BS EN 14106 Fat and oil derivatives - Fatty Acid Methyl Esters (FAME) - Determination of free glycerol content
5 Reagents
Use only reagents of recognized analytical grade, unless otherwise specified.
5.1 Hexane.

5.2 1,4 Butanediol, 99 % minimum.

5.3 Ethyl alcohol, 95 % minimum.

5.4 Formic acid, 99 % minimum.

5.5 Glycerol, 99 % minimum.

5.6 Carrier gas: helium, GLC grade.

5.7 Auxiliary gases:
- hydrogen, purity 99 % minimum, free from moisture and organic compounds;
- air, free from organic compounds.

5.8 Internal Standard Solution : weigh approximately 80 mg (accuracy +/- 0.0001 g) of 1,4 Butanediol in a volumetric flask, 100 ml capacity. Dissolve in some ml of distilled water, add 1 ml of formic acid and fill up to the mark using distilled water. The so prepared solution is stable for 24 h if stored at ambient temperature.

6 Apparatus
6.1 Gas chromatograph equipped by the following devices:
6.1.1 Thermostatic oven for column, able to maintain the set temperature with an accuracy of +/- 1 °C.

6.1.2 Thermostatic injection port, equipped for capillary column in split-splitless mode or, alternatively, for packed column.

6.1.3 Flame ionisation detector (FID) and converter/amplifier device.

6.1.4 Recorder/integrator, capable to run with the converter/amplifier with a response time of 1 s maximum and variable chart speed.

6.1.5 Capillary column, type PoraPLOT Q, length = 10 m, diameter 0.32 mm, film thickness = 10 µm (Note) or, alternatively packed column filled with CHROMOSORB 101, diameter 4 mm, length = 1 m.

NOTE Some problems and discrepancies on capillary column behaviour from different suppliers have been noticed. Some Authors refer also the possibility to use capillary columns coated with FFAP (Free Fatty Acid Phase) or polyethyleneglycol phases instead of the suggested stationary phase. Nevertheless the choice of column should be based on the following criteria:
- the separation between glycerol and internal standard peaks should be complete (baseline nearly to zero between two peaks);
- the analysis time should not exceed 1 5 min;
- columns with response factor for glycerol, calculated such as 9.3, higher than 2.5 are not suitable.

6.1.6 Flow controllers, for carrier and auxiliary gases.

6.2 Microsyringe for gas-chromatography, 5 µl or 10 µl.

6.3 Volumetric flasks, 50 ml and 100 ml capacity.

6.4 Two mark precision pipettes, 1 ml capacity.

6.5 Volumetric pipettes, 5 ml capacity.

6.6 Analytical balance, accuracy +/- 0.0001 g.

6.7 Glass conical test tubes, 10 ml capacity.

6.8 Centrifugal machine, able to run at the speed of 2000 revolutions per minute.

7 Sampling
Sampling is not part of the method specified in this European Standard. A recommended sampling method is given in EN ISO 5555.

Sampling procedures shall take into account that free glycerol contained in FAME has a strong affinity for glass. Therefore samples shall be collected and stored avoiding the use of glass.

8 Preparation of test sample
Prepare the test sample in accordance with EN ISO 661. The test sample shall not be heated and/or filtered.