Test Principle behind the test Expected positive result of the test

The test involves a reaction between an amino group and ninhydrin, an

Ninhydrin Test oxidizing agent. Amino acids undergo oxidative deamination, releasing
Deep blue/ violet colour indicates the presence of
ammonia, CO2, and reduced ninhydrin (hydrindantin). This reaction amino acids
produces a blue substance called Ruhemann's complex.
Biuret Test The Biuret test is used to find out if a deep blue/purple color
a sample contains any proteins. The test is based
on the hypothesis that the peptide bonds in
proteins can form a compound with copper ions in
an alkaline solution, changing the color from blue
to violet.
Xanthoproteic Test The reaction occurs due to nitric acid forming The appearance of a dark yellow or orange-colored
nitro modifications in amino acids like tyrosine solution represents a positive test.
and tryptophan, but phenylalanine is not affected.
Million’s Test Tyrosine when reacted with ormation of a red or pink colored
acidified mercuric sulphate solution gives yellow precipitate.
precipitate of mercury-amino acid complex. On
addition of sodioum nitrate solution and heating,
the yellow complex of mercury-amino acid
complex converts to mercury phenolate which is in
red color.
Hopkins-Cole Reaction The test is based on the principle that the layering of A positive result is represented by the formation
concentrated sulfuric acid over a mixture of tryptophan-
containing proteins with the Hopkin's Cole reagent results in
of a purple-colored ring at the junction of two
the formation of a violet ring at the interface. layers. This indicates the presence of tryptophan-
containing proteins.
Bromine Water Test Alkene groups react with bromine water in the yellow to colourless.
dark condition and undergo an addition reaction to
give a decolourised solution.
Pauly Reaction Tyrosine or histidine can only be found using this Red presence of histidine and tyrosine
test. Sulphanilic acid is dissolved in hydrochloric
acid and used as the test reagent. A diazonium salt
is produced when sulphanilic acid is diazotized in
the presence of sodium nitrite and hydrochloric
acid. In an alkaline media, the diazonium salt
produced interacts with either tyrosine or histidine
to produce a red chromogen (azo dye).
 Lead Acetate Reaction The sulfur-containing amino acid such as cysteine, Formation of black precipitate indicate the
cysteine, and methionine (sulfhydryl/thiol group) presence of sulfur-containing amino acid
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 (S2-) in the
form of H2S (hydrogen sulfide). The sulfide ions
can react with lead (II) acetate to form a brownish-
black precipitate.
Nitroprusside Reaction Sodium nitroprusside reacts with compounds Red color
containing sulphahydryl groups produce an
intensely red but somewhat unstable color.
Sakaguchi Reaction Arginine and other guanidyl derivatives Red color indicates the presence of an arginine or
(glycocyamine, methylgyanidine etc) react with guanidinium compound.
hypo bromide and alpha napthol to give a red
colored product.
Schiff’s Test . A positive test is indicated by a change from pale
yellow (colourless) to deep red-violet
Folin’s McCarthy Sullivan Test Imino acids such as Proline and hydroxyproline Red color
condense with isatin reagent under alkaline
condition to yield blue colored adduct. Addition to
sodium nitroprusside[Na2Fe(CN)5NO] to an
alkaline solution of methionine followed by the
acidification of the reaction yields a red colour.
Heat Denaturation Heat coagulation alters protein structure by positive outcome of the heat coagulation test can
changing temperature and pH, causing denature in be manifested by the creation an extremely dense
acidic environments. Coulometric proteins form coagulum on the top of the mixture. The lower
uncoiling masses, with weight varying depending portion of the solution functions as a buffer.
on particle size and protein level. Chlorophenol
red and acetic acids aid in breaking peptide bonds.
Isolelectric Precipitation Isoelectric precipitation occurs when proteins' positively charged at low pH and negatively
solubility decreases at acidic pHs, forming charged at high pH.
aggregates that precipitate and can be separated.
Concentrated Mineral Acids
Organic Solvents
Salts of Heavy Metals Heavy metals (e.g. Hg2+, Pb2+, Cu2+) are high
molecular weight cations. The positive charge of
these cations counteracts the negative charge of
the carboxylate group in proteins giving a
precipitate
Alkaloidal Reagents Alkaloidal reagents (e.g. tannate &
trichloroacetate) are high molecular weight anions.
The negative charge of these anions counteracts
the positive charge of the amino group in proteins
giving a precipitate.
Salting Out Salting out takes place because the salt ions break the
hydrogen bonds that are stabilizing the individual protein
molecules. This in turn causes the aggregation of the
protein molecules, which leads to precipitation.
To 1 ml of protein solution in a test tube, add 1
drop of lead acetate; a white ppt is obtained. 2-
To 1 ml of protein solution in a test tube, add 1
drop of 10% copper sulfate; a blue ppt is
obtained.
2- To 1 ml of protein solution in a test tube, add 1
ml of trichloroacetic acid (TCA); a white ppt is
obtained.
Half Saturation
 The formation of a white precipitate in the
solution under half-saturation indicates a
positive result. This result confirms the
presence of globulin in the solution.
Full Saturation
 The formation of a white precipitate in the
solution under full saturation indicates a
positive result. This result confirms the
presence of albumin in the solution.