College of

Nursing
Pharmacy &
QUALITATIVE
Allied
Health TEST FOR LIPIDS
Sciences
 Lipids
Department of Pharmacy

are a class of compounds

distinguished by their insolubility
in water and solubility in non-
polar solvents.
 A. Solubility Test
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This test is used to know the

solubility of lipids in some solvents.

Lipids are insoluble in polar solvents

and soluble in non-polar solvents like
chloroform, benzene and boiling
alcohol.
 How is it performed?
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1. In two clean dry test tubes add 1 ml of oil

for each tube.
2. Add for the first one, 1ml of chloroform.
3. Add for the second tube, 1ml of distilled
water.
4. Shake both tubes vigorously for 2 mins.
5. Allow the tube to stand and observe.
Department of Pharmacy
 B. Translucent Spot Test
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Is a preliminary test for the lipids,

which is characterized by a
translucent and greasy spot.
The lipid will not wet the filter
paper, unlike water. The lipids will
form a greasy or translucent spot
 What are the methods?
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Take a filter paper.

Add one drop of water at one end
and a drop of oil or lipid at the other
end.
Observe the appearance of a
translucent spot on the filter paper.
Department of Pharmacy
 C. Saponification Test
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This test is used to form soap by

saponification process which includes
hydrolysis of fat or oil with alkaline to
yield glycerol and salt of fatty acid
(soap).
 What are the procedure?
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1. In clean dry large test tubes add 2 ml

of oil.
2. Add 4 ml of 20% alcoholic potassium
hydroxide.
3. Shake well and boil the mixture in
boiling water bath for 5 mins.
Department of Pharmacy

4. Make sure after the 5 mins. that the

soap is formed, all of the fat is
converted to soap and an alcohol is
evaporated. If not continue boiling.

5. The solid material formed is soap.

Department of Pharmacy
 D. Unsaturation Test
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This test is used to detect the unsaturated

fatty acids or double bond in a lipid
sample.
Fatty acids in animal fat are usually
saturated.
Vegetable oils are generally unsaturated.
 What are the procedure?
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1. Take 5 ml of chloroform and 5 ml of

Huble’s iodine reagent in a beaker, giving
pink colour to the solution.
2. Add lipid sample drop by drop and shake
vigorously, until pink colour disappears.
3. Count the number of drops added to
chloroform and Huble’s iodine solution until
pink colour disappears.
Department of Pharmacy
 E. Burchard Test
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It was first given by the scientist

Liebmann to detect the presence of
cholesterol.
Cholesterol reacts with the strong
concentrated acid, i.e. sulphuric acid and
acetic anhydride.
 Methods
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1. Take crystals of cholesterol in a test tube.

2. Then, add 2 ml of chloroform to dissolve
the cholesterol.
3. Add 10 drops of acetic anhydride in a
solution and 2-3 drops of concentrated
sulphuric acid.
4. Observe the test tube for the appearance of
a bluish-green colour.
Department of Pharmacy
 F. Emulsification Test
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Emulsification test is used to detect the

presence of lipids.
It is a procedure that uses emulsifying
agents to stabilize a water-oil emulsion.
Emulsifying agents emulsify lipids,
causing them to appear as tiny droplets
suspended in the solution.
 Methods
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1. Take two test tubes and label it as test tube

A and test tube B.
2. Add oil to each of the test tubes.
3. Shake the test tube and allow it to stand for
about two minutes.
4. Observe the test tube for the appearance of
tiny droplets in the suspension of liquid.
Department of Pharmacy
 G. Sudan IV Test
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Sudan IV test is used to detect the

presence of lipid in a solution.
The principle of lipid binding and
solubility in non-polar compounds
underpins this test.
Sudan IV does not stain or bind to the
polar compounds.
 Methods
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1. Take 1 ml of the lipid sample in a test

tube.
2. Then add 1-2 drops of Sudan IV to the
solution.
3. Observe the tubes for the appearance of
red-orange colour in the solution.
Department of Pharmacy
 H. Acrolein Test
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Acrolein test is used to detect the

presence of glycerol and fat.
The dehydration reaction, in which
water molecules are removed from
glycerol by adding the reagent potassium
hydrogen sulphate, is the basis for this
test.
 Methods
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1.Take 1 ml of the lipid sample in a test

tube.
2.Add crystals of potassium hydrogen
sulphate.
3.Heat the solution for a few minutes.
4.Smell the test tube for the pungent
smell.
Department of Pharmacy
 I. Dichromate Test
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Dichromate test is also used to detect

the presence of glycerol.
It is based on the principle of an
oxidation reaction.
 Methods
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1.Take 2-3 ml of a sample in a test tube.

2.Then, add a few drops of 5% potassium
dichromate solution.
3.After that, add 5 ml of concentrated
nitric acid.
4.Observe the test tube for the appearance
of a blue colour.
Department of Pharmacy
 J. Tests for the Free Fatty Acids
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This is based on the neutralization

reaction, in which the alkali neutralizes
the lipids by introducing free fatty acids.
 Methods
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1.Add phenolphthalein solution in a test

tube.
2.Then, add dilute alkali to the above
solution (gives a pink colour).
3.At last, add a lipid sample.
4.Observe the tube for the disappearance
of the pink colour after the addition of
lipid.
Department of Pharmacy
 REFERENCE:
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