GROUP II
EXPERIMENT
NOTE: If Nitrogen is present carry out the tests for Amides and if Nitrogen is absent carry out the tests for Carbohydrates
TESTS FOR
CARBOHYDRATES
MOLISCH TEST
0.1g sample dissolved in 1ml water +
two dropsMolisch Reagent. Mix
thoroughly. + Add carefully 1ml
Conc.H2SO4 along the side of the test
tube
FEHLING’S TEST
Mix 1ml of Fehling’s A and 1ml of
Fehling’s B solutions in a test tube +
1ml sample solution. Heat on a boiling
water bath for five minutes
OBSERVATION INFERENCE
Reddish Violet ring at the junction
No Reddish Violet ring at junction
Reddish Brown precipitate
No precipitate
Carbohydrate present
Carbohydrate absent
Reducing sugar
(may be glucose, fructose,
mannose, lactose etc)
Non-reducing sugar
(may be sucrose, starch,
cellulose etc)
PRINCIPLE AND REACTIONS
Molisch Test is a general test for carbohydrates. Most carbohydrates are
dehydrated with concentrated sulfuric acid to form furfural These furfurals react
with the α-naphthol in the test reagent to give apurple product.
Fehling’s solution (alkaline cupric tartrate complex)
Fehling’s reagent is freshly prepared bymixing equal amounts of Fehling’s
solution A and Fehling’ssolution B. Fehling’s reagent deteriorates on
keeping,whileFehling’ssolutions A and B are quite stable. Fehling’s solution A
is anaqueous copper sulphate solution while Fehling’s solution B isan alkaline
solution of sodium potassium tartarate (Rochelle’s salt).The reagent contains
Cu2+ion complexed with tartarate ions. Thestructure of the complex is given
below. Complex formation decreases the cupric ion concentrationbelow that
necessary for precipitation of cupric hydroxide.
Monosaccharides and those disaccharides that have a potential aldehyde group
(ex: maltose) will reduce Fehling’s solution to produce a red precipitate of
copper(I) oxide. Disaccharideswhichdo
notcontainpotentialaldehydegroups(e.g.,sucrose)andalsopolysaccharides(e.g.,sta
rchandcellulose)donotreduceFehling’s solution.
BENEDICT’S TEST
1ml Sample solution in water + 2ml
Benedict’s Reagent. Heat on a boiling
water bath for five minutes.
TOLLENS TEST
0.1g sample + 2ml Freshly prepared
Tollens Reagent. Warm the test tube in a
warm water bath for five minutes.
OSAZONE TEST
1ml Sample solution in water + 2ml
Phenylhydrazinereagent. Heat on a
boiling water bath for ten minutes.
Reddish Brown precipitate
No precipitate
Silver mirror on walls of the test
tube or a grey black precipitate
No Silver mirror on walls of the
test tube or a grey black precipitate
Yellow Crystalline Precipitate
Benedict modified the original Fehling’s test by using a singlesolution which is
more convenient for the test. Benedict’s solutionis more stable than Fehling’s
Reducing sugar reagent and can be stored for a longtime. It is an alkaline solution containing a
(may be glucose, fructose, mixture of coppersulphate and sodium citrate (2Na3C6H5O7.11H2O).
mannose, lactose etc)
In Benedict test also, Cu2+ions are reduced to Cu+ions in thesame manner as in
the case of Fehling’s reagent.
Non-reducing sugar
(may be sucrose, starch, Monosaccharides and those disaccharides that have a potential aldehyde group
cellulose etc) (ex: maltose) will reduce Benedict’s solution to produce a red precipitate of
copper(I) oxide. Disaccharideswhichdo
notcontainpotentialaldehydegroups(e.g.,sucrose)andalsopolysaccharides(e.g.,sta
rchandcellulose)donotreduce Benedict’s solution
Reducing sugar Tollen’s reagent is an alkaline solution of silver cationcomplexedwith ammonia,
(may be glucose, fructose,
mannose, lactose etc) Monosaccharides and those disaccharides that have a potential aldehyde group
(ex: maltose) will reduce Tollens reagent to give a precipitate ofsilver metal.
Disaccharideswhichdo
notcontainpotentialaldehydegroups(e.g.,sucrose)andalsopolysaccharides(e.g.,sta
rchandcellulose)donotreduceTollens reagent.
Non-reducing sugar
(may be sucrose, starch,
cellulose etc)
Carbohydrates react with excess Phenylhydrazine to give phenylhydrazone
which is then oxidized to give osazones. An osazone can be isolated as a
Reducing sugar derivative and its melting point determined. Glucose,fructose and mannose give
(may be glucose, fructose, identical osazones.Osazones also have characteristic crystalline appearance.
mannose, lactose etc)
 EXPERIMENT
BARFOED’S TEST
1ml Sample solution in water + 2ml
Barfoed’s Reagent. Heat on a boiling
water bath for five minutes.
SELIWANOFF’S TEST
1ml Sample solution in water + 2ml
Seliwanoff’s Reagent. Heat on a boiling
water bath for five minutes
BIAL’S TEST
1ml Sample solution in water + 2ml
Bial’s Reagent. Boil on a Bunsen burner
flame
IODINE TEST
1ml Sample solution in water + 2 drops
Iodine solution
OBSERVATION
Red precipitate formed within two
minutes
Red precipitate formed after two
minutes
Dark Red colour or precipitate
within five minutes
No change in colour
Blue green colour
Bluish Black colour
INFERENCE PRINCIPLE AND REACTIONS
Barfoed’s test distinguishes reducing monosaccharides and reducing
disaccharides bya difference in the rate of reaction. The reagent consists of
Monosaccharide copper(II) ions, likeBenedict’s reagent. In this test, however, Barfoed’s reagent
(Glucose, Fructose, reacts with reducing monosaccharides to produce copper(II) oxide faster than
Galactoseetc) with reducing disaccharides.
Disaccharide or
Oligosaccharide
(Sucrose, Lactose, Maltose
etc)
Seliwanoff’s test depends on the relative rates of dehydration of
carbohydrates.A ketohexose reacts rapidly by equation 2 to give 5-
Ketose hydroxymethylfurfural, whereasan aldohexose reacts more slowly by equation 3
to give the same product. Once 5-hydroxy-methylfurfural is produced, it reacts
with resorcinol to give a dark redcondensation product.
Aldose
Bial’s test is used to differentiate pentose sugars from hexose sugars. Pentose
sugars yield furfural on dehydration in acidic solution. Furfural reacts with
Pentose orcinoland ferric chloride to give a blue-green condensation product. Hexose
sugars give5-hydroxy-methylfurfural, which reacts with the reagent to yield
colors such asgreen, brown, and reddish-brown.
Polysaccharide (Starch) Starch forms a typical blue color with iodine. This color is due to the absorption
ofiodine into the amylose molecules present in starch
 EXPERIMENT OBSERVATION INFERENCE PRINCIPLE AND REACTIONS

