Laboratory Determination of Fat in Milk

The following is straight from my college nutrition lab book. Please leave comments if you have questions and cannot find answer on internet. I'll try to help.

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Milk is a colloidal dispersion and contains water-soluble solutes as well as insoluble material. Fatty compounds are generally insoluble in water but soluble in organic solvents like hexane, chloroform, etc. The determination of fat in milk will require extraction of fat in an organic solvent. Direct treatment of such an organic solvent generally produces an emulsion at the junction pf twp solvents (water from milk and organic solvent.)

The problem can be solved in various ways. One of the simple methods is to first precipitate the proteins and then filter them out. Proteins can be precipitated out by trichloroacetic acid, acetic acid (vinegar), and heavy metals or by adding large amount of salt.

REQUIREMENTS: Milk sample to be analyzed, suitable reagent for precipitation of proteins, organic solvent to extract fatty compounds, filter equipment or a centrifuge, evaporating dish or beaker, separatory funnel, balance (scale).

PROCEDURE:

1. Weight a 250 ml size beaker. Place 100 ml of milk sample in it and record the total weight. From these weights find out the weight of milk.

2. Add 40% solution of trichloroacetic acid (be careful) solution or acetic acid solution (vinegar from a supermarket) whichever is provided to you. Stir continuously and continue the addition of a reagent until the precipitation is complete. If a pH meter is available check the pH of the milk sample. You may use pH paper. The pH should be 4.0 or less than 4.0. The pH of normal milk sample is generally close to neutral pH (i.e., pH 7.0).

3. If a centrifuge is available, centrifuge the sample carefully in the pre-weighed centrifuge tubes for 25 minutes at 4000 rpm.

Pour the supernatant into a clean beaker. If a centrifuge is not available, filter the ppt (precipitate) using pre-weighed Whatman 52 or fine grade filter paper.

4. Now add approximately 20 ml of hexane or petroleum ether or the solvent recommended by your instructor to the filtrate or the supernatant (liquid separated after centrifugation).

Stir well and pour the contents into a separatory flask. Allow the two liquid layers to separate as clearly as possible. You may add approximately 4 ml of methanol if you see emulsion or scum like cloudy or milky interface.

5. Separate the organic layer. Depending on the density of the solvent used, the organic solvent will be either upper or lower layer. Collect the organic layer in weighed evaporating dish or a small beaker.

6. The water layer should be treated with 20 ml of the fresh organic solvent for the extraction of fat. Then separate clear organic solvent.

7. Repeat the extraction two more times. Collect all the extracted fat solutions in the same container.

8. If air current is available dry over such air jets and weigh the beaker/evaporating dish. Otherwise, dry it over a boiling water bath.

CALCULATIONS:

Weight of beaker:

Weight of milk and beaker:

WEight of milk used: (in other words, net weight)

WEight of dish:

Weight of dish plus dried fat:

Weight of fat: (in other words, net weight)

% fat content = [weight of fat X100]/Weight of milk sample

%fat content = [weight of fat X 100]/Weight of milk used

SAMPLE CALCULATIONS:

Wt. of beaker: 25.5 g
Wt of beaker + milk: 115.0 g
Wt. of milk = 115.0 - 25.5 = 89.5 g
Wt of dish:35.8 g
Wt of dish and dried fat: 39.8 g
Wt of fat= 39.8 g - 35.8 g = 4.0 g
% fat content = [4.0 g X 100]/89.5 g = 400/89.5 = 4.47%. This might be a homogenized milk sample.

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"Laboratory Manual for Introductory Course in Nutrition," by Kris Dhawale, Professor of Chemistry, IU East, Richmond, IN. 2003The following is straight from my college nutrition lab book. Please leave comments if you have questions and cannot find answer on internet. I'll try to help.

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