Islamic University_ Gaza
Faculty of Health Sciences.
Medical Technology Department.
MEDI 2130
Qualitative
amino acid tests
Objectives:
• General information about amino acids.
• Qualitative tests of amino acids.
Introduction
 Food are divided into three classes :
1- Carbohydrate
Source of energy.
2- Lipid
Principal of energy reserve.
3- Proteins
Energy for growth and cellular maintance.
Amino acid structure
(Building blocks of proteins which linked to peptide bond )
 Each amino acid consists of :
1. Central carbon atoms
2. An amino acid
3. Carboxyl group
4. Side chain (All amino acids found in proteins have this
basic structure, differing only in the structure of the Rgroup or the side chain.)
Classification of amino acids according
source
• Essential amino acids: Humans incapable of forming
requisite and must be Required in diet.
• Non essential amino acids: Not required in diet.
• The simplest, and smallest, amino acid found
in proteins is glycine for which the R-group is
a hydrogen (H).
• Proline It is unique among the 20 proteinforming amino acids in that the amine
nitrogen is bound to not one but two alkyl
groups, thus making it a secondary amine.
• There are 20 natural amino acids that are
found within proteins . All of them are L-α
amino acids.( except glycine, is achiral
carbon) because it has four different groups
bonded to it. ).
Classification of amino acids according to
their (polarity) in water
1- Non-polar (Hydrophobic amino acid) : are amino
acid that contain C,H in their side chain (hate
water, normally buried inside the protein core)
2- Uncharged polar.
3-polar amino acids: amino acid that contain in their
side chain O,N and they can dissolve in water (
like dissolve like ) hydrophilic (love water),tend to
found on surface
A-Basic polar (positively charged).
B- Acidic polar (negatively charged).
Note :
Polar amino acids are more soluble
in water than non-polar.
At acidic pH, the carboxyl
group is protonated
and the amino acid is
in the cationic form
At
neutral
pH,
the
carboxyl
group
is
deprotonated but the
amino
group
is
protonated. The net
charge is zero; such
ions
are
called
Zwitterions
At alkaline pH, the amino
group is neutral –NH2
and the amino acid is
in the anionic form.
 Iso electric point (PI) : It is the pH
value at which concentration of anionic
and cationic groups are equal (i.e. the
net charge of this molecule equals
zero). Each amino acid have a different
PI
Qualitative test for amino acids
• There number of test to detect the presence
of amino acid
• This is largely depend on the natural of side
chain
Amino acids analysis
A. Solubility test.
B. The presence of proteins in a solution is often
detected by general tests, such as biuret or
specific tests that depend on the presence of a
specific amino acid.
1. Ninhydrin test: for α-L amino acids
2. Xanthoproteic test: for Aromatic amino acids
3. Lead sulfite test: detection of amino acids
containing sulfhydral group (- SH)
4. Millon's test: for amino acids containing
hydroxy phenyl group
5. Sakaguchi Test.
6. Hopkins-Cole (Glyoxylic Acid Reaction)
7. biuret test
Solubility test.
• Objective:
To investigate the solubility of selected amino acid in various
solutions
-Principle:
• Polar amino acids are soluble in polar solvent, and vice versa.
• The solubilities of amino acids in water are highly
variable.
• The solubility of amino acids and proteins is
largely dependent on the solution pH.
• The structural changes in an amino acid or protein
that take place at different pH values alter the relative
solubility of the molecule.
• In acidic solutions, both amino and carboxylic
groups are protonated.
• In basic solutions, both groups are deprotonated.
Amino acids are essentially soluble in water.
Their solubilities in water, dilute alkali and dilute
acid vary from one compound to the other depending on
the structure of their side chains.
Solubility of Amino Acids
•
You will already have dissolved a number of amino acids in
water for the previous experiments. In this test try to dissolve
tyrosine and cystine in water and note the result.
Procedure
•
Put 2.0 mL (34 drops) of water in a clean test tube. Take a
small portion of the contents of the vial marked “tyrosine” on
a spatula and add it to the water. Shake well. Note if the
tyrosine has dissolved. If it has not, then heat the test tube
and note if the tyrosine dissolves. Is tyrosine readily
soluble/sparingly soluble/insoluble in water? Repeat this
procedure with cystine.
Ninhydrin
Principle:
• 1.Ninhydrin (triketohydrindene hydrate) degrades amino acids into
aldehydes (on pH range 4), ammonia and CO2 though a series of
reactions.
• 2. The reducon product obtained from ninhydrin (hydrindantin) then
reacts with NH3 and excess ninhydrin to produce an intensely
blue or purple pigment, sometimes called ruhemann's
purple(diketohydrin).
• This reaction provides an extremely sensitive test for amino acids.
• alpha-amino acid + 2 ninhydrin ---> CO2 + aldehyde + final complex(BlUE) +
3H2O
• The imino acids proline and hydroxyproline also react with ninhydrin, but
they give a yellow colored complex instead of a purple one.
