Lab 5: Qualitative Analysis Test for Carbohydrates
Benedict's Test
(positive for reducing sugars)
•
Principle:
sodium citrate, sodium carbonate , CuSO4. 5H2O solution.
cupric ions, which in an alkaline environment, oxