The bound fat may be broken down by hydrolysis or by other chemical treatments to yield free fat. Hence, the total fat can be extracted from food products by Rose Gottleib method.
Bound fat can be made free if the food sample is dissolved completely prior to extraction with polar solvents. Dissolution of the food can be achieved by acid or alkaline hydrolysis.
In Rose Gottleib method, the material is treated with ammonia and alcohol and the fat is extracted with diethyl ether-petroleum ether mixture.
The alcohol precipitates the protein, which dissolves in the ammonia and the fat is then extracted with ether. Petroleum ether is then added for extraction thereby impurities such as water and non-fatty substances e.g. sugar do not get extracted along with the total fat content.
Fat extraction apparatus
Hot air oven-set to operate at 98 to 100ºC.
- Conc. ammonia solution (sp-gr 0.88), AR Grade
- Ethyl Alcohol – 95 to 96 per cent (v/v).
- Diethyl Ether, AR Grade
- Light Petroleum (boiling range 40 to 60°C).
- Mixed Solvent – prepared by mixing equal volumes of the ether and light petroleum.
Weigh accurately 10 to 11 g in case of liquid sample and 2-3 g in case of solid sample into the extraction tube, add 1 ml of conc. ammonia solution and mix well.
Add 10 ml of alcohol and again mix well. Complete extraction of the fat is dependent on satisfactory mixing at each stage.
Add 25 ml of ether, close the tube with the cork (or stopper), which is wetted with water before insertion, and shake vigorously for one minute.
Remove the cork and with 25 ml of light petroleum, wash the cork and neck of the tube so that the washings run into the tube.
Replace the cork again, wetted with water, and shake vigorously for 30 seconds.
Note : It is essential that the cork (or stopper) be wetted with water before each insertion and washed with solvent during each removal, Also, before each removal, to avoid spurting of the solvent, a slightly reduced pressure should be induced in the tube by cooling.
Rubber stoppers shall not be used. Allow the tube to stand until the ethereal layer is clear and completely separated from the aqueous layer, usually for not less than 30 minutes.
Remove the cork and insert the siphon (or wash-bottle) fitting so adjusted for length that the inlet is 2 to 3 mm above the interface between the ethereal and aqueous layers, and transfer the ethereal layer to a suitable flask.
Add 5 ml of mixed solvent to the extraction tube, using it to wash the siphon or wash-bottle fitting which is raised sufficiently to permit this but not removed, and the inside of the tube.
Lower the fittings and transfer the solvent, without shaking, to the flask. Repeat this operation with a further 5 ml of mixed solvent.
Wash the tip of the siphon fitting into the flask with mixed solvent. Remove the siphon fitting, and repeat the extraction of the milk residue, using 15 ml of ether and 15 ml of light petroleum, and repeat the subsequent operations, as before.
Use the ether to wash the inner limb of the siphon (or wash-bottle) fitting during its removal from the tube. Finally, repeat the extraction once more with 15 ml each diethyl ether and petroleum ether.
Distil carefully the solvents from the flask and dry the residual fat in the oven at 98 to 100O C for one hour taking precautions to remove all traces of volatile solvent and cooling the flask to room temperature in a desiccator charged with an efficient desiccant.
Repeat this procedure for periods of half an hour until successive weighings do not show a loss in weight by more than 1 mg.
Make a blank determination using the specified quantities of reagents and water in place of sample and deduct the value found, if any, from the apparent weight of fat.
M1 = mass, in g, of the empty conical flask,
M2 = mass, in g, of the conical flask along with dried residue, and
M = mass, in g, of the material taken for the test.
RESULTS AND INFERENCE
Results of fat content should be reported to the nearest 0.1% (m/m). The difference between two single and independent results found by two analysts working in different laboratories on identical test material should not exceed :
For food products having fat content:
≥ 10% (m/m) = 1% of fat content
4-10% (m/m) = 0.06% of fat content
1-4% (m/m) = 0.04% of fat content
≤1% (m/m) = 0.03% of fat content
The method is suitable for the total fat content (i.e. free fat and bound fat) in food products viz., dairy products. The method is applicable to both solid and liquid food.
• The tubes shall be provided with good quality bark corks or stopper of other
materials (PTFE, Silicone rubber) unaffected by the reagents used.
• Ensure no loss of fat content occurs during sample preparation.
• Allow sufficient time for the complete separation of layers to avoid discrepancies
in the results.