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1. Ninhydrin Reaction
Spot the solutions of amino acids provided
on a filter paper, add a drop of ninhydrin
reagent to each spot and hold the paper over
a bunsen flame to dry. Note and comment on
the colors produced.
Expected results: Amino acids give purple
colors with ninhydrin reagent. But proline (an
imino acid) gives yellow color with ninhydrin.
2. Xanthoproteic Reaction
Add 1 ml of conc. nitric acid to 1 ml of the
amino acid solution. Warm the mixture in a
boiling water bath and allow to cool. Note the
formation of precipitates and colors. Add
sufficient 40% NaOH to make the solution
strongly alkaline.
Expected result: A yellow color in acid
solution which turns bright orange in alkali
indicates the presence of one or more of the
aromatic amino acids: tryptophan,
phenylalanine, and tyrosine.
3. Millons Reaction
Add 5 drops of Millons reagent to 1 ml of the
test solution and warm the mixture in a
boiling water bath for 10 minutes. Cool to
room temperature and add 5 drops of sodium
nitrate solution.

Expected result: The formation of a brick-red

color indicates the presence of a phenolic
amino acid, i.e., tyrosine or its derivatives.
Phenols and naphthols also give positive
4. Violet Ring Test
Add 2 ml of glacial acetic acid that has been
exposed to light to 2 ml of the test solution.
Pour 2 ml of conc. sulfuric acid carefully
down the sides of the test tube in a sloping
position so as to form two layers.

Expected result: A violet ring at the interface

indicates the presence of tryptophan.
5. Nitroprusside Test
Mix 0.5 ml of a fresh solution of sodium
nitroprusside (C5FeN6Na2O or Na2Fe(CN)5NO) with 2
ml of the test solution. Add 0.5 ml of ammonium
hydroxide. Note the color formed.

Expected result: Thiol groups (cysteine) react with

sodium nitroprusside in the presence of excess
ammonia to give a red color.
6. Sagakuchi Reaction
Mix 1 ml of 40% sodium hydroxide with 3 ml
of arginine solution and add 2 drops of -
naphthol. Mix thoroughly and add about 4
drops of bromine water. Note the color

Expected result: The guanidine group of

arginine reacts with -naphthol and an
oxidizing agent such as bromine water to
give a red color.
7. Biuret Test
Biuret Test for Proteins: Biuret reagent is a light blue
solution which turns purple when mixed with a
solution containing protein. When the copper ions of
the Biuret Reagent react with peptide bonds in the
polypeptide chains, a purple color complex is
To 2 ml of the test solution add 5 drops of biuret
reagent. Mix thoroughly and note the colors formed.
This is a test for the presence of two or more peptide
bonds in solution. The intensity of the color
produced is proportional to the number of peptide
bonds present.
Determination of Protein
Concentration: Biuret Method
You are provided Test (ml) Blank (ml)

with protein
samples, e.g., Test Sample 0.1 -

serum. Place the

following reagents 3%Sodium 0.4 0.4
into various test
tubes. Distilled 0.5 0.6

Biuret 4.0 4.0

Mix the contents of the test tubes by gentle shaking
and let stand for 20 minutes for color development.
Then read the absorbance of each test solution
against the blank at 540 nm.
You are also provided with a standard solution of
albumin (bovine serum albumin) with a
concentration of 10 mg/ml. Prepare a standard curve
using various concentrations of the albumin. This
can be done by replacing the test sample in the table
above with various amounts, i.e., 0.1, 0.2, 0.3, 0.4,
0.5, and 0.6 ml, of the albumin. Adjust the amount of
distilled water accordingly in order to keep the total
volume constant at 5ml. Read the absorbance of the
solution in each tube after 20 minutes and plot a
graph of absorbance against concentration of
albumin. Estimate the concentrations of the protein
samples provided from your graph.