Millon's Test: Reactions

Millon’s Test
REACTIONS:
• After adding 2 drops of millon’s raegent
it turned into cloudy white solution and a precipitate
formed which settle down in the test tube
• Mix and heat
When millon’s reagent is added to a protein, a
white ppt is formed, which turn brick red on
heating.
• Responsible for this color reaction:

Millon's Reagent-a solution that is usually made
by dissolving mercury in concentrated nitric acid
and diluting with water and that when heated
with phenolic compounds gives a red coloration
What amino acids gives positive MILLON’S test?

Tyrosine is one of the 20 amino acids that are used by
cells to synthesize proteins .
any protein containing tyrosine will give a positive
test of a pink to dark-red color
Xanthoproteic Test
REACTIONS:
• After adding 5 drops of conc. Nitric acid
There was a white precipitate formed
• Heat and observe color change

The solution went from being white into yellow in color
• Cool and add 5 drops of ammonium hydroxide

There was a yellow ppt. formed and the solution remains yellow as well
• What responsible for the color change?

The amino acids tyrosine and tryptophan contain activated benzene rings which
can readily undergo nitration and will change the solution color to yellow in the
xanthoproteic test..
Hopkins-Cole Test
REACTIONS:
• Allow 10 drops of pure conc. Sulfuric acid
The solution became cloudy in appearance.
• What is the color produced at the point of contact of

the 2 liquids?
There was a violet color produced at the point of
contact. Tryptophan has an indole nucleus which is
responsible for the violet ring found at the junction
between the two layers.
• What is the cause for this color change?

The glyoxylic/oxalic acid reacts with this indole ring to
form a voilet product .

Biuret Test
 The biuret test is a chemical test used for
detecting the presence of peptide bonds.
 The Biuret Reagent is made of sodium

hydroxide and copper sulfate.
 An aqueous sample is treated with an

equal volume of 10% strong base (sodium or
potassium hydroxide) followed by a few drops
of aqueous copper(II) sulfate. If the solution
turns from blue to purple, protein is present.
 Albumin is an intact protein/peptide so there should be a positive result.

Ninhydrin Test
Ninhydrin is a powerful oxidizing agent reacts with amino
acids.
In Ninhydrin test, the resulting color is
blue violet.
Ninhydrin reagent is reduced to
hydrindantin during reaction with amino
acids.
The amino acid in turn is converted into an aldehyde.

Ammonia Carbon dioxide is evolved.
Hydrindantin and ammonia interact with another molecule

of ninhydrin to form Ruhemann’s purple colored complex.
It also indicates the presence of α

amino acid.
Sakaguchi Test
• The Sakaguchi test is a specific
qualitative test for the detection of
a specific type of protein with the
amino acid containing the
guanidinium group. In basic
conditions, alpha naphtol and
sodium hypobromite/chlorite react
with the aforementioned
compound to form red-orange
complexes.
Precipitation reaction by concentrated
mineral and organic acids
• Salt bridges result from the neutralization of an acid
and amine on side chains. The final interaction is ionic
between the positive ammonium group and the
negative acid group. Any combination of the various
acidic or amine amino acid side chains will have this
effect.
• As might be expected, acids and bases disrupt salt
bridges held together by ionic charges. A type of
double replacement reaction occurs where the positive
and negative ions in the salt change partners with the
positive and negative ions in the new acid or base
added.
Precipitation by metallic salts
• Proteins are precipitated by salts of heavy
metals, such as mercuric chloride, zinc sulfate,
etc. in weak alkaline solutions, protein
molecules carry negative charges and
combine with positively charged metal ions to
form insoluble salts which precipitate from
the solution. The precipitated proteins are
denatured and this process is irreversible.
Precipitation by alkaloidal reagents
• In acid environment, the positively charged
protein molecules can react with the
negatively charged acid radicals of organic
acids or alkaloidal reagents to form
precipitate. The proteins thus precipitated are
denatured. The commonly used organic acid
are trichloroacetic acid, sulfosalicylic acid, and
alkaloidal reagents are tungstic acid, picric
acid.
Precipitation by Alcohol
• First tube contained acidified ethanol. second tube
contained alkaline ethanol and the third test tube
contained only ethanol. On adding egg albumin to all
the three tubes. More precipitation occurs as albumin
is less soluble in ethanol. Since albumin contains
proteins which are soluble in acids as well as in alkalies
so less precipitation of albumin will take place. Protein
of albumin has more solubility in alkaline solution so
least precipitation occurs in alkaline solution.
• It will become clearer as you will study more about the

properties of proteins.
Coagulation by Heat
• Egg-white is faintly alkaline. Complete
precipitation takes place only in faintly acid
solution. The temperature at which
coagulation takes place depends to a large
extent on the amount of acid and of salts
present.
Test for denatured/Coagulated
proteins
• (Biuret test) Excess of copper sulphate must be avoided in making the
biuret test, since the color of the salt prevents the recognition of the color
produced in the reaction. The presence of ammonium salts interferes with
the test. In applying the reaction to solutions containing these salts a large
excess of sodium hydroxide must be present. Compounds which give the
biuret test must contain at least two -CO-NH- groups. The color formed in
the reaction varies in shade with the complexity of the molecules.
• (Xanthoproteic) The color is produced as the result of the formation of

nitro-derivatives of the compounds which contain a benzene ring, for
example, tyrosine.
• (Hopkins-cole test) The color produced is due to the formation of a

compound from the glyoxylic acid in the reagent and the tryptophane in
the protein. A similar color is produced when sulphuric acid is added to a
protein solution in the presence of a trace of formaldehyde. The reaction
is used as a test for formaldehyde in milk.

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Millon’s Test

• Responsible for this color reaction:

What amino acids gives positive MILLON’S test?

• Heat and observe color change

• Cool and add 5 drops of ammonium hydroxide

• What responsible for the color change?

• What is the color produced at the point of contact of

• What is the cause for this color change?

form a voilet product .

 The Biuret Reagent is made of sodium

 An aqueous sample is treated with an

 Albumin is an intact protein/peptide so there should be a positive result.

The amino acid in turn is converted into an aldehyde.

Hydrindantin and ammonia interact with another molecule

It also indicates the presence of α

• It will become clearer as you will study more about the

• (Xanthoproteic) The color is produced as the result of the formation of

• (Hopkins-cole test) The color produced is due to the formation of a

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