BS EN 14105 Fat and oil derivatives - Fatty Acid Methyl Esters (FAME) - Determination of free and total glycerol and mono-, di-, triglyceride contents
8 Determination of results
8.1 Integration of the peaks
In each family of glycerides, there exist small peaks (see Annex A) which have to be integrated. This method therefore calculates the percentage of mono-, di- and triglycerides (8.4) by summing the area peaks for each family. It is advised to integrate jointly the two diglyceride peaks containing 36 atoms of carbon, major compounds of this family, on account of an insufficient resolution which may induce quantification errors if the two peaks are integrated separately. The presence of a double peak at the level of the glycerol retention time shall lead to the verification of the sylilation stage, which is probably incomplete (presence of water in the samples).
8.2 Glycerol calibration function
The calibration function is given by the following equation, obtained from the experimental data using the linear regression method as in Annex B and according to:
Mg/Mei = ag(Ag/Aei) +bg
where
Mg is the weight of glycerol (mg);
Mei is the weight of internal standard 1,2,4-butanetriol (mg);
Ag is the peak area of glycerol;
Aei is the peak area of the internal standard 1,2,4-butanetriol;
ag, bg are the regression coefficients of the calibration function for glycerol.
The calibration function shall be regarded as correct only if the correlation coefficient, calculated according to Annex B, is equal or greater than 0.9 (see Annex C for a worked example).
8.3 Free glycerol
Calculate the mass percentage of free glycerol (G) in % (m/m) in the sample using the equation:
G = [ag(Ag/Aei1) + bg] x (Mei1/m) x 100
where
Aei1 is the peak area of internal standard 1,2,4-butanetriol;
Mei1 is the weight of internal 1,2,4-butanetriol;
m is the weight of sample (mg).
8.4 Glycerides
Calculate the mass percentage of the mono-, di- and triglycerides in % (m/m) using the following equations:
M = (AMono/AMonoC19) x (MMonoC19/m) x 100
D = (ADi/ADiC38) x (MDiC38/m) x 100
T = (ATri/ATriC57) x (M TriC57/m) x 100
where
M, D, T are the mono-, di- and triglyceride concentration in the sample respectively;
AMono, ADi, ATri are the sums of the peak areas of the mono-, di- and triglycerides respectively;
AMonoC19 is the peak area of internal standard Mono C19;
MMonoC19 is the weight of internal standard Mono C19 (mg);
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);
m is the weight of sample (mg).
8.5 Total glycerol
Calculate the percentage of total glycerol in the sample (GT) in % (m/m) using the following equation:
GT = G + 0.255M + 0.146D + 0.103T
9 Expression of results
Free and total glycerol content is expressed to the nearest 0.001 % (m/m).
All glyceride contents are each expressed to the nearest 0.01 % (m/m).
