QUALITATIVE ANALYSIS

PROTEINS AND LIPIDS

Important Points:
1. All the tests and reactions described here are not quantitative and volumes are approximate,
despite these facts some tests do not work if quantities greatly in excess of those stated are used.
2. DO NOT place/change your pipettes/dropper in reagent/sample tube as this leads to
contamination.
3. In most tests, it is important to apply a negative control test using water instead of the test
solution under examination. If you are in doubt about the result of a test, perform the reaction
with a suitable known compound/sample.
4. In all experiments, unknown samples are given in their liquid state. To perform each
procedure, you should not prepare your own test solutions.
5. When you need to boil your sample in a test tube, prepare a hot water in a large beaker and put
your test tube inside the beaker or you can request for a water bath.

LIPIDS
a) Solubility test
Procedure:
i. Take clean test tubes and add 1ml of oil samples into each tube.
ii. Add for the first one 1 ml of chloroform.
iii. Add for the second tube 1 ml of distilled water.
iv. Shake both tubes vigorously for 2 minutes.
v. Allow the tubes to stand and note the formation of homogenous solution with chloroform
indicating that the lipid is dissolved and the formation of two layers with water as control
indicating that the lipid is insoluble in water.

b) Grease Spot Test

Procedure:
i. Place 1 drop of oil sample on a piece of brown paper.
ii. Place 1 drop of any (chloroform or ethanol or water as control) on a second piece of brown
paper.
iii. Wait a few minutes for the spots to dry.
iv. Hold the papers up to the light and determine if each spot is translucent.

c) Iodine test (Unsaturated Fatty Acid)

Principle.
This test is used to test the degree of unsaturation of fatty acids. Fatty acids in animal fats are
usually saturated, whereas those in vegetable oils are generally unsaturated. Halogens like iodine
or bromine when added to unsaturated fatty acid the double bond will be saturated and
decolorize the iodine or bromine, the decolorization indicates the presence of unsaturated fatty
acids. Iodine test is used for distinguish between saturated and unsaturated fatty acids as well as
between oils and fats.

Procedure.
i. In clean dry test tube add 1 ml of oleic acid (unsaturated).
ii. In other test tube add 1 ml of stearic acid (saturated).
iii. Add for each tube 5 drops of hubl’s reagent.
iv. Observe that the color of halogen in the reagent will be disappeared when it is added to
the oleic acid until we reach a certain point after which the color of the reagent will
persist indicating that all of the double bonds are saturated by halogens.
v. Observe that the color of halogen in the reagent does not change when it is added to
stearic acid because this fatty acid is saturated.

c) Chloroform-Ethanol Test (Unsaturated Fatty Acid-Double Bond)

Procedure:
i. Pipette 1ml of Chloroform-Ethanol (1:1) mixture into all the test tubes as labeled.
ii. Add 2 drops of distilled water into the control tube and 2 drops of oil sample(s) into the
other tube(s), then mix well.
iii. Add 2ml of acetic acid and 2 drops of 10% KI then mix and leave the experiment
undisturbed at room temperature for 5mins.
iv. Compare the iodine colour in each test tubes with the control.

PROTEINS
Biuret Test
Procedure
i. Take three clean and dry test tubes.
ii. Add 1ml of the test solution, egg albumin, and deionized water as control into their
respective test tubes.
iii. Add 1ml of Biuret reagent to all the test tubes.
iv. Shake well and allow the mixture to stand for 5 minutes.
v. Observe for any color change.

Sakaguchi’s Test (Test for Amino Acid)

Principle
Molisch reagent is α-naphthol in alcohol. Sodium hydroxide provides alkaline Ph. At the alkaline
pH guanidino group of arginine combines with α-naphthol to form bright red color. The protein
solution contains amino acid with guanidino group (Arginine).
Procedure
i. Add 5 drops of 5% sodium hydroxide solution to 1ml of protein samples as well as negative
control and shake the tubes vigorously.
ii. Add 4 drops of Molisch’s reagent.
iii. A bright red color develops.