiC38 is the weight of internal standard Di C38 (mg);
ATriC57 is the peak area of internal standard Tri C57;
MTriC57 is the weight of internal standard Tri C57 (mg).

The results of the calculation of RRF shall be lower than 1.8. For higher values, the gas chromatography system is not suitable for analysis and shall be verified in order to improve triglyceride detection.