1. (a) Identification of sugars by Molisch’s test.
Molisch’s test is a general test for the identification of all carbohydrates
(monosaccharide, disaccharide, and polysaccharide) and glycoprotein. In this test conc. sulphuric
acid added to the solution which hydrolyzes the all glycosidic linkage in the sugar molecules
(disaccharide, and polysaccharide) to yield monosaccharide, which in the presence of an acid get
dehydrated to form furfural and its derivatives. This is very reactive, and condenses with α-
naphthol to give a purple or violet colored product.
Prepareed 5% α-naphthol reagent in ethanol. Added 2 to 3 drops of α-naphthol reagent to
2ml of sugar solution in the test tube. Incline the test tube and very gently added few drops of
conc. H2SO4 along the side of test tube. violet colored ring indicates the presence of
carbohydrates in the solution.
Violet rings formed at the junction which indicated the presence of carbohydrates
in the sample.
1. (b)Identification of reducing sugar by using Fehling’s test
Fehling’s test is a specific test for the identification of reducing sugar. When
Fehling’s reagent react with reducing sugar it reduces into brownish-red precipitate
Oxidation of carbonyl group take place which give aldonic acid. Formation Brownish-red
precipitates indicate the presence of reducing sugar in the sample. Fehling’s reagent
consists of two solution Fehling’s solution A (CuSO4) and Fehling’s solution B (KOH+
Na-K tartrate). Mix both solutions before adding the sugar sample.
CuSO4 + 2KOH Cu (OH) 2 + K2SO4 ; Cu (OH) 2 CuO + H2O
Prepared Fehling’s solution A by dissolving 7.0g CuSO4.7 H2O in distilled water, and
then adjusting the total volume to 100ml. For Fehling’s solution B, dissolved 24g of KOH and
34.6 of Na-K tartrate in distilled water and make up the volume to 100ml. mix solution A & B in
1:1 ratio.Added 5-8 drops of sugar samples to 5ml of Fehling’s solution and boil for 2-3 min.
The precipitate of light brown appeared which show the presence of reducing
sugar in the sample.
1. (c) To detect reducing sugar by performing the Osazone test.
When phenyl hydrazine reacts with reducing sugars at a boiling temperature,
osazone is formed. Phenyl hydrazine reacts with carbonyl compound in neutral or slightly
acidic condition to give phenyl hydrazone, which is highly soluble. When hydrazone
reacts with further phenyl hydrazine molecules, the condensation products formed are
insoluble which precipitate out as crystals.
Mixed 2g phenyl hydrazine and 3g sodium acetate and transfered 0.3g of this mixture
separately in six cleans test tubes.Added 5ml of 1% glucose, fructose, maltose, lactose, sucrose,
and distilled water separately into these test tubes.Incubate the tubes in a boiling water bath for
30-45 min.Cool and observed test tubes for the shape of different sugar osazone crystal formed
by using a light microscope having magnification of 10x. For fructose and glucose needle shaped
crystals of osazone can be observed with unaided eye as well as by using microscope. Maltose
and lactose formed flower shaped crystals whereas no osazone formation for sucrose.
Disaccharide, namely lactose and maltose yield osazone crystals whish are soluble in hot water,
and hence, they separate out as crystal only on cooling.
1. (d)Estimation of reducing sugar by performing Benedict’s test
Benedict test is just like Fehling’s test the only difference is that in benedicts test is
carried out weak alkaline condition by the sodium carbonates, whereas the reduction
reaction in Fehling’s test is brought about in strong alkaline condition by using KOH.
The reagent used for Benedict’s test contains CuSO4.7 H2O in alkaline citrate solution.
Reducing sugars can reduce the cupric ions into red color cuprous oxide, and turns
oxidized into sugar acid.
Na2CO3 + 2H2O 2NaOH +H2CO3
2NaOH +CuSO4 Cu (OH) 2+ Na2SO4 ; Cu (OH) 2 CuO + H2O
D-glucose + 2CuO + Heat D-dluconic acid + Cu2O
Dissolved 17.3g sodium citrate and 10g sodium carbonate in 85ml of distilled water..Dissolve
1.73g CuSO4.7 H2O in 10ml distilled water and while stirring, add slowly to the alkaline citrate
solution.Adjust the total volume to 100ml using distilled water.To the 5ml of the reagent, add 8
drops of the sugar solution. Boil 2-3 min and observe the change. Similarly, for the control, add
distilled water to Benedict’s reagent instead of sugar.
the brown precipitate were formed which indicate the presence of reducing sugar in the
1. (e) Detection of Ketose sugars by performing Seliwanoff’s test.
Seliwanoff’s test is a specific test for the detection of ketose sugars namely fructose.
Aldose, however, failed to respond to this test. Ketoses undergo dehydration to give
furfural derivatives, which then condense with resorcinol to form a red colored complex.
Seliwanoff’s test is responded positively by the sucrose as it gets hydrolyzed to give
glucose and fructose due to the action of conc. HCl present in the reagent. Fructose being
a keto sugar gives red colored complex when react with Seliwanoff’s reagent.
Prepare Seliwanoff’s reagent by dissolving 50mg resorcinol in 100ml of 3N HCl.Added 8 drop
of a sugar sample to 5ml of the reagent and boil for exactly 30 sec. keep a parallel control that
contain distilled water instead of a sugar solution.Observe the tube for the presence of a red
colored complex, which indicates the presence of a keto sugar.
Lab manual in biochemistry by Arti Nigam.