CLASSSIFICATION

TEST Liebermann Nitroso

CLASS TESTED FOR  Test for Phenols
 Confirmatory Test for Secondary Amines
PRINCIPLE Phenols having a free para-position undergo this reaction.

Both aliphatic and aromatic amines react with nitrous acid (HNO2) to form
N-nitroso derivatives which are liquids, insoluble in the reaction mixture.

Therefore, when a secondary amine reacts with nitrous acid, an oily N-

nitrosamine separates from the reaction mixture
The N-nitrosamine obtained by the reaction of a secondary amine with
HNO2 is heated with phenol in the presence of concentrated sulfuric acid.

The formed p-nitrosophenol undergoes further reaction. [see first figure]

MATERIALS GLASSWARES REAGENTS
Beakers (50-ml) 5% Sodium nitrite (NaNO2)
Test tubes Conc. Sulfuric acid (H2SO4)
Test tube holder 5% Sodium hydroxide (NaOH)
Alcohol lamp Hydrochloric acid
Droppers 5% Sodium nitrite (NaNO2)
Spatula Sodium nitrite (NaNO2)
Aspirator Conc. Sulfuric acid (H2SO4)
Pipet Ether
Stirrer Dilute urea solution
Volumetric flask Anhydrous calcium chloride (CaCl2)
Phenol
5% Sodium hydroxide (NaOH)
SAMPLE The organic compound to be tested should be pure. Purification step is
PREPARATION necessary for compounds with impurities.
PROCEDURE Take the compound (100 mg) in a dry test tube, add sodium nitrite (NaNO2)
(few crystals) and concentrated sulfuric acid (H2SO4) (1 ml), mix well and
heat gently. A blue color is obtained. The solution turns red on dilution with
water (H2O) and blue on basification with a dilute solution of sodium
hydroxide (NaOH).
Treat the solution of secondary amine with HCl with sodium nitrite solution
(NaNO2). Extract the N-nitrosamine with ether. Wash the ether extract with
dilute urea solution to remove HNO2. Then dry it over anhydrous CaCl2.
Filter and evaporate ether (caution, ether is highly flammable). Heat the
residue with phenol (few crystals), cool, add concentrated H2SO4. (For
details of subsequent treatment see “Test for Phenols” above)
POSITIVE TEST It gives green to blue solutions which turned to red or violet, yellow or
brown, respectively, after dilution with water. Alkalization of these
solutions changed the color to green or blue, but also to yellow or brown,
respectively.
COMPLICATIONS  Except for 4-iodophenol and hydroquinone, phenols substituted in the p-
position will not react with either nitrite or p-nitrosophenol. Negative
reactions will be obtained with 2- and 3-nitrophenols, 2- and 3-
hydroxybenzoic acids and 2,6-dichlorophenol, although these phenols have
a free p-position. The sensitivity is much poorer than that of other reactions
for detection of phenols, e.g. the Emerson or Gibbs reactions. The
Liebermann test is often recommended as a generic test for phenols, but its
importance, selectivity and sensitivity seem to have been considerably
overestimated. Application of the reaction for spectrophotometric
determination of phenols is impracticable, because of the very poor
reproducibility, the small yield of reaction product and the inconvenience of
working with concentrated sulfuric acid media. (Fraenkl et al, 1986)
CLEANING UP The obtained mixture of reagents after the test should be stored in waste
bottle properly labelled and be subjected for treatment.
REFERENCES Ahluwalia, V.K., & Dhingra, S. (2004) Comprehensive Practical Organic
Chemistry: Qualitative Analysis. Telangana, India: Universities Press.

Fraenkl, M., Svobodová, D., & Gasparič, J. (1986) A critical investigation

of the liebermann colour test: The formation and behaviour of
phenolindophenol in strong acid media. Microchimica Acta, 90(2), 367–
386.
EDITED BY Diana V. Montecastro
BS Chemistry 4
CLASSSIFICATION
TEST Ninhydrin
CLASS TESTED FOR  Test for Amino Acids
PRINCIPLE In the pH range of 4-8, all α- amino acids react with ninhydrin
(triketohydrindene hydrate), a powerful oxidizing agent to give a purple
colored product (diketohydrin) termed Rhuemann’s purple. All primary
amines and ammonia react similarly but without the liberation of carbon
dioxide. 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.

MATERIALS GLASSWARES REAGENT

Beakers (50-ml) Ninhydrin (2%W/V) in acetone
Test tubes
Test tube holder
Alcohol lamp
Droppers
Spatula
Aspirator
Pipet
Stirrer
Volumetric flask
SAMPLE Dissolve 20 mg of the compound in 1 ml water or appropriate solvent.
PREPARATION
PROCEDURE Heat a small amount of the aqueous solution of the organic compound (20
mg in 1 ml water) with a ninhydrin solution (1 ml) for 15-20 seconds.
POSITIVE TEST The appearance of a blue-violet color indicates the presence of an α- or a β-
amino acid.
COMPLICATIONS Proline, hydroxyproline, o-, m-, and p-amino benzoic acids do not give a
positive test but produce a yellow color instead.
Ammonium salts give a positive test.
Some amines, such as aniline, yield orange to red colors, which is a
negative test.
CLEANING UP The obtained mixture of reagents after the test should be stored in waste
bottle properly labelled and be subjected for treatment.
REFERENCES Ahluwalia, V.K., & Dhingra, S. (2004) Comprehensive Practical Organic
Chemistry: Qualitative Analysis. Telangana, India: Universities Press

vlab.amrita.edu, (2011). Qualitative Analysis of Amino Acid. Retrieved 13

January 2018, from vlab.amrita.edu/?sub=3&brch=63&sim=1094&cnt=1

http://kimiagar2010.blogspot.com, (2010). Detachment and Identify of

Organic Compound. Retrieved 13 January 2018, from
kimiagar2010.blogspot.com/2010/05/procedure-add-about-2-mg-of-sample-
to-1.html
EDITED BY Diana V. Montecastro
BS Chemistry 4