OBSERVATIO INTERPRETATION
SOLUTION N
1%  Violet Presence of Protein
albumin
1% casein Violet  Presence of Protein

1% Pink Low concentration of

peptone peptide bonds

  OBSERVATION INTERPRETATION
SOLUTION
Urea with Violet Peptide bonds are
distilled present
water

MILLON’S REACTION
Purpose: To detect amino acid containing phenol group (hydroxyl group
attached to benzene ring) i.e. Tyrosine
Principle: Compounds containing hydroxybenzene radical react with Millon’s
reagent to form red complexes. The only amino acid having hydroxybenzene ring
is tyrosine. Thus, this test is specific for the amino acid tyrosine and the protein
containing this amino acid. Tyrosine when reacted with acidified mercuric
sulphate solution gives yellow precipitate of mercury-amino acid complex. On
addition of sodium nitrate solution and heating, the yellow complex of mercury-
amino acid complex converts to mercury phenolate which is in red color.
Reagents: Millon’s reagent (Acidified mercuric sulphate)
 and 1% phenol
Results: Positive Millon’s test: Brick red color (Tyrosine and phenol solution)
Negative Millon’s test: no red color 
**all phenol give positive Millon’s test
RESULTS

 SOLUTION w/ millon’s reagent OBSERVATION INTERPRETATION

1 % Albumin Brick red (Tyrosine and phenol solution)
1 % Gelatin No red color  (absence of phenol solution)
1 % Casein Brick red (Tyrosine and phenol solution)

 SOLUTION OBSERVATION INTERPRETATION

20 drops of 1% phenol Brick Red all phenol give positive

Millon’s test

XANTHOPROTEIC REACTION
Purpose: This test is performed to identify the amino acid having benzene ring in
them like tyrosine, tryptophan, and phenylalanine.
Principle: Treating aromatic amino acids with the concentrated nitric acid leads
to the nitration of the aromatic ring and formation of yellow nitro-products (nitro
derivatives). When the strong basic solution is added the color of obtained
products turns darker (from yellow to orange). The positive result of
xanthoproteic reaction gives: tyrosine, tryptophan and phenylalanine (only after
extended heating time). It is difficult to nitrate phenylalanine under normal
conditions so it does not respond to this test without extended heating.
Reagents: Conc. HNO3 (Nitric acid), Conc. NH4OH (Ammonium Hydroxide) and
1% phenol solution
Results:
Positive : Color changes to yellow and orange 
indicating the presence of aromatic amino acids
Negative – color does not change
RESULT
SOLUTION w/ OBSERVATON INTERPRETATION
HNO3 and NH4OH
1 % Albumin  Orange (Color changes to orange indicating the
  presence of aromatic amino acids)
1 % Gelatin No color (Color does not change to yellow indicating
change the absence of aromatic amino acids)
 
1 % Casein Orange (Color changes to orange indicating the
  presence of aromatic amino acids)

  OBSERVATIO INTERPRETATION
SOLUTION N
10 drops of 1% ORANGE (Color changes to orange indicating the
phenol presence of aromatic amino acids)

TEST FOR SULFUR

Purpose: to detect amino acid containing sulfhydryl group (-SH) i.e. cysteine 

Principle: Proteins containing sulfur (like: cysteine) give a black deposit of lead
sulfide (PbS) when heated with lead acetate in alkaline medium. When cysteine is
heated with strong alkali like NaOH, some of the sulphur is converted to sodium
sulphide (Na2S) which can be detected by precipitation as lead sulphide (PbS) 
from alkaline solution.

Reagents: 5% Lead acetate and  10% NaOH

Results: Positive – Black precipitate
Negative- no black ppt

Sulfur-containing protein ----> NaOH----> Na2S- ----+Pb2----> PbS

RESULT
 SOLUTION OBSERVATION INTERPRETATION
Albumin  Black ppt Presence of sulfur

Solid Gelatin No black ppt Presence of Methionine

Peptone Black ppt Presence of sulfur

HOPKIN’S-COLE REACTION
Purpose: It is also known as the glyoxylic acid reaction. This test used for
detecting the presence of tryptophan in proteins.
Principle: The indole group of tryptophan reacts with glyoxylic acid in the
presence of conc. H2S04 to give a purple color. Glyoxylic acid is prepared by
reducing oxalic acid with magnesium powder or sodium amalgam. Glacial acetic
acid which has been exposed to the sunlight also contains glyoxylic acid and can
thus be used for this test.
Reagents: Hopkins Cole Reagent and conc. H2SO4
Results: Positive – Violet ring formed between the two layers due to presence
of tryptophan
Negative – No appearance of violet ring
RESULT
  OBSERVATIO INTERPRETATION
SOLUTION N
1% Violet ring (Violet ring appears between the two layers due to
Albumin   presence of tryptophan)
1 % Gelatin No Violet ring (No appearance of a violet ring between the two
  layers due to absence of tryptophan)
1 % Casein Violet ring (Violet ring appears between the two layers due to
  presence of tryptophan)

NINHYDRIN REACTION
Purpose: Used to determine the presence of free amino acids in a solution
Principle: Reaction of amino acids with ninhydrin leads to their decarboxylation,
deamination (formation of CO2 and ammonia) and formation of aldehyde which
has one carbon atom less in its structure. The ninhydrin undergoes reduction to
form hydrindantin. The reduced ninhydrin condenses with ammonia and non-
reduced ninhydrin molecule which leads to the formation of the blue-purple
condensation product called Ruhemann’s purple.
Reagents: Freshly prepared ninhydrin solution
Results: Positive test: Blue-purple reaction products positively identify free
amino groups on amino acids and proteins.
 Negative test: No change (absence of amino acids and Proteins)

RESULT
  OBSERVATION INTERPRETATION
SOLUTION w/ ninhydrin solution
Albumin solution Blue-purple Presence of amino acids

  OBSERVATION INTERPRETATION
SOLUTION
Ammonia Water Pale Purple NO presence of Amino Acids

0.2% Urea Pale Purple NO presence of amino acids

0.2% Glycine Blue - Purple Presence of Amino acids

1. Why do all proteins give a positive in Biuret test?

 It gives positive result to any compound containing two carbonyl groups
attached to a nitrogen or carbon atom. Thus, it may not be completely protein-
specific. 

2. Why does nitric acid strain the skin with a yellow color?
Proteins and amino acids that contain phenyl rings form a yellow colored
compound when treated with concentrated nitric acid. When nitric acid comes in
contact with the skin, it reacts with the protein, keratin to produce yellow color
nitrated products called xanthoproteic acid. 
3. Will methionine give a positive sulfur test? Support your explanation with the
chemical structure of methionine.
No, Methionine does not give lead sulfide test because sulfur in methionine
does not split in presence of alkali. Sulphur in methionine is inert, it does not
readily react with NaOH to form sodium sulfide which would form black/brown
ppt. with lead acetate. Getting this ppt. is the positive response in sulfur test for
amino acids. 
4. What grouping in amino acid or proteins is responsible for the ninhydrin
reaction?
Ninhydrin is used in many bioanalytical techniques particularly for amino
acid analysis method. Ninhydrin reacts with the α-amino group of primary amino
acids producing 'Ruhemann's purple'.

5. What color will be produced by the proline with ninhydrin? Why? 

Proline, a secondary amine, gives a yellow-orange product. Proline has
aliphatic side chains with a distinctive cyclic structure. Proline does not give the
ninhydrin reaction as this reagent requires free alpha amino group (-NH2) but
proline has an imino group (-NH). For the amino acids which have a free –NH2
group, ninhydrin test is positive but is negative for proline because it only has –NH
group.