Ethanol Emulsion Test for Fats and Oils

The Ethanol Emulsion Test is a food test which determines the presence of a broad group of naturally occurring compounds known as lipids. Lipids consist of fats and oils. Other lipid tests include the Grease Spot Test and the Sudan Stain Test. The Grease spot test is performed on fats - lipids which are solid at room temperature. Sudan stain colours lipids red, but is a less common bench reagent than ethanol. The Ethanol Emulsion Test is the most common test amongst the three.

Procedure
Soild sample : 1.Crush the food sample and place in a dry test tube. 2.Add ethanol to about 2 cm3 above the level of the sample and shake thoroughly. 3.Allow the solid to settle (about 3 min) to allow the lipid to be extracted. 4.Decant the ethanol into another test tube. 5.Add 2 cm3 of deionized water to the second test tube. 6.Make observations.

Liquid sample: 1.Add a few drops of the liquid food sample to a dry test tube. 2.Add 2 cm3 ethanol and shake it thoroughly 3.Add 2 cm3 of deionized water. 4.Make observations.

Results and Interpretation

OBSERVATION
POSITIVE TEST

INTERPRETATION

A layer of cloudy white suspensionforms at the top of the solution. (Upon close inspection you can see the tiny globules of fat suspended in the solution. This an emulsion. Foods Lipids are present with high lipid content have a higher layer than foods with less). Solution remains colourless. No emulsion is formed. Lipids are not present

NEGATIVE TEST

Principle of the Ethanol Emulsion Test

The solubilities of lipids and ethanol are exploited in this test. Lipids are non-polar organic compounds. Hence they are soluble in organic solvents such as ethanol (alcohol), but insoluble in water. Ethanol is an organic substance and so dissolves other organic substances; it is frequently used as an organic solvent. However ethanol is also miscible in water due to the presence of the hydroxyl (OH) functional groups and the shortness of its chain (2C). The hydroxyl group participates in hydrogen bonding with water (see below).

The hydrophobic interaction of the carbon in the short chain with water is not great and is overcome by the hydrogen bonding. Ethanol extracts the lipid from the crushed solid sample. As ethanol is miscible with lipids no change is seen upon its addition to the solid and liquid samples. The lipid spontaneously comes out of solution when water is added and is dispersed as micelles (small droplets) throughout the solution of ethanol and water.( This happens as hydrophobic portion of the lipid molecules project inwards and excludes the aqueous environment; the hydrophilic portion (-COOH) group faces the aqueous environement.) A layer is formed at the top as lipids are less dense than water. The droplets diffract light, appearing cloudy white.

A Conclusive Test
A positive test shows conclusively that lipids are present - and not the other major biological molecules: 1. Carbohydrates Reducing sugars and non- reducing sugars - slightly soluble in ethanol and soluble in water Starch insoluble in both ethanol and water

2. Proteins insoluble in ethanol (the addition of ethanol is used to precipitate proteins) 3. Nucleic Acids are insoluble in ethanol and soluble in water No change is seen in a negative test as there are no lipids to come out of solution.

Experimental Skills
The Ethanol Emulsion Test procedure is suitable for assessment of the Measurement and Manipulation Experimental skill. Below are some criteria your teacher may use to assess you. He will be scrutinizing the steps you take to prevent contamination , including cross contamination from other food samples. CRITERIA Preparation of a Solid Sample: 1. Completely crushes adequate amount of food with a clean and dry mortar and pestle. 2. Transfers an adequate amount of crushed food sample to a clean test tube. 3. Adds 2cm3 ethanol above the food sample. 4. Shakes the test tube thoroughly and safely (using a vortex mixer if available). 5. Allows the solid to settle for about 3 min. 6. Decants the ethanol carefully, into a clean, dry test tube without transference of food particles. 7. Adds 2cm3 of de-ionized water to the test tube. 8. Compares each result with the negative control. 9. Compares the heights of the emulsion layer of all the samples.