10 May / June 2016
Final temperature: 115°C Ramp: Stirrer:
Hold time: Power:
4 minutes Strong
15 minutes 200 Watt
After the digestion and extraction, the solvent is transferred in a previously weighed aluminium bowl in order to perform a cold extraction. The digestion in the vial is treated again with 5 mL n-hexane and then stirred on a magnetic stirrer for one minute. Afterwards, this solvent will be transferred in the aluminium bowl. This cold extraction is repeated three to four times. The solvent with the extracted fat is dried in the heating oven for twenty minutes at 105°C and the bowl finally weighed. Calculation of the total fat content is done by following Equation 1:
Figure 5: Chromatogram of fatty acids in a sausage sample, derivatisation according to microwave procedure and identified FAMEs.
F(%)= mL hydrochloric acid (25 weight-%).
The glass beaker is covered with a glass lid, and the sample is then boiled for 30 to 60 minutes. After the digestion process, the hot sample is diluted with 100 mL of water and filtered in a 2 layer round filter which has been wetted with before hot water. Afterwards, the filters and the residue are washed with neutral with hot water. Finally, the filter paper and residue are dried in an oven for one hour at 105°C. The dried filters are then transferred in the extraction sleeve and finally positioned in a 250 mL extractor (Soxhlet-Apparatus).
The round bottom flask of the Soxhlet apparatus (250 mL) is filled with solvent at a volume corresponding to 1.5 times the volume of the Soxhlet apparatus. The temperature of the solvent petrol ether is 40 – 60°C. The reflux condenser is then set, the solvent is boiled and the fat is extracted over 3 to 6 hours. After extraction, the remaining liquid is fully evaporated in the rotation evaporator. Afterwards, the open flask is further dried for 1 hour at 105°C in order to remove the extraction liquid completely. After cooling, the flask is weighed and the total fat content determined.
ISO based fatty acid derivatisation for GC analysis:
The extracted fat is transferred in a 10 mL round flask and treated with 2 mL (0.2 mole/L) sodium methanolate in methanol
(8 g sodium hydroxide are dissolved in 1,000 mL methanol). Boiling under reflux is performed until the solution is clear (5 to 20 minutes). The boiling time depends on the chain length of the fatty acids, the longer the fatty acid, the longer the boiling takes. Afterwards the flask is removed from the heating source and two drops of phenolphthalein solution are added.
Sulphuric acid (1 mole/L) is then added in methanol solution until the solution is clear; another 0.2 mL are then added. The cooler is reinstalled, and the solution is boiled under reflux for 5 minutes. The solution is again removed from the heating source and cooled down in water. 4 mL of a saturated sodium chloride solution are now added and shook well. Afterwards, 1 mL n-hexane is added and the flask is shook again for 15 seconds. The solution is left to stand until both phases are separated. Saturated sodium chloride solution is again added until the aqueous phase reaches the lower level of the flask neck, approx. 2 – 3 mL. The upper phase now contains the FAMEs generated.
Microwave method: Extraction method:
0.5 g of sample is weighed in an 80 mL glass vial and treated with 3.5 mL hydrochloric acid (37 weight-%), 7.5 mL water and 5 mL n-hexane. A stirrer chip is added. The following parameters are set on the Discover SP-X®.
m2 E * 100 - m1
m2 = Mass in g empty bowl m1
Equation 1
= Mass in g bowl with dried fat E = Mass in g weight sample
For the derivatisation (MDM), one or two drops of the dried fat are treated in a glass vial with 10 mL water-free methanolic potassium hydroxide solution (2.5 weight-%), and a stirrer chip is added.
The alkaline derivatisation requires the following parameters on the Discover SP-X®
Final temperature: 90°C Ramp:
Hold time: Stirrer: Power:
5 minutes 10 minutes Medium 200 Watt
After finishing the alkaline derivatisation, the vial is cooled down in a water bath. 14.25 mL Methanol and 0.75 mL hydrochloric acid (37 weight-%) are added, and the acidic derivatisation is generated with following parameters on the Discover SP-X®
Final temperature: 120°C Ramp:
Hold time: Stirrer: Power:
5 minutes 6 minutes Medium 200 Watt
After finishing the derivatisation, the vial is cooled down in a water bath. 10 mL
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