19
Methods Conventional ISO procedure:
The conventional standard procedure is based on the ISO extraction method (ISO 8262) for total fat determination in milk and milk products according to Weibull-Berntrop and the derivatisation of fatty acids in fatty acid methyl esters (FAMEs) based on the ISO method (DIN EN ISO 12966-2).
The ISO 8262 method serves as a base and correlates with the extraction method of Weibull-Stoldt for the determination of total fat content in food samples.
The ISO extraction method according to Weibull-Stoldt is based on a saponifi ed digestion using hydrochloric acid and water, which releases fat bound to proteins in food samples. In the following hot fi ltration, the fat released remains in the fi lter. The fi lter including the fat is washed to neutral and then dried. Finally, the fat is extracted with a proper solvent in a Soxhlet extractor, taking several hours. As soon as the extraction is fi nished, the solvent is removed using a rotation evaporator and the remainder is dried. Determination of total fat is performed by weighing the dried fat.
Based on the ISO procedure on sample preparation for GC analysis, the transformation of fatty acids in fatty acids methyl esters (FAMEs) requires a sequence of alkaline and acidic derivatisation in water-free methanolic environment.
The alkaline derivatisation generates the FAMEs out of the fatty acids bound as Triglycerides and the saponifi cation of free fatty acids. The following acidic derivatisation fi nally transfers the remaining saponifi ed fatty acids in FAMEs.
The reaction equations are displayed in Figure 1 and Figure 2.
Microwave procedure: The new microwave procedure for determination of fat has been developed for the microwave system Discover SP-X®
Experimental Part ISO procedure: Extraction method:
The food sample is weighed in a 600 mL glass beaker, in the expectation that a total fat weight of 2 to 3 g will be present. The sample is treated with 100 mL water and 150 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 fi ltered in a 2 layer round fi lter which has been wetted with before hot water. Afterwards, the fi lters and the residue are washed with neutral with hot water. Finally, the fi lter paper and residue are dried in an oven for one hour at 105°C. The dried fi lters are then transferred in the extraction sleeve and fi nally positioned in a 250 mL extractor (Soxhlet-Apparatus).
The round bottom fl ask of the Soxhlet apparatus (250 mL) is fi lled 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 refl ux 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 fl ask is further dried for 1 hour at 105°C in order to remove the extraction liquid completely. After cooling, the fl ask is weighed and the total fat content determined.
ISO based fatty acid derivatisation for GC analysis: (CEM, Kamp
Lintfort). It consists of a microwave extraction method (MEM) and a microwave derivatisation method (MDM).
The MEM has been developed based on the Weibull-Stoldt method and extracts the total fat in a food sample in a closed system under defi ned conditions using microwave radiation simultaneously.
In a closed system (including MDM), derivatisation of the extracted fatty acids is performed under microwave radiation. Transformation of fatty acids in fatty acids methyl esters (FAMEs) also requires a sequence of alkaline and acidic derivatisation in water-free methanolic environment for subsequent GC analysis.
GC analysis:
Analysis of FAMEs of the food samples has been done using a gas chromatograph GC 2010 Plus AF with FID detector (Shimadzu Europa GmbH, Duisburg, Germany).
Infl uence of the method of sample preparation and derivatisation on the content of unsaturated fatty acids in the total fat content of food samples needs to be investigated. In order to compare the contents of unsaturated and saturated fatty acids in both methods, the total amounts of all fatty acid peak areas are calculated (= 100%). Peak areas of unsaturated and saturated fatty acids are then summarised, and the ratio of unsaturated vs. saturated fatty acids is calculated. This procedure has been applied for both the ISO procedure and the microwave procedure.
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®
.
Final temperature: 115°C Ramp: Stirrer:
Hold time: Power:
4 minutes Strong
15 minutes 200 Watt
The extracted fat is transferred in a 10 mL round fl ask and treated with 2 mL (0.2 molmole/L) sodium methanolate in methanol (8 g sodium hydroxide are dissolved in 1,000 mL methanol). Boiling under refl ux 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 fl ask 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 refl ux 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 shaken well. Afterwards, 1 mL n-hexane is added and the fl ask is shaken 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 fl ask neck, approx. 2 – 3 mL. The upper phase now contains the FAMEs generated.
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