TESTS FOR AMIDES

TESTS FOR AMIDES
AMMONIA EVOLUTION TEST
Sample + 2ml dil.NaOH. Boil and
expose a wet red litmus paper to the
vapors.
A primary amide can be hydrolysed by NaOH to give salt of carboxylic acid
and ammonia gas which turns red litmus paper blue.
Red litmus turns blue Amide

HYDROXAMIC ACID TEST Amides combine with hydroxylamine to give hydroxamicacid . The solution is
then treated with ferric chloride to produce the ferric hydroxamate complex,
which has a characteristic burgundy or magenta color.
Sample + 1ml Hydroxylamine
Magenta color Amide
hydrochloride solution in ethanol
+ 1ml dil.NaOH. Boil, Cool and
acidify with 2ml dil.HCl. Then
add 2 drops neutral ferric chloride
solution
When aliphatic diamide (ex: Urea) is heated at a temperature above its melting
BIURET TEST FOR UREA point, ammonia is evolved and a crystalline compound called biuret is formed.
In a dry test tube 0.2g sample. Gently Purple or violet colour Urea present This biuret
uret in alkaline medium gives a violet colour with a drop of copper
heat until the sample just melts then sulphate solution.
solidifies. Ammonia gas is evolved and a
white solid remains which is known as
Biuret. Dissolve in 1ml warm water and
1ml dil.NaOH. Cool and add two drops
copper sulphate solution

UREA NITRATE TEST

2ml sample solution in water + 1ml Crystalline precipitate Urea
Conc.HNO3