• Besides amino acids, other complex structures such as peptides, peptones
and proteins also react positively when subjected to the ninhydrin
reaction.
Ninhydrin_ethanol reagent is flammable. Toxic, and irritant. Keep
away from Bunsen burner flames prevent eye, skin, clothing contact.
Avoid inhaling the vapors or ingesting the reagent.
With all amino acid will give purple or deep blue with
exception Proline gives yellow not violet (why)?
The secondary amino group (imino group) of
proline residues is held in rigid conformation
that reduces the structural flixibility of
polypeptide regions containing proline. Proline
does not give ninhydrin reaction as this
reagent requires free alpha amino group but
proline have imino group
• Procedure
• To 1 mL solution add 5 drops of 0.5% ninhydrine
solution
• Boil over a water bath for 2 min.
• Allow to cool and observe the blue color formed.
Results:
Xanthoproteic test :
Objective:
•
to differentiate between aromatic amino acids which give positive results [yellow
color] and other amino acids.
Principle:
•
Concentrated nitric acid react with aromatic nucleus present in the amino acid
side chain [nitration reaction] giving the solution yellow color.
Note:
•
•
Amino acids tyrosine and tryptophan contain activated benzene rings [aromatic
nucleus] which are easily nitrated to yellow colored compounds.
The aromatic ring of phenyl alanine dose not react readily with nitric acid despite
it contains a benzene ring, but it is not activated, therefore it will not react
Procedure
• To 2 mL amino acid solution in a
boiling test tube, add equal
volume of concentrated HNO3.
• Heat over a flame for 2 min and
observe the color.
• Now COOL THOROUGHLY and
CAUTIOSLY run in sufficient 3ml
NaOH (why)
• Observe the color of the nitro
derivativative
of
aromatic
nucleus.
Hopkins-Cole (Glyoxylic Acid
Reaction)
Aim: Specific for tryptophan (the only amino acid containing indole
group)
Principle:
• 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.
Procedure
1. In a test tube, add to 2 ml of the
solution 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
4.Millon's test
Objective:
• This test is specific for tyrosine. Because it is the only
amino acid containing a phenol group.
• Note: phenol group, a hydroxyl group attached to
benzene ring.
Millon’s reagent contains mercury and
HNO3 and is very toxic, corrosive a
strong oxidant, an irritant, and can
cause burns
Principle:
The phenol group of tyrosine is first nitrated by nitric acid in
the test solution. Then the nitrated tyrosine complexes
mercury ions in the solution to form a brick-red , appearance
of red color is positive test.
Note:
all phenols (compound having benzene ring and OH attached
to it) give positive results in Millon’s test.
Procedure
• To 2 ml of protein solution in a test
tube, add 3 drops of Millon’s reagent.
• Mix well and heat directly on a small
flame. BWB 5 min
• A white ppt is formed with albumin
and casein (but not gelatin);
• the ppt gradually turns into brick red.
Sakaguchi Test.
• Objective:
detection of amino acid containing gauanidium
group. In other words it’s a test for, arginine.
• Principle
In alkaline solution, arginine react with αnaphthol and sodium hypobromite /chlorite as
an oxidize agent, to form red complexes as a
positive result.
• 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 %
sodium hypobromite solution
(NaOBr).
• 2. The formation of a red color
indicates the presence of a
guanidinium group in the
compound under examination.
Lead Sulfite Test
• Objective:
• This test specific for–SH [sulfhydral group ]
containing amino acid (Cysteine).
• Principle:
• - Sulfur in cystine, is converted to sodium
sulfide by boiling with 10% NaOH.
• - The Na2S can be detected by the
precipitation of PbS (lead sulfide) from an
alkaline solution.
• when adding lead acetate Pb (CH3COO)2.
Methionine and cysteine contains sulfur group
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.
Biuret test
 Biuret structure:
it is result of condensation of two molecule of urea
• Principle:
• The biuret reagent (copper sulfate in a strong base)
reacts with peptide bonds in proteins to form a blue
to violet complex known as the “biuret complex”.
• This
color
change
is
dependent
on
the number of peptide bonds in the solution, so the
more protein, the more intense the change.
• The NaOH is there to raise the pH of the solution
to alkaline levels; the crucial component is
the copper II ion (Cu2+) from the CuSO4.
• When
peptide
bonds
are
present
in
this alkaline solution, the Cu2+ions will form a
coordination complex with 4 nitrogen atoms from
peptide bonds.
• N.B. Two peptide bonds at least are required for the
formation of this complex.
A chelate is a chemical compound composed of a metal ion and a chelating
agent. A chelating agent is a substance whose molecules can form several
bonds to a single metal ion. In other words, a chelating agent is a
multidentate ligand.
Procedure
• To 2 ml of protein solution in a test tube, add
4ml of reagent incubation 30 min
• Result :
Observations
Interpretation
No change ( solution remains
blue )
The solution turns from blue to
violet( purple)
The solution turns from blue to
pink
Proteins are not
present
Proteins are present
Peptides are present (
Peptides or peptones
are short chains of
amino acid residues)