Test for CARBOHYDRATES/FATS/PROTEINS/

EXPERIMENT : To test the given food stuff for the presence of

carbohydrates/proteins/fat .
S NO EXPERIMENT OBSERVATION INFERENCE
1 Molisch’s test : 5 drops of The formation of a confirms the
given food stuff + Add 2-3 purple ring at the presence of
drops of alcoholic solution of interface of the two carbohydrates.
1% 1-naphthol-[ Molisch layers
reagent] then pour 2 mL conc.
H2SO4 down the sides of the
test-tube so that it forms a
separate layer at the bottom of
the test tube.
2 Fehling’s test [ Mix 1 mL The formation of a the presence of
each of Fehling’s solutions A green ,yellow & finally reducing
and B in a test tube and add orange-red sugarconfirmed
the mixture to test tube precipitate of Cu2O
containing given food stuff . indicates the
Heat the content of the test presence of reducing
tube on a water bath. sugar.

RCHO + 2 Cu2+ + 5OH– -----→ RCOO– + 3H2O ++ Cu2O (orange-red precipitate)

D-Maltose + Cu2+(tartarate) Na OH (aq) D-Maltobionic acid + Cu2O(orange-red ppt)
Sucrose (disaccharide)+ Fehling's solution  No reaction
3 Benedict test Add 1 mL of The formation of a the presence of
Benedict’s reagent to test orange-red reducing
tube containing given food precipitate due to the sugarconfirmed.
stuff . Heat the content of the formation of copper
test tube on a water bath and (I) oxide indicates
heat the mixture to boiling in a
water bath for 2 minutes.
RCHO + 2 Cu2+ + 5OH– -----→ RCOO– + 3H2O + Cu2O (orange-red precipitate)
D-Glucose + Cu2+ (citrate) +Na2CO3 (aq) D-Gluconic acid + Cu2O (orange-red ppt )
4. Tollen’s reagent Test: to test Formation of silver presence of reducing
tube containing given food mirror on the walls of sugar confirmed
stuff Add tollen’s reagent to the test tube
the sugar solution contained in
a test tube and warm the
reaction mixture on a water
bath.
shows
RCHO + 2 [Ag (NH3)2]+ + 2OH–→ 2Ag + RCOONH4+ H2O + 3NH3

Result: The given foodstuff was found to contain carbohydrate.

MOLISCH’S REAGENT : [alcoholic solution of 1% 1-naphthol ]
On adding concentrated sulphuric acid to the aqueous solution of carbohydrate
containing alcoholic solution of 1-naphthol, a deep violet colour appears at the junction
of the two liquids. Concentrated sulphuric acid hydrolyses glycosidic bonds of
carbohydrate to give monosaccharides which are dehydrated to an aldehyde known as
furfural which undergoes reaction with 1–naphthol to give a unstable condensation
product of deep violet colour. This test may be given by some other organic compounds
also. The following reaction takes place :

A. Fehling’s test and Benedict test

Black copper (II) oxide is formed on heating a suspension of copper hydroxide in
alkaline solution.
Cu(OH)2 ----------→ CuO + H2O
Copper (II) oxide
(black)
If some reducing agent is present in the reaction medium, then orange-red copper (I)
oxide is precipated.
2Cu(OH)2 -------→ Cu2O + 2H2O + [O]
Copper (I) oxide
(Orange-red)

Reducing sugars contain aldehydic group or α-hydroxy ketonic group, therefore in

alkaline medium reduce Cu2+ ions. But if the reaction is carried out directly in the
presence of an alkali then,copper (II) hydroxide gets precipitated. To overcome this
problem,copper (II) ions are complexed with tartarate ions (Fehling’s reagent) or citrate
ions (Benedict’s solution). Both the complex ions are soluble in alkaline medium and
yield Cu2+ ions in such a low concentration that solubility product of cupric hydroxide is
not reached.
Reducing sugars react with Fehling’s reagent according to the following reaction:
RCHO + 2 Cu2+ + 5OH– -----→ RCOO– + Cu2O + 3H2O
(orange-red precipitate)
The discharge of blue colour due to Cu2+ ions and appearance of
orange-red precipitate of Cu2O, indicates the reducing property of
sugars.
D-Glucose + Cu2+ (citrate) +Na2CO3 ( aq) D-Gluconic acid +
Cu2O
(orange-red precipitate)
D-Maltose + Cu2+(tartarate) Na OH (a q) D-Maltobionic acid +
Cu2O
(orange-red precipitate)
Sucrose (disaccharide)+ Fehling's solution  No reaction
Sometimes, the cuprous precipitate comes down as yellow cuprous hydroxide,
but on warming this is converted to orange red copper (I) oxide.
In some cases, this reaction may be used as a quantitative analytical process for
the determination of reducing sugars in blood and urine etc.
1.All monosaccharides are reducing sugars.
2.Disaccharides which contain a free hemi-acetal group ( ) are also mild reducing
sugars. Most naturally occurring disaccharides are reducing sugars (sucrose is an
exception).

B. Tollen’s test
Preparation of Tollen’s reagent: Prepare Tollen’s reagent by adding sodium
hydroxide solution dropwise to 1 mL aqueous silver nitrate solution to get the precipitate
of silver oxide. Now add ammonium hydroxide solution while shaking the mixture so that
initially formed silver oxide precipitate dissolves.

