The sample is treated with ammonia and ethyl alcohol; the former to dissolve the protein and the latter to help precipitate the proteins. Fat is extracted with diethyl ether and petroleum ether. Mixed ethers are evaporated and the residue weighed. This method is considered suitable for reference purposes. Strict adherence to details is essential in order to obtain reliable results.
- i) Mojonnier fat extraction flask or any other suitable extraction tube (as per ISI specification).
- ii) Bark Cork or ground glass stopper are usually unaffected by the fat solvents.
iii) 100 ml flat bottom flask with G/G joint or stainless steel or aluminum dishes of 5.5 cm height and 9 cm diameter or glass bowl.
- a) Concentrated Ammonium Solution- Specific gravity 0.8974 at 16°C
- b) Ethyl alcohol (95%-96% v/v)
- c) Diethyl ether, peroxide-free-specific gravity 0.702
It may be maintained free from peroxides by adding wet zinc foil (approximately 80 cm / L, cut in strips long enough to reach at least half-way of the container) that has been completely immersed in dilute acidified copper sulphate for 1 minute and subsequently washed with water.
- d) Petroleum ether- boiling range 40-60°C
- e) Mixed Solvent
Prepared by mixing equal volumes of the ether and light petroleum.
Weigh accurately about 10 g of sample (liquid milk), transfer to extraction tube. Add 1.0 ml of ammonia sp. gr. 0.8974; mix well in the lower bulb thoroughly. Add 10 ml ethyl alcohol and mix by allowing the liquid to flow backwards and forwards between the two bulbs. Allow the tube to cool in cold, running water or by immersing in chilled water. Add 25 ml of diethyl ether (peroxide free), close with bark cork or glass stopper which is wetted with water before insertion, and shake vigorously for 1 minute. Open the tube and then add 25 ml light petroleum ether, close the tube, and shake again vigorously for a minute. Let the tube stand on the flat bottom of the lower bulb until the upper ethereal layer is clear and separated completely from the aqueous layer, usually not less than 30 minutes, or centrifuge until clear. If there is a tendency to form emulsion, a little alcohol may be added to help separation of the layers.
Decant off as much as possible of the supernatant layer into a suitable flask by gradually bringing the cylindrical bulb of the tube into a horizontal position. When as much as possible has been poured off, wash the outside of the neck of the tube and the cork or stopper with mixed solvent, collecting the rinsing in the flask. With the Mojonnier tube in a vertical position, wash the inside of the neck with 4 to 5 ml of mixed solvent, and decant. Repeat twice extraction of the liquids remaining in the extraction tube using 15 ml of ether and 15 ml of petroleum solvent every time. Distil carefully the solvents from the flask and dry the flask containing the residual milk fat in an air oven at 102 ± 2°C for two hours, cool in a desiccator and weigh. Heat the flask again in the oven for 30 min. Cool in desiccator and weigh. Repeat the process of heating and cooling and weighing until the difference between two successive weights does not exceed 1 mg. Wash out the fat from the flask with petroleum ether carefully leaving any insoluble residue in the flask. Dry the flask in the oven, cool and weigh as before. The difference in weights represents the weight of fat extracted from the milk.
Make a blank determination using the specified quantities of reagent throughout, and water in place of milk, deduct the value found. If reagent blank is more than 0.5 mg purify or replace reagents. Difference between duplicate determinations obtained simultaneously by the same analyst should not be more than 0.03 gm fat/100 gm product.
Fat percent w/w = Weight of fat x 100/Weight of Milk
Ref: – IS: 1479 (Part II) – 1961 Methods of test for dairy industry. A.O.A.C, 17th edn, 2000 Official method 905.02 Fat in milk,)