Biuret test
Principle of Biuret test:
Biuret test is a general test for 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.
This test is given by compounds containing two or more peptide bond (CO-NH group). Since
all proteins and peptides possessing at least two peptide linkage ie. tripeptide gives positive
biuret test.
The principle of biuret test is conveniently used to detect the presence of proteins in
biological fluids.
Alkaline CuSO4 reacts with compounds containing two or more peptide bonds to give a
violet colored product which is due to formation of co-ordination complex of cupric ions with
un-shared electron pairs of peptide nitrogen and O2 of water.
Biuret Test Uses
It can be used to detect the amount of protein in the urine.
Biuret reaction with protein is applicable to the quantitative determination of total protein by
spectrophotometric analysis.
The biuret test uses an alkaline mixture, or reagent, composed of potassium hydroxide and copper
sulfate. The normal color of biuret reagent is blue. The reagent turns violet in the presence of peptide
bonds -- the chemical bonds that hold amino acids together.
Biuret reagents:
Copper sulfate (CuSO40
Sodium hydroxide (NaOH)
Sodium potassium tartarate (commonly known as Rochelle salt)
Ninhydrin Test: Principle, Requirements,
Procedure and Result
What is the Ninhydrin Test?
The ninhydrin test is 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 colour indicates the presence of ammonia, primary/secondary amines, or amino acids in the
analyte.Ninhydrin Test Principle
The amino group belonging to a free amino acid undergoes a chemical reaction with ninhydrin, which
behaves as an oxidizing agent. When exposed to ninhydrin, the amino acid undergoes oxidative
deamination, resulting in the liberation of CO2, NH3, and an aldehyde along with hydrindantin (which is
a reduced form of ninhydrin).
Now, the ammonia goes on to react with another ninhydrin molecule to form diketohydrin (which is
also known as Ruhemann’s complex). This complex is responsible for the deep blue colour. When the
analyte contains Imino-acids like proline, a yellow coloured complex is formed. When asparagine is used,
the colour of the resulting complex is brown.
/For ammonia, primary/secondary amines, and amino acids, deep purple colour is obtained.
/For hydroxyproline and proline, a yellow colour is obtained.
/For asparagine, brown colour is obtained.
If no colour change is observed, the analyte does not contain amino acids, amines, or ammonia.
Adamkiewicz reaction (Hopkin’s-cole
test): Objective, Principle, Reagents,
Procedure and Result
Objective:
to detect amino acid tryptophan present in protein
It is also known as the glyoxylic acid reaction. This test used for detecting the presence of tryptophan in
proteins.
Principle:
The indole group of tryptophan reacts with glyoxylic acid in the presence of cone H2S04 to give a purple
colour. 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.
Positive test: A purple ring appears between the two layers due to presence of tryptophan.
Negative test: No appearance of a purple ring between the two layers due to absence of tryptophan.
Sakaguchi test: Objective, Principle,
Reagents, Procedure and Result
Objective:
to detect amino acid gauanidium group [R-NH-C= (NH2)2+-NH2]. i.e arginine
Principle of Sakaguchi test:
This test is specific for arginine because this reaction is given by guanidinium compound. The arginine
reacts with α – napththol and an oxidizing agent such as bromine water or sodium hypochlorite/sodium
hypobromite to give a red colored product. The other guanidinium containing compounds other than
amino acid also give this reaction.
Reagents:
test solution: 1 % arginine, 1 % glycine, 5 % egg white (albumin)
1 % α naphthol in alcohol
Sodium hypochlorite
1 % urea solution
Dry test tubes
Pipettes
Procedure of Sakaguchi test
Take 1ml test solution in dry test tube.
Similarly, take 1ml distilled water in another test tube as control.
Add 2 drops of α naphthol and mix well.
Now add 2ml sodium hypochlorite to all test tubes.
Immediately add 1ml of urea solution to establish the red complex formed.
Result interpretation:
Positive sakaguchi test: Red color ( Arginine)
Negative sakaguchi test: no red color ( glycine, albumin)
Xanthoproteic test: Objective, Principle,
Reagents, Procedure and Result
Objective:
this test is used to differentiate aromatic amino acids which give positive result from other
amino acids
Principle:
Xanthoproteic test is used to detect amino acids containing an aromatic nucleus (tyrosine, tryptophan
and phenylalanine) in a protein solution which gives yellow color nitro derivatives on heating with conc.
HNO3. The aromatic benzene ring undergoes nitration to give yellow colored product. Phenylalanine
gives negative or weakly positive reaction though this amino acid contains aromatic nucleus because it is
difficult to nitrate under normal condition. On adding alkali to these nitro derivative salts, the color
change for yellow to orange.
Reagents:
test solution: 1 % tyrosine, 1 % tryptophan, 1 % phenylalanine, 5 % egg white (albumin).
Nitric acid
40 % NaOH
