BIURET TEST

General test for intact proteins and protein hydrolates

(Test for peptide linkages: at least tripeptide)
 REAGENT: 2.5 NaOH and 0.1M CuSO4
 PRINCIPLE: Formation of coordination
complex of copper and four nitrogen atoms
 POSITIVE RESULT: pink-violet to blue color
If hydrolysis of the protein is complete, a negative
result will be obtained from Biuret test.

NINHYDRIN TEST
General test for amino acids with a-amino group
 REAGENT: 1,2,3-indanetrione monohydrate
or triketohydrindene hydrate and ethanol
 PRINCIPLE: oxidative decarboxylation and
deamination followed by condensation
 POSITIVE RESULT: blue to blue-violet;
proline: yellow

XANTHOPROTEIC TEST
General test for aromatic amino acids
 REAGENT: conc. Nitric acid and NaOH
 PRINCIPLE: nitration of the aromatic rings via
SEAr
 POSITIVE RESULT: yellow solution/ppt with
conc. HNO3; Orange with excess NaOH

MILLON’S TEST
Detects phenolic ring containing amino acids
 REAGENT: Millon’s rgt. (mercury
containing compound)
 PRINCIPLE: complexation (mercuration
& nitration)
 POSITIVE RESULT: old rose to fleshy red

HOPKINS-COLE TEST
Detects indole group containing amino acid
 REAGENT: Glyoxylic acid (Mg Powder,
Oxalic acid and Acetic acid) and Sulfuric
acid
 PRINCIPLE: reduction of oxalic acid to
glyoxylic acid and acid-catalyzed
condensation of two tryptophans with
glyoxylic acid
 POSITIVE RESULT: pink to violet interface