Lab Activity 1
Qualitative Tests for Amino Acids
IUG, 2013
Dr. Tarek M. Zaida
Main Classes of food ?
• Carbohydrates: (readily available energy resources)
• Lipids: (principal energy reserves)
• Proteins: (Source of energy for growth & cellular maintenace)
Proteins are….
• The most important cell content after water
• Are either functional or structural
• Macromolecules made up of amino acids,
connected together by peptide bonds.
Peptide bond: Amide bond, formed between
COOH & -NH2 of 2 adjacent amino acids.
Amino acids
Proteins are made up of 20 A.A. All of them have the same general
structural formula shown above, however they are different in the
R- group (side chain).
Classification of amino acids
• Non-essential..
Are synthesized by the body
• Essential..
(Valine, Leucine, Isoleucine, Methionine, Threonine, Tryptophan,
Phenylalanine, Lysine, Histidine)
What amino acids chemical reactions are due to?
1. Amphoteric nature
2. R-group or side chain

The accessibility of certain functional groups to the reagent will
determine the intensity of the product color.
 The color intensity varies among proteins and is proportional to the
number of reacting functional, or free groups present.
A.A in acidic, neutral, and basic solutions
Experiments
• A.A can be characterized qualitatively by using
several dyes that will react with certain groups
of the A.A.
 Seven Tests:
1. Ninhydrin
2. Biuret
3. Millon’s
4. Xanthoproteic
5. Hopkin’s- Cole
6. Sulfur
7. Sakaguchi
1. Ninhydrin Test
• For amino acids containing a free NH2 & free
COOH.
• Reaction with ninhydrin to produce a colored
product.
1. When NH2 is attached to α-C on the amino acid’s carbon
chain, the amino group’s N is part of a blue-purple product.
2. Amino acids that have N-H (a secondary amino group (e.g.
proline) also react with ninhydrin, but they yield a yellow
product.
Reaction of A.A with Ninhydrin
Procedure..
1.
Label 6 cleaned, drained test tubes with the names of the
following solutions: 2 % glycine, 1 % tyrosine, 2 % proline, 2 %
casein, 2 % gelatin, 2 % albumin.
2. Add 15 drops of each solution in the corresponding test tube.
3. To each of the test tubes add 5 drops of 0.5 % ninhydrin reagent
solution.
4. Place the test tubes into the boiling-water bath for 5 minutes.
Remove the test tubes from the water bath and place then in a
test tube rack.
Record your observations!
2. Biuret
• For detecting peptide bonds (hence peptides or
proteins)..
• How it works?
• The copper atoms of Biuret solution (CuSO4 ) in a
basic environment will react with peptide bonds
(-CO ---NH) to form a chelate of a deep violet
color, indicating the presence of proteins.
• A light pink color indicates the presence of
peptides..
Biuret complex with proteins…
Procedure..
1. To 1 ml of a solution containing protein add
4 ml of a biuret reagent.
2. Mix well, then let to stand at RT for about
30 min.
3. Record your abservations!
3. Millon’s
• Any compound containing a phenolic hydroxyl
group will give a positive result with Millon’s
reagent.
• Cosequently..
• Proteins containing tyrosine will give a
positive test of a pink to dark-red color
• Note: Some proteins will initially form a white
precipitate that will turn red when heated.
Procedure..
1. 2 ml of 2% casein, 2% egg albumin, and
0.1 M tyrosine add 3 drops of Millon's
reagent.
2. Immerse the tubes in a boiling water bath
for 5 minutes.
3. Cool the tubes down. Record the colors
formed.
4. Xanthoproteic
 For detection of aromatic groups, derivative
of benzene, (hence aromaric amino acids).
These aromatic groups can undergo reactions
characteristic of benzene, and its derivatives.
One such a characteristic reaction for benzene
is: Nitration
 The amino acids tyrosine and tryptophan contain activated
benzene rings and readily undergo nitration, while
phenylalanine does not contain a readily activated benzene
ring.
 a. Act. tyrosine
 b. Act. Tryptophan
Procedure...
1. Add 1 ml of a concentrated HNO3 in a test
tube containing 2 ml of a protein solution.
2. The formed white precipitate, will turn
yellow upon heating, and finally will dissolve
giving a yellow color to the solution.
3. Cool the solution down. Carefully add 3 ml
of 6 N NaOH. The yellow color turns orange.
5. Hopkins-Cole (Glyoxylic Acid Reaction)
• Specific for tryptophan (the only
amino acid containing indole group)
• Reacting with a glyoxylic acid in the
presence of a strong acid, the indole ring
forms a violet cyclic product.
• The protein solution is hydrolyzed by
conc. H2SO4 at the solution interface.
• Once the tryptophan is free, it reacts with
glyoxylic acid to form violet product.
Indole
Glyoxylic acid
Procedure..
1. In a test tube, add to 2 ml of the solution
under examination, an equal volume of
Hopkins- Cole reagent and mix thoroughly.
• Incline the tube and let 5 to 6 ml of conc.
H2S04 acid flow slowly down the side of the
test tube, thus forming a reddish - violet ring
at the interface of the two layers. That
indicates the presence of tryptophan.
6. Sulfur Test
• For the detection of sulfur-containing
amino acids such as cysteine.
• Is done by converting S to an
inorganic sulfide ( S2-) through
cleavage by a base.
• When the resulting solution is
combined with lead acetate
(CH3COOPb), a black precipitate of
lead sulfide is formed.
Sulfur-containing protein ----> NaOH----> S2- ---Pb2+----> PbS
Cysteine
Procedure..
1. Place 1 ml of 2% casein, 2% egg albumin, 2%
peptone, 2% gelatine and 0.1 M cysteine into
separate, labeled test tubes.
2. Add 2 ml of 10 % aqueous sodium hydroxide.
Add 5 drops of 10 % lead acetate solution.
3. Stopper the tubes and shake them. Remove
the stoppers and heat in a boiling water bath
for 5 minutes. Cool and record the results.
7. Sakaguchi
• For detection of the amino acid
containing the guanidinium group
(e.g. arginine).
• In basic conditions, α- naphthol
and sodium hypobromite/chlorite
react with the guanidinium group
to form red orange complexes.
Guanidinium
group
Arginine
Procedure
1. Add 1 ml of 3 N NaOH solution to 1 ml of the
protein solution, followed by addition of 0.5
ml of 0.1 % α- naphthol solution, and a few
drops of 2 % hypobromite solution (NaOBr).
2. The formation of a red color indicates the
presence of a guanidinium group in the
compound under examination.