SOP: Total Fat Extraction by Chloroform–Methanol

GIFSQ — Global Institute for Food Safety & Quality

Principle

The fat is extracted from the sample at room temperature using a mixture of chloroform and methanol. The extract is diluted with the calculated amount of water so that the two phases separate; the lower chloroform layer, which contains the fat, is separated, washed with dilute NaCl solution to remove the extracted proteinaceous material, and dried using anhydrous sodium sulphate. The chloroform extract is then evaporated to dryness in a tared dish and the fat residue weighed. The method is applicable to all food products when further characterisation of fat is required; it is more precise than the petroleum-ether crude-fat method.

Apparatus required

• Boiling tube (20 cm long)
• Whirl mixer
• Sintered funnel of porosity No. 1
• Fine-bore pipette attached to a suction line
• Centrifuge
• Sintered crucible of No. 4 porosity
• Tared dish
• Water bath
• Oven, at 100°C
• Desiccator

Reagents

a. Chloroform.

b. Methanol.

c. 20% magnesium chloride aqueous solution (w/v).

d. 0.1% aqueous NaCl solution.

e. Anhydrous powdered sodium sulphate.

f. Glass beads.

Procedure

Weigh 5 g of the sample into a boiling tube (20 cm long); add a few glass beads, 5 ml of chloroform, 10 ml of methanol and 0.05 ml of magnesium chloride solution

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Mix thoroughly using a whirl mixer for 2 min; add another 5 ml of chloroform and mix again for 2 min

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Add sufficient volume of water so that the total water content, including the moisture content of the sample, is 9 ml (for example, if the moisture content of the sample is 60%, 5 g of sample contains 3 ml of water, hence add 6 ml of water); mix again for 30 seconds

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Filter the extract through a sintered funnel of porosity No. 1 under mild suction into a boiling tube — do not filter through filter paper, as phospholipids are absorbed by the paper; wash the tube in which the sample was taken and the funnel three times with 2.5 ml portions of chloroform

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Swirl the contents and centrifuge at 1500 rpm for 5 min; with a fine-bore pipette attached to a suction line, remove the top aqueous layer as much as possible without disturbing the chloroform layer

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To the chloroform extract, add 10 ml of 0.1% NaCl solution and mix by gentle inversion 6 times — do not shake vigorously, as an emulsion will form (the salt solution removes the protein that might have been extracted)

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Centrifuge again for 5 min; remove the top aqueous layer as before

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To the chloroform layer, add 1–2 g of anhydrous powdered sodium sulphate and shake vigorously to dry the chloroform; filter using a sintered crucible of No. 4 porosity into a dry receiver; wash the tube and the funnel three times with 2.5 ml portions of chloroform

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Transfer the dried chloroform extract to a tared dish, using small portions of chloroform for washing the residue; evaporate the chloroform on a water bath and dry the dish containing the fat residue in an oven at 100°C for 5 min; cool in a desiccator and weigh

Calculation

                   Wt of residue
% Total fat = ────────────────────── × 100
                   Wt of sample

References

Ranganna, S., Handbook of Analysis and Quality Control for Fruit and Vegetable Products, 2nd edition.

Folch, J., M. Lees & G.H.S. Stanley, J. Biol. Chem., 226, 497 (1957) — cited in the book.