Experiment II-A- Millons Test

Objectives:
To observe the color changes in the protein solution upon addition of Millons
reagent
To understand the principle behind Millons Test .
Procedure:
Drops of Millons reagent were added in each protein solutions (gelatin,
casein, and albumin) in the test tube. It was mixed and boiled gently until
the white precipitate formed upon the addition of reagent turned red
Result:
Millons test is used to detect the presence of soluble proteins. In the
Experiment, color of 3 solutions (gelatin, albumin, and casein) changes from
white precipitate to reddish brown coloration.

Millons reaction is given by any compound containing a phenolic hydroxy group. Consequently, any
protein containing tyrosine will give a positive test of a pink to dark-red colour. Compounds

containing in their molecules a hydroxyl group bound to an aromatic ring, yield a

complex mercuric salt with the Millons reagent.

Millons test is used to detect the presence of hydoxyphenyl group (benzene ring with -OH). It is a specific
test for tyrosine (since tyrosine is the only amino acid that has a hydroxyphenyl group) and gives a red
precipitate or a red solution as a positive result. This happens when the phenol group of tyrosine is
nitrated and then the nitrated phenol complexes with mercury (I) and mercury (II) ions. Nevertheless, it is
important to make sure that no other phenols aside from the ones from tyrosine are present because the
Millons test gives a positive result for any compound with phenol groups. Phenols containing more than
one hydroxyl group do not give the typical red color. With a few drops of Millons reagent, the aqueous
solution of monacid phenol even in the cold, or when warmed if only traces of phenol are present takes on

a light to dark red color. This reaction takes place when one or more hydrogen atoms of the benzene ring
of monacid phenols are replaced by hydrocarbon radicals. But such a substituent as chlorine, bromine,
iodine or carbon free radicals prevent it. The fact that aniline and other primary aromatic amines give this
test is due to the presence in the reagent of free nitric and nitrous acid. The red color observed in this test
is due to a mercury salt of nitrated tyrosine.

References:
Walsh, Edward O'Farrell (1961). An Introduction to Biochemistry. London: The English Universities Press Ltd.
pp. 406407. OCLC 421450365.