9 Operating procedure

9.1 Extraction of the analytes

9.1.1 Method A: liquid-liquid extraction

Extract approximately 20ml of oil sample, weighed to the nearest 0.1g, with 5ml of acetonitrile, measured by a pipette, in a 25ml stoppered cylinder, shaking the samples on a mechanical shaker for at least 2min.

Allow the samples to stand until the solvent and oil layers separate completely. The extract can then be analysed as described in 9.2.

Note The time period necessary for phase seapration tends to increase for oils with higher acid values.

9.1.2 Method B: solid-liquid extraction

Weigh exactly 10g±0.1g of oil sample and dissolve it in 10ml analytical grade n-pentane.

Rinse a new silica cartridge with 2ml n-pentane and discard the eluate. While the silica is still wet, immediately pass the sample solution through the cartridge under a slight vacuum at a maximum flow of 3ml/min followed by 20ml n-pentane. Discard all eluates.

Dry the cartridge by suction by maintaining the vacuum for at least 5min.

Release the vacuum and elute the retained material with the same eluent used in the first minutes of the chromatogram (see 9.2), using a 10ml syringe as teh dispenser.

Collect the first two millilitres in a 2ml volumetric flask.

Note Annex A describes a method to assess the efficiency of the silica cartridge.

9.2 Analysis of the extract

9.2.1 Adjust the liquid chromatograph in accordance with teh manufacturer's instructions and establish suitable operating conditions.

9.2.2 The following set of conditions has been found satisfactory but some adjustments may be appropriate depending upon specific features of the instrumenation and local conditions.

- isocratic doncitions;

- eluent: water 60% to 80% in volume; methanol or acetonitrile 20% to 40% in volume;

- flow rate: 0.5 to 2.0ml/min.

Note In some cases, an elution gradient may be useful to improve the separation. Also the additon of a small amount of acetic acid (0.01%) may improve the separation of the more polar compounds.

9.2.3 The UV detector shall monitor the following wavelengths:

220nm for 2-furfurylalcohol.

275nm to 280 nm for all otehr compounds listed in clause 8.

Note Maximum light absorption occurs at approximately the following wavelengths:

- 5-hydroxymethyl-2-furfural 280nm

- 2-furfurylalcohol 215nm

- 2-furfural 274nm

- 2-acetylfuran 272nm

- 5-methyl-2-furfural 288nm

Selection of tthe above wavelengths will enhance the sensitivity of the analysis for specific compounds.

9.2.4 When the chromatograph and detector are equilibrated inject an aliquot of the extract and record the detector response. Examples of typical chromatograms are shown in Figure 2.

9.2.5 After elution of the last peak of interest (5-methyl-2-furfural with specified columns) switch the mobile phase to 100% methanol or acetonitrile and increase the flow rate until all oil residues have been flushed out of the column.