Caution!Never heat the test tube on direct flame as it may cause explosion.

Tollen’s reagent is ammoniacal solution of silver nitrate. A reducing sugar, reduces

silver ion to metallic silver which gets deposited on the inner surface of the test tube in
the form of silver mirror.

EXPERIMENT : To test the given food stuff for the presence of

carbohydrates/proteins/fat .
Observations:

S NO EXPERIMENT OBSERVATION INFERENCE

1 Molisch’s test : 5 drops of The formation of a confirms the
given food stuff + Add 2-3 purple ring at the presence of
drops of alcoholic solution of interface of the two carbohydrates.
1% 1-naphthol-[ Molisch layers
reagent] then pour 2 mL conc.
H2SO4 down the sides of the
test-tube so that it forms a
 separate layer at the bottom of
the test tube.
2 Iodine test :Make a The appearance of the presence of
suspension of (0.1 g) starch in blue colour due to the starch. Confirmed
5 mL water and pour it in 10 formation of a
mL boiling water to get an complex known as
aqueous colloidal solution. To starch iodide complex
this .
add a few drops of aqueous
iodine solution.

Result: Result: The given foodstuff was found to contain starch.

EXPERIMENT : To test the given food stuff for the presence of

carbohydrates/proteins/fat .
Observations:

S EXPERIMENT OBSERVATION INFERENCE

NO
1 Biuret test for peptide bonds Appearance of violet protein confirmed
Take 2-3 mL of the solution of colour due to the
given food stuff and add about formation of a
2 mL of 10% sodium hydroxide complex
solution to it. Add a few drops of species of Cu2+ ions
copper reagent and warm the with - CONH – group
mixture for about 5 minutes.
2 Ninhydrin reaction : Take 2-mL of Appearance of protein confirmed .
an aqueous solution of given food blue colour indicates
stuff in a test tube.Add 3-drops of the presence of
ninhydrin solution to it and heat. protein.

3 XANTHOPROTIC TEST : Take 1 A yellow colour protein confirmed

 mL of an aqueous solution of given appears. Cool
food stuff in a test tube and add a the test tube under
few drops of concentrated nitric running tap and add a
acid. Heat the reaction mixture for a few drops of 10M
few minutes on a Bunsen flame. sodium hydroxide
solution,

Result: The given foodstuff was found to contain protein.

I. Biuret test for peptide bonds: Alkaline copper sulphate reacts with compounds
containing two or
more peptide bonds to form complexes of violet colour.The name of the test comes from
the name of the compound, biuret, which gives this test. The reaction is not absolutely
specific for peptide bond because many compounds containing two carbonyl groups
linked through nitrogen or carbon atoms give a positive result.

II . Ninhydrin reaction
Ninhydrin is a powerful oxidizing agent and reacts with proteins to give a blue-violet
compound called Rhumann’s purple
.Note : Ammonia, primary amines, amoniacids and peptides also react with ninhydrin.

III. Xanthoproteic reaction

Aromatic groups of either the free aminoacid or protein, undergo nitration on heating
with concentrated nitric acid. The salts of these derivatives are orange in colour.
 EXPERIMENT : To test the given food stuff for the presence of
carbohydrates/proteins/fat .
Observations:
S NO EXPERIMENT OBSERVATION INFERENCE
1 Transleuscent spot test: put The change from Confirms the
a drop of oil/ fat on the filter opaque to presence of fat.
paper & warm gently transleuscent spot
which grows in size
on heating
Test for Lipids :(“brown paper bag test” for the presence of lipids)
 The sample of on the left is the result of the lipid test on tap water (control)
 The sample on the right is a positive test for the presence of lipids tested on vegetable
oil (note the translucent spot on the paper)
2 Emulsion test: Add 2cm3 of A cloudy white Confirms the
give food stuff in a china dish suspension. presence of fat.
containing 2cm3of absolute
ethanol. Dissolve the lipid by
shaking vigorously. Add an
equal amount of cold water..
Basis of test: Lipids are immiscible with water. Adding water to a solution of the lipid in
alcohol results in emulsion of tiny droplets in the water which reflect light and give a
white , opalescent appearance.
3. ACROLEIN TEST. Add a few A pungent smell due Confirms the
crystals (0.5g) of dry potassium to acrolein formation . presence of fat.
hydrogen sulphate to 3 mL of
given food stuff taken in a
china dish and heat the
contents gently.
Result: Result: The given foodstuff was found to contain FAT.
Theory: These are the esters of glycerol and long chain fatty acids and are known as
triglycerides.

 Triglycerides which are liquids at room temperature are oils and those that are solids
are called fats
 . Oils are of plant origin and fats are of animal origin.
 Triglycerides in which three acyl groups are same are called simple triglycerides and a
triglyceride in which three acyl groups are different is called mixed triglyceride.
 Many naturally occurring fatty acids contain two or three double bonds. The fats from
which these come are called polyunsaturated fats or oils.
 while oils are glycerides of unsaturated fatty acids.
 Fats and oils are insoluble in water.
On heating with potassium hydrogen sulphate, oils and fats give characteristic
odour of acrolein. This is the test for glycerol present either free or combined as an
ester. On heating pungent smell due to acrolein formation confirms the presence of an
oil or a fat..with potassium hydrogen sulphate, glycerol is dehydrated and acrolein is
formed which has a

1-Palmitoleoyl-2-linoleoyl- 3-stearoylglycerol