Qualitative Tests on Amino Acids

A. Solubility test
The solubility of proteins is the proportion of nitrogen present in the protein sample, which is in a soluble
state under specific conditions. Amino acids present in proteins are generally soluble in water but are insoluble in
non-polar organic solvents. The presence of amino and carboxyl groups in amino acids enables the amino acids
and proteins to accept and donate protons to the aqueous solution. The solubility of a protein in a solvent is
influenced by various factors like the molecular size, pH of the medium, hydration of the proteins, and the salting-
in process. In the salting-in technique, the addition of salts generates an interaction between the surface ionic
charges on the proteins and the electrolyte, preventing the aggregation of protein particles. Depending on all these
factors, the solubility of the protein in different solvents might differ.

Positive result: The positive result in the solubility test is demonstrated by the appearance of a clear
solution with no precipitation of cloudiness.
Negative result: The negative result in the solubility test is demonstrated by the appearance of a cloudy
solution with precipitation.
Uses of Solubility Tests of Proteins
• The solubility tests of proteins help to understand the pattern of solubility of proteins which then
helps in the detection and differentiation of such proteins.
• The solubility of proteins provides insight into the solubility of proteins during the development and
testing of new protein composition.
• Solubility testing of proteins enables the selection of proteins for their use in liquid foods and
beverages.
• The solubility of proteins also provides information about the amino acid composition and molecular
weight.
Limitations
• The observation in solubility tests must be carried out carefully as chances of false-negative results
might be prevalent.
 • The test tubes should be allowed to stand for some time to provide some time for solubility.
Theoretical Results on Solubility Test
Samples
Hot distilled water Amino acids will show high degree of solubility
Concentrated HCl Polar and acidic amino acids will show high degree of solubility
NaOH solution Polar and basic amino aids will show high degree of solubility
Chloroform Some amino acids are insoluble to non-polar solvents

B. Ninhydrin Test
A chemical test which is used to check whether a given analyte contains amines or α-amino acids. In this test,
ninhydrin (a chemical compound with the formula C9H6O4; IUPAC name: 2,2-dihydroxyindane-1,3-dione) is added to a
test solution of the analyte. The development of a deep blue or purple color complex indicates the presence of ammonia,
primary amines, or amino acids in the solution.

The amino group of the analyte forms a compound similar to diketohydrin. This compound has a deep blue colour,
often referred to as Ruhemann’s purple. However, proline will form a yellow color complex due to the amino group found
in its aliphatic ring considered as secondary amines.
Theoretical Results
Sample Observations

Distilled water No reaction

Glutamine Purple complex

Tyrosine Purple complex

Valine Purple complex

Proline Yellow complex

Cysteine Purple complex

C. Xanthoproteic test
A test uses a nitration reaction to determine the presence of proteins in a solution. When the sample is
treated with a hot, concentrated nitric acid it reacts with aromatic amino acids such as phenylalanine, tyrosine and
tryptophan and forms a yellow-colored product known as Xanthoprotein. With the addition of strong base such as
NH3 or NaOH, it further changes to deep-orange color. So, this test gives a positive result in those proteins which
contain amino acids that have aromatic rings in their side chains.

The aromatic side chains are nitrated upon the addition of HNO3 acid which gives the yellow color
complex. However, for phenylalanine, there would be no reaction since its benzene ring is a stable form of
aromatic ring.
Theoretical Results

Observations
Samples Addition of HNO3 Addition of NaOH
Phenol gives a yellow color complex gives a yellow-orange color complex
Tyrosine gives a yellow color complex gives a yellow-orange color complex
Glutamine No reaction No reaction
Proline No reaction No reaction
Valine No reaction No reaction
Cysteine No reaction No reaction
D. Millon’s test
A test used to confirm the presence of tyrosine or amino acids containing phenols. Millon’s reagent is the
solution containing mercuric nitrate that is dissolved in nitric acid and is used to identify the presence of phenolic
amino acids such as tyrosine and its derivatives. By mixing the sample with this reagent and heating it gently results
in the formation of reddish-brown color precipitate signaling the presence of tyrosine. Some of the proteins
containing a phenolic hydroxyl group initially form a white precipitate and changes to red when heated which is also
considered as a positive result.
 The phenol group of tyrosine is nitrated by nitric acid. Then, this product forms a complex with mercury (I)
and mercury (II) which gives brick red color to the solution.
Theoretical Results:
Amino Acids Observations
Glutamine No reaction
Tyrosine Brick red to brownish color
Valine No reaction
Proline No reaction
Phenol Brick red to brownish color
E. Sakaguchi Test
A test is positive for the amino acid containing the guanidine group in Arginine. Guanidine group present in the
amino acid reacts with α-Naphthol and alkaline hypobromite to give a red-colored complex.

Arginine reacts with α-naphthol in presence of an oxidizing agent such as bromine water or sodium
hypobromite to give a red colored product.
Theoretical Results:
Samples Observations
Glutamine No reaction
Tyrosine No reaction
Chicken strips Red color complex
 Egg white Red color complex

Qualitative Tests for Proteins

A. Biuret test:
A general test for proteins or compounds having a peptide bond. Biuret is a compound formed by heating
urea to 180° C. When biuret is treated with dilute copper sulfate in alkaline condition, a purple-colored compound
is formed. This is the basis of biuret test widely used for identification of proteins and amino acids.

The copper (II) present in the biuret reagent binds itself to the nitrogen atoms that are present in the protein
peptides. Now the copper (II) undergoes reduction and is converted to copper(I). The reaction between the copper
(II) ions and the nitrogen belonging to the peptide bonds results in the displacement of peptide hydrogens (as long
as the environment is sufficiently alkaline).

Now, four nitrogen atoms donate lone pairs to form coordinate covalent bonds with the cupric ion (illustrated
above), resulting in the formation of a chelate complex. This chelate complex has the ability to absorb light with a
wavelength of 540nm, which imparts a purple color to it.
 Theoretical Result:
Samples Observations

2 ml gelatin solution Dark blue green color

2 ml raw egg white solution purple color
small chicken strips purple color
2 ml of milk purple color

B. Lead Acetate/SulfideTest
The sulfur-containing amino acid such as cysteine, cysteine, and methionine (sulfhydryl/thiol group) reacts
with lead acetate under alkaline conditions to form a brown precipitate. These sulfur-containing amino acids are
degraded in strongly alkaline media to release sulfide ion (S 2-) in the form of H2S (hydrogen sulfide). The sulfide
ions can react with lead (II) acetate to form a brownish-black precipitate also called lead sulfide (PbS).

Theoretical Result:
Observations
Samples + Lead acetate After water bath
2 ml gelatin solution No reaction No reaction
2 ml raw egg white solution Precipitation of Lead salt (white) Black or brown ppt
small chicken strips Precipitation of Lead salt (white) Black or brown ppt
2 ml of milk Precipitation of Lead salt (white) Black or brown ppt

C. Hopkins-Cole test
It is also known as the glyoxylic acid reaction. This test used for detecting the presence of tryptophan in
proteins. The indole group of tryptophan reacts with glyoxylic acid in the presence of cone 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.
 Preparation of the Hopkins-Cole Reagent Reaction

The glyoxylic acid added to the sample combines two tryptophan molecules by acting on the indole ring
of the tryptophan molecules. The condensation product thus formed undergoes dehydration to form a violet-
colored pigment. The color developed in the test is due to the indole group of the tryptophan molecule, which is
converted into a colored compound by oxidation brought about by the aldehyde group of glyoxylic acid. The
H2SO4 added to the reagent helps to stabilize the glyoxylic acid and prevent its decomposition and the release
of carbon dioxide.
Theoretical Results:
Samples Observations
2 ml gelatin solution No reaction
2 ml raw egg white Purple color complex
solution
small chicken strips Purple color complex
2 ml of milk Purple color complex

D. Precipitation of Proteins by Acids

o This experiment is used to precipitate different proteins using strong acid solution. Each protein can be
precipitated at specific acid concentration. The main purpose of protein precipitation is to separate the
protein from the solution either to eliminate interferences or to purify them.
o This method relies on the changes of a solution pH. Every protein has a defined isoelectronic point or pI
value, little changes in a medium pH have an impact on the protein structure. Adding acids to the solution,
lowers the pH and leads to positively charging the protein, due to the proton capture by amino groups. In
 an aqueous solution the hydration sphere surrounding a protein is disrupted; occurred imbalance in
structure leads to precipitation.
o The use of 1m NaCl solution as dissolving medium allows the disruption of hydrogen bonds in the protein
molecules resulting to a change in shape and form.

The addition of a strong acid causes dehydration to a protein structure resulting to removal of charge and
formation of precipitate.
Theoretical Result:
Observations
Samples Addition of Nitric Acid After the water bath
2 ml gelatin solution No reaction No reaction
2 ml raw egg white White and cloudy precipitate coagulation
dissolve in 1M NaCl
solution
2 ml of milk White and cloudy precipitate coagulation
E. Precipitation of Protein by Heavy Metals:
• Heavy metal salts usually contain Hg+2, Pb+2, Ag+1 Tl+1, Cd+2 and other metals with high atomic weights.
Since salts are ionic, they disrupt salt bridges in proteins. The reaction of a heavy metal salt with a protein
usually leads to an insoluble metal protein salt. At pH 7 and above, proteins are usually negatively charged,
the positively charged metal ions neutralize the charges of protein causing precipitation of the protein.
• Precipitation by heavy metals is therefore most effective at neutral to slightly alkaline pH value. NOTE: The
solution must not be too alkaline; otherwise, there is a risk of precipitation of metal hydroxides.
• Addition of NaOH as dissolving medium for protein is needed to increase the alkalinity of protein and
disrupting hydrogen bonds so heavy metals can easily form insoluble salts.
 Metallic ions form a complex with an alkaline protein
Theoretical Result:

Observations
Samples Lead Acetate Silver Nitrate
2 ml gelatin solution No precipitation No precipitation
2 ml raw egg white White precipitate White precipitate
dissolve in 1M NaOH
solution
2 ml of milk (casein White precipitate White or grayish precipitate
dissolve in 1M NaOH
solution)

References:
https://microbenotes.com/solubility-tests-of-proteins/
https://byjus.com/chemistry/ninhydrin
http://www.magdyelnashar.com/new/images/pdf/Practical.Proteins.and.Amino.Acids.Identification.pdf
https://www.onlinebiologynotes.com/biuret-test-principle-requirements-reagents-preparation-procedure-and-result/
http://ecoursesonline.iasri.res.in/mod/page/view.php?id=53516