CARBOHYDRATES – REACTIONS OF IDENTIFICATION
REDUCING PROPERTIES OF SUGARS
1. FEHLING’S TEST FOR REDUCING SUGARS
All monosaccharides and many disaccharides reduce weak oxidizing agents like Cu2+ ion.
These carbohydrates are called reducing sugars. In the presence of reducing sugars Fehling’s
solution, that contains Cu2+, changes colour from blue to red/brown.
Prepare three test tubes. Place 1 ml of glucose into first tube, 1 ml of fructose into second one
and 1 ml of maltose into third one. Add 1 ml of Fehling’s I reagent (CuSO4) and Fehling’s II
solution (alkaline solution of sodium and potassium tartrate) and mix well. Place all three test
tubes in the boiling water bath for five minutes. Observe any changes.
2. BENEDICT’S TEST FOR REDUCING SUGARS
Benedict’s reagent contains Cu2+in the solution of sodium citrate. Reducing sugar reduces
Cu2+into Cu1+, and appearance of red or orange precipitate is observed.
Prepare four test tubes and place 1 ml of Benedict’s reagent in all of them. Then add 0.5 ml of
glucose to the 1st tube, 0.5 ml of fructose to the 2nd tube, 0.5 ml of maltose to the 3rd tube and
0.5 ml of sucrose to the last one. Place all the test tubes in the boiling water bath for five
minutes. Observe any colour changes.
3. NYLANDER’S TEST FOR REDUCING SUGARS
Nylander’s reagent contains alkaline bismuth nitrate. Reducing sugars reduce Bi3+into
metallic Bi, which precipitates in form of black precipitate.
Place 2 ml of a reducing sugar in the test tube. Add 1 ml of Nylander’s reagent and mix well.
Then heat the test tube in the water bath for ten minutes. Record any changes.
4. BARFOED’S TEST TO DISTINGUISH BETWEEN REDUCING MONO- AND
DISACCHARIDES
Barfoed’s reagent contains copper acetate in acetic acid and it is not as reactive as
Benedict’s reagent. Obtaining a light red precipitate is a positive result of the reaction.
Monosaccharides produce the red precipitate in 2 to 3 minutes; disaccharides produce the
precipitate in 10 minutes.
Prepare three test tubes and place 1 ml of glucose in the 1st tube, 1 ml of lactose in the 2nd tube
and 1 ml of maltose in the 3rd tube. Then add 0.5 ml of Barfoed’s reagent to all three tubes,
mix well and put into the water bath for about 3 min. Record any changes.
REACTIONS OF IDENTIFICATION
5. MOLISCH TEST - A GENERAL TEST FOR CARBOHYDRATES
This test is used to detect carbohydrates in several substances. In this test, Molisch reagent is
mixed with a dilute solution of carbohydrate. Then concentrated sulphuric acid is introduced
carefully, and a purple colour develops at the interface if a carbohydrate is present.
Carbohydrates undergo dehydration reactions (loss of water) in the presence of concentrated
sulphuric acid. Pentoses and hexoses form five member oxygen-containing rings on
dehydration. The five-membered ring, known as furfural, further reacts with Molisch reagent
to form coloured compounds.
Place 1 ml of the carbohydrate solution in a clean test tube. Add 2 drops of Molisch reagent
and mix well. Incline the test tube and slowly add 0.5 ml of concentrated sulphuric acid
(H2SO4) in drops along the sides of the test tube. Record any changes.
6. SELIWANOFF’S TEST FOR KETOHEXOSES
Seliwanoff’s test is used to distinguish aldohexoses from ketohexoses. A ketohexose like
fructose forms a deep red colour with Seliwanoff’s reagent while an aldohexose shows only a
light pink colour and it takes a longer time to develop the colour.
Prepare two test tubes. Place 1 ml of glucose into first tube and 1 ml of fructose into second
one. Then add 1 ml of concentrated HCl and few drops of Seliwanoff’s reagent to both tubes,
and mix well. Place the test tubes in the boiling water bath for five minutes. Observe colour
changes.
7. BIAL’S TEST FOR PENTOSES
This test is used to distinguish between pentoses and hexoses. Pentoses give a positive test
with Bial’s reagent. In the presence of concentrated HCl, pentoses form a five-membered
ring, known as furfural. The formation of furfural is indicated by a blue-green colour in the
presence of Bial’s reagent (contains resorcinol and ferric ions). A positive test is the
formation of a bluish-green colour within 5 minutes without the formation of a precipitate.
The colour of hexoses solutions becomes yellow.
Prepare two test tubes. Place 1 ml of arabinose in first tube and 1 ml of glucose in the second
one. Then add 1 ml of HCl and few drops of Bial’s reagent, and mix well. Heat the test tube in
the boiling water bath until solution boils. Observe changes of solutions colours.
8. MOORE’S TEST TO DISTINGUISH BETWEEN REDUCING MONO- AND
DISACCHARIDES
Concentrated bases cause translocation of enol bond into other positions of carbon chain of
carbohydrates. This bond may be broken down giving various derivatives that undergo
polymerization. Sugar solution, colourless at the beginning, becomes yellow and even red-
brown. Non-reducing sugars, like sucrose or starch, give negative test with Moore’s reagent
(the solutions remain colourless).
Prepare four test tubes and place 1 ml of glucose to the 1st tube, 1 ml of maltose to the 2nd
tube, 1 ml of sucrose to the 3rd tube and 1ml of starch to the 4th one. Then add 1 ml of 20%
KOH to all of them, mix well and place all the four test tubes in the boiling water bath for two
minutes. Observe changes of solutions’ colour.
9. IODINE TEST FOR STARCH AND OTHER POLYSACCHARIDES
Starch is a polysaccharide that can be easily identified by the iodine test. The many glucose
units in starch trap the I2 molecules and form a dark blue-black complex. Mono and
disaccharides are too small and they are unable to form a complex with I2. Cellulose, a
polysaccharide, does not form coloured complexes with I2, either.
Place 1 ml of starch solution in a test tube, add 2 drops of Lugol’s reagent (solution of I2 in
KI) and stir well. Observe the solution’s colour.
