BS EN 14106 Fat and oil derivatives - Fatty Acid Methyl Esters (FAME) - Determination of free glycerol content
9 Procedure
9.1 Gas chromatographic conditions
The following general operating conditions have been shown to be satisfactory.
Oven temperature: 210 °C;
injector temperature: 230 °C;
detector temperature: 250 °C;
split ratio: approximately 50:1 ;
carrier gas flow: 1 ml/min to 2 ml/min.
Conditions for packed column:
oven temperature: 200 °C;
injector temperature: 230 °C;
detector temperature: 250 °C;
carrier gas flow: 20 ml/min to 30 ml/min.
9.2 Peaks identification
The identification of peaks belonging to internal standard and glycerol can be achieved by retention time comparison of suitable standards analysed under the same analytical conditions. An illustrative chromatogram is reported below.
9.3 Determination of response factor
Weigh approximately 100 mg +/- 0.1 mg of glycerol and 100 mg +/- 0.1 mg of 1,4 butanediol in a 100 ml volumetric flask. Dissolve in 50 ml of ethyl alcohol (5.3) and fill up to the mark with water. Consider the heating and the volume diminution due to the water/alcohol mixing. Inject at least three times in the chromatograph 1 µl of this solution using the experimental conditions listed above, in order to calculate the response factor. The solution is stable for some weeks even if stored at room temperature. Using the obtained chromatograms the response factor Fr can be calculated as follows:
Response factor (Fr) = (A1/M1)/(A2/M2)
where
A1 is the peak area of internal standard;
A2 is the peak area of glycerol;
M1 is the mass of 1,4 butanediol in response factor solution, expressed in mg;
M2 is the mass of glycerol in response factor solution, expressed in mg.
The response factor Fr, such as calculated above shall be 2.5 or lower (see note in 6.1.5).
9.4 Quantitative analysis
9.4.1 Sample preparation
Weigh approximately 3.5 g +/- 0.0001 g of sample (corresponding to about 4 ml) into a 10 ml test tube. Add 1 ml of ethyl alcohol (5.3) and gently shake to ensure mixing. Add exactly 1 ml of internal standard solution (5.8) and 4 ml of hexane. Tightly plug and shake vigorously the tube for five minutes. Centrifuge the sample for 15 min. Use the lower phase for gas chromatographic analysis.
9.4.2 Gas chromatography analysis
Take approximately 1 µl of the lower phase using the GC micro-syringe and draw enough air to empty the needle ("hot needle" technique). Carefully clean body and needle of syringe using a paper towel. Introduce the needle through the membrane of injection port, wait for 5 s, then rapidly inject the sample. After 5 s draw off the needle. Take note of injection moment on chromatogram. Continue elution some minutes after the complete detection of glycerol peak.
