EXPERIMENT NO.

- 01

Aim: Qualitative analysis of carbohydrates

INTRODUCTION

Carbohydrates may be classified based upon the number of polyhydroxy aldehyde

or ketone (saccharide) units found in their structure. The basic carbohydrate unit
is a single polyhydroxy aldehyde or ketone, or monosaccharide. A disaccharide can
be viewed as two monosaccharides from which water is removed to join the units.
The bond that forms between the two sugar units is known as a glycosidic bond or
linkage. Oligosaccharides consist of 3-10 sugar units covalently joined together,
and polysaccharides are large biomolecular polymers of covalently bonded
monosaccharide Units.

Carbohydrate Chemical Reactions

The chemical reactions of carbohydrates is largely that of the hydroxy and carbonyl
groups. The aldehyde and β-hydroxy ketone (a ketone that has an OH on a
carbon next to the carbonyl group) structural units in sugars undergo mild
oxidation to carboxylic acids. In solution, the ring structure of sugars are able to
open at the anomeric carbons to form the open-chain aldehyde or -hydroxy
ketone form, which undergoes oxidation in the presence of a mild oxidizing agent.
Such sugars are referred to as reducing sugars. However, when the anomeric ring
carbon is involved in a glycosidic linkage, the ring is locked in place and cannot
open for oxidation to occur. All monosaccharides are reducing sugars since no
glycosidic linkages exist in their structures. The disaccharides maltose and lactose
are reducing sugars since only one of the C1 anomeric carbons is involved in a
glycosidic linkage, while sucrose is a nonreducing sugar since both the C1 and C2
anomeric carbons participate in the glycosidic linkage. Starch and glycogen are
also nonreducing sugars because of their size.
A reducing sugar is any sugar that, in a solution, has an aldehyde or a ketone
group. The enolization of sugars under alkaline conditions is an important
consideration in reduction tests. The ability of a sugar to reduce alkaline test
reagents depends on the availability of an aldehyde or keto group for reduction
reactions. A number of sugars especially disaccharides or polysaccharides have
glycosidic linkages which involve bonding a carbohydrate (sugar) molecule to
another one, and hence there is no reducing group on the sugar; like in the case of
sucrose, glycogen, starch and dextrin. In the case of reducing sugars, the presence
of alkali causes extensive enolization especially at high pH and temperature. This
leads to a higher susceptibility to oxidation reactions than at neutral or acidic pH.
These sugars, therefore, become potential agents capable of reducing Cu+2 to Cu+,
Ag+ to Ag and so fort. Most commonly used tests for detection of reducing sugars
are Fehling’s Test, Benedict’s Test and Barfoed’s Test.

PRINCIPLE
Molisch Test
The Molisch test is used to determine the presence of carbohydrates, regardless of
structure. The Molisch reagent provides the condensation reagent, -naphthol in
alcohol. Concentrated sulfuric acid acts as the dehydrating reagent. It also serves
as a catalyst for the hydrolysis of di- and polysaccharides. The presence of a purple
ring at the interface between the carbohydrate solution and concentrated sulfuric
acid is a positive indication of a carbohydrate structure. Monosaccharides react
more quickly than di- and polysaccharides, which must slowly undergo hydrolysis
before they react with the Molisch reagent.
Benedict's Test
Benedict's reagent reacts with reducing sugars to form inorganic precipitates which
are readily detected by visual observation. In the reaction between a reducing sugar
and Benedict's reagent, copper(II) ion is reduced to copper(I) by the aldehyde
functional group: The formation of a red to orange precipitate indicates the
presence of a reducing sugar.
Barfoed's Test
Barfoed's reagent also uses the reduction of copper(II) ion to red Cu2O as an
indication of a reducing sugar. However, it not as reactive as Benedict's reagent,
and the rate at which the red precipitate forms can be used to distinguish between
mono-saccharides and disaccharides. The appearance of red Cu2O within 2-3
minutes is a positive test for a monosaccharide; disaccharides produce the
precipitate in approximately 10 minutes.

Seliwanoff Test
The Seliwanoff test differentiates between ketohexoses and aldohexoses. It also
uses concentrated HCl for the dehydrating agent, but the condensation agent is
resorcinol. Ketohexoses are dehydrated to 5-hydroxymethylfurfural, which
undergoes condensation with resorcinol within two minutes to form a red-colored
product.

FLOW CHART FOR IDENTIFICATION OF CARBOHYDRATES

PROCEDURE
1. MOLISCH TEST:
2ml of Carbohydrate Soln.+ 2-3 drops of Molisch reagent. Mix well and Add
Slowly.2ml of Conc. H2SO4 along the sides of the test tube.
Observations: Reddish Violet ring at the junction of the two liquids.
2. IODINE TEST:
2ml of Carbohydrate Soln.+ 2-3 drops of Iodine Soln.
Observations: Blue colour for starch. Red colour for Dextrin
3. BENDEDICT’S TEST:
0.5 ml of Carbohydrate Soln+ s.5 ml of Benedict’s qualitative reagent. Mix
well and boil for 2 mins on a small flame.
Observations: Green ppt.: 0.5 % Yellow ppt.:1.0%
Reddish Yellow ppt.:1.5% Brick Red ppt.: 2.0%
4. FEHLING’S TEST:
1ml of Carbohydrate Soln.+ 5ml of Fehling’s Soln. (A+B). Mix well and boil
for 2 mins.
Observations: Yellow or Brick red ppt.
5. PICRIC ACID TEST:
2ml of Carbohydrate Soln.+1ml of picric acid (1%0 + 1ml of NaOH Soln. Mix
well and boil.
Observation: Red colour
6. BARFORD’S TEST:
2ml of Carbohydrate Soln.+1 ml of Barford’s reagent. Mix well and boil for 2
mins. Allow to stand.
Observation: Red ppt. Settling to the bottom
7. SELVINOFF’S TEST:
1 ml of Carbohydrate Soln. +3 ml of Selvinoff’s reagent. Mix well and boil of
1min
Observations: Red colour for Fructose (and Sucrose)
8. OSAZONE TEST:
5 ML OF carbohydrate Soln. (2%) + 10 drops of glacial acetic acid + a pinch
of ( 10mg) of phenyl hydrochloride +20 mg Sodium acetate crystals, Mix well
and keep in a boiling water bath for 20 mins. Allow the tube to cool in cold
water for 30 to 40 mins.
Observe the crystals under microscope and draw the diagram of crystals.
 EXPERIMENT NO 1A

QUALITATIVE ANALYSIS OF CARBOHYDRATES: GLUCOSE

TEST OBSERVATION INFERENCE

1. MOLISCH TEST

2. IODINE TEST

3. BENEDICT’S TEST

4. FEHLING’S TEST

5. PICRIC ACID TEST

.BARFORD’S TEST

7. OSAZONE TEST

Picture of Osazone Test :

Report: Given Sample of the carbohydrate is confirmed as Glucose.

 EXPERIMENT NO 1B

QUALITATIVE ANALYSIS OF CARBOHYDRATES: FRUCTOSE

TEST OBSERVATION INFERENCE

1. MOLISCH TEST

2. IODINE TEST

3. BENEDICT’S TEST

4. FEHLING’S TEST

5. PICRIC ACID TEST

.BARFORD’S TEST

7. OSAZONE TEST

Picture of Osazone Test :

Report: Given Sample of the carbohydrate is confirmed as Fructose.

 EXPERIMENT NO 1C

QUALITATIVE ANALYSIS OF CARBOHYDRATES: LACTOSE

TEST OBSERVATION INFERENCE

1. MOLISCH TEST

2. IODINE TEST

3. BENEDICT’S TEST

4. FEHLING’S TEST

5. PICRIC ACID TEST

.BARFORD’S TEST

7. OSAZONE TEST

Picture of Osazone Test :

Report: Given Sample of the carbohydrate is confirmed as Lactose.

 EXPERIMENT NO 1D

QUALITATIVE ANALYSIS OF CARBOHYDRATES: MALTOSE

TEST OBSERVATION INFERENCE

1. MOLISCH TEST

2. IODINE TEST

3. BENEDICT’S TEST

4. FEHLING’S TEST

5. PICRIC ACID TEST

.BARFORD’S TEST

7. OSAZONE TEST

Picture of Osazone Test :

Report: Given Sample of the carbohydrate is confirmed as Maltose.

 EXPERIMENT NO 1E

QUALITATIVE ANALYSIS OF CARBOHYDRATES: SUCROSE

TEST OBSERVATION INFERENCE

1. MOLISCH TEST

2. IODINE TEST

3. BENEDICT’S TEST

4. FEHLING’S TEST

5. PICRIC ACID TEST

.BARFORD’S TEST

7. OSAZONE TEST

Report: Given Sample of the carbohydrate is confirmed as Sucrose.

 EXPERIMENT NO 1F

QUALITATIVE ANALYSIS OF CARBOHYDRATES: STARCH

TEST OBSERVATION INFERENCE

1. MOLISCH TEST

2. IODINE TEST

3. BENEDICT’S TEST

4. FEHLING’S TEST

5. PICRIC ACID TEST

.BARFORD’S TEST

7. OSAZONE TEST

Report: Given Sample of the carbohydrate is confirmed as Starch.