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Activity 11

Qualitative Tests for Bile

Objectives of the Activity


bile's components and how will

they react with the tests.


the presence of cholesterol in bile.

the bile salts/pigments, and their

reactions to the tests.

Gmelin's Test for Bile Pigments

This test is for bile pigments. After adding nitric acid to the
solution, different colored rings are formed if bile pigments are
present as they are oxidized by the nitric acid.

Blue, green and violet rings are visible if biliburin (bile

pigment) is present and is oxidized.

Biliburin: is the yellow breakdown product of normal heme

catabolism, caused by the body's clearance of aged red blood
cells which contain hemoglobin. It is responsible for the yellow
color of bruises and the yellow discoloration in jaundice. It is
also responsible for the brown color of feces (via its conversion
to stercobilin), and the background straw-yellow color of urine.

Pettenkofer's Test for Bile Salts

This test is for the presence of bile salts. Consists of the

introduction to the solution of a drop of sucrose (strong)
and then of sulfuric acid. If bile salts are present, a
cherry red coloration is developed. Bile salts react with

Hydroxymethylenephurphural: is the product of certain

sugars being dehydrated - in this case, sulfuric acid is the
dehydrating agent.

Cholesterol in Bile: 2 Tests

Salkowski Test

This test is for cholesterols. The reaction principle of this test indicates
the presence of double bond in one cholesterol ring is responsible for
its ability to form colored products in the presence of concentrated
inorganic acids and it should yield a layering of colors. When concentrated
sulfuric acid is added to a chloroform solution of cholesterol, the
chloroform layer shows a red to blue color and the acid layer shows a
green fluorescence.

Leibermann-Burchard Test:

This is also a test for cholesterols. Theoretically, our results must be in

deep green color. This is due to the hydroxyl group of cholesterol which
reacts with the reagent which is acetic anhydride and sulfuric acid
and also due to the increasing conjugation or the transformation of a
substance to a hydrophilic state.

Results and Observations

Analysis and Discussion

Gmelin's Test: We had a negative result which might be caused by

experimental errors and such but theoretically, biliburin is present in bile
and it should be oxidized by conc. sulfuric acid, yielding multi-colored rings.

Pettenkofer's Test: We had a positive result. It yielded a cherry red solution

which was by the reaction of hydroxymethylenephurphural to the bile salts.

Cholesterol in Bile:
Salkowski Test: We had a positive result. We had a bluish chloroform layer
with tints of red and a green acid layer which is an indication of the
presence of the double bond on one cholesterol ring, which proves
cholesterol is present in bile .
Leibermann-Burchard Test: We had a positive result. The experiment
yielded a deep green solution after it reacted with the reagents, which is an
indicator of the hydroxyl group of cholesterols, therefore proving that
cholesterol is present in bile.

Bile as a part of our body has many components.
Knowing and understand that, may help us
become aware of our health status. In the
biochemistry side, bile isn't just a simple liquid
that is produced by the liver, but it is a complex
mixture of substances that react with different
substances, too. These qualitative tests we
performed helped us prove their presence and this
activity enabled us to understand how each of
them react.