EXPERIMENT 2 1 September 2020

2020

 Objective: To confirm the presence of carbohydrates in the given sample

 Materials:
Equipments: Test tube holder, Dropper, Steam/Water Bath, Tongs
Glassware: Beaker, Conical Flask, Test Tubes, Nessler’s Cylinder
Chemicals: Samples, Distilled Water, Molisch’s Reagent, Conc. H2SO4,
Fehling’s Solution A & B, Benedict’s Reagent, Iodine Solution

 Theory:

Carbohydrates are polyhydroxy aldehydes and ketones or substances that

hydrolyse to yield polyhydroxy aldehydes and ketones.  Aldehydes (–CHO)
and ketones (= CO) constitute the major groups in carbohydrates.
Carbohydrates are mainly divided into monosaccharides, disaccharides,
and polysaccharides. The commonly occurring monosaccharides includes
glucose, fructose, galactose, ribose, etc. The two monosaccharides
combine to form disaccharides which include sucrose, lactose, and
maltose.  Starch and cellulose fall into the category of polysaccharides,
which consist of many monosaccharide residues.

Some tests that are used to differentiate and identify the carbohydrates
are as follows:

 Solubility Test
Due to the presence of free polyhydroxy groups in monosaccharides
and disaccharides, they are readily soluble and show positive solubility
test while in case of polysaccharides like starch the polyhydroxy group
are not free to dissolve in water and hence they are insoluble in water.

1|Page
  Iodine Test
Iodine (iodine-potassium iodide, I2KI) staining distinguishes starch (a
polysaccharide) from monosaccharides, disaccharides, and other
polysaccharides. The basis for this test is that starch is a coiled polymer
of glucose. Iodine interacts with these coiled molecules and becomes
bluish black. Other non-coiled carbohydrates do not react with iodine.
Therefore, a bluish black colour is a positive test for starch, and
a yellow-ish brown colour (i.e., no colour change) is a negative test for
starch. Glycogen, the common polysaccharide in animals, has a slight
difference in structure and produces only an intermediate colour
reaction. 

 Molisch’s Test
Carbohydrates when treated with concentrated Sulphuric acid
undergoes dehydration to give furfural derivatives. These compounds
condense with a-naphthol to form cooler products. Pentoses yield
furfural while hexoses yield 5- hydroxy methyl furfurals.

2|Page
  Benedict’s Test
Benedict’s test is performed by heating the reducing sugar solution with
Benedict’s reagent. The presence of the alkaline sodium carbonate
converts the sugar into a strong reducing agent called enediols. During
the reaction, enediols decrease the cupric particles (Cu2+) present in
Benedict’s reagent to cuprous particles (Cu+) which appear as red
copper oxide (Cu2O) which is insoluble in water. When Benedict’s
reagent solution and reducing sugars are heated together, the solution
changes its color to orange-red/ brick red precipitate. The red-colored
cuprous oxide is insoluble in water and hence, separate out from the
solution. When the concentration of the reducing sugar is high in the
solution, then the color becomes more intense (reddish) and the
volume of the precipitate increases in the test tube making it clearly
visible.

 F
e
h
l
i
n
g
’s Test
3|Page
 Fehling A is aqueous copper sulphate solution. Fehling B is alkaline
sodium potassium tartrate (Rochelle salt). Rochelle salt present in the
reagent acts as the chelating agent in this reaction. On heating an
aldehyde or reducing sugar with Fehling's solution, it gives reddish
brown precipitate (cuprous oxide).

Procedure

 Solubility Test
Take a small amount of the given sample in a test tube and add 3 ml of
distilled water into it and shake the test tube well.

 Iodine Test
Take a small amount of aqueous solution of the sample in a test tube
and add 1-2 drops of iodine solution into it.

 Molisch’s Test
Take about 2 ml of aqueous solution of the sample in a test tube and
add few drops of Molisch’s reagent into it. Pour 1 ml conc. H2SO4 slowly
along the side of the test tube.

 Benedict’s Test
Take 1-2 ml of aqueous solution of the sample in a test tube and add 1-
2 ml of Benedict’s reagent. Keep the test tube in a boiling water bath.

 Fehling’s Test
4|Page
 Take 2 ml of aqueous solution of the sample in a test tube and add 1-2
ml each of Fehling’s solution A and Fehling’s solution B into it. Keep the
test tube in a boiling water bath.

 Observation Table

 Solubility Test
SI No.  Sample  Observation
1  Glucose  Soluble
2  Sucrose  Soluble
3 Maltose Soluble
4 Lactose Soluble
5  Starch  Insoluble

 Iodine Test
SI No.  Sample  Observation
1  Glucose  No characteristic change.
2  Sucrose  No characteristic change.
3 Maltose No characteristic change.
4  Lactose  No characteristic change.
5  Starch  A blue colour is formed.

 Molisch’s Test
SI No.  Sample  Observation
 A red violet (purple) ring is
1  Glucose formed at the junction of two
layers.
 A red violet (purple) ring is
2  Sucrose formed at the junction of two
layers.
3 Maltose A red violet (purple) ring is
formed at the junction of two

5|Page
 layers.
A red violet (purple) ring is
4 Lactose formed at the junction of two
layers.
 A red violet (purple) ring is
5  Starch formed at the junction of the
two layers.

 Benedict’s Test
SI No.  Sample  Observation
 A reddish precipitate is
1  Glucose
formed.
 No formation of reddish
2  Sucrose
precipitate.
Orange red precipitate
3 Maltose
formed
Orange red precipitate
4 Lactose
formed
 No formation of reddish
5  Starch
precipitate.

 Fehling’s Test
SI No.  Sample  Observation
 A reddish precipitate is
1  Glucose
formed.

 No formation of
2  Sucrose
reddish precipitate.

 A reddish precipitate is
3 Maltose
formed.

A reddish precipitate is
4 Lactose
formed.

 No formation of
3  Starch
reddish precipitate.

 Result

 Solubility Test

6|Page
 Glucose and sucrose are soluble in water whereas starch is insoluble in
water.
 Iodine Test
Appearance of blue colouration indicates the presence of starch.
 Molisch’s Test
Presence of carbohydrate is confirmed due to the presence of Red-
violet ring.
 Benedict’s Test
Reddish precipitate indicates the presence of a reducing sugar.
 Fehling’s Test
Reddish precipitate indicates the presence of a reducing sugar.

 References

 http://vlab.amrita.edu/?sub=3&brch=63&sim=631&cnt=1
 https://www.phywe.com/en/the-solubility-of-
carbohydrates.html#:~:text=Carbohydrates%20are%20hydrophilic
%20substances%20which,is%20examine%20in%20this%20experiment.
 https://byjus.com/chemistry/tests-of-carbohydrates/
 http://amrita.olabs.edu.in/?sub=73&brch=8&sim=209&cnt=582
 https://microbenotes.com/benedicts-test/
 https://msu.edu/course/lbs/145/luckie/Lab1.html#:~:text=Iodine
%20(iodine%2Dpotassium%20iodide%2C,molecules%20and
%20becomes%20bluish%20black.
 https://www.onlinebiologynotes.com/tests-for-specific-
carbohydrates-seliwanoffs-test-bials-test-and-iodine-test/

***

7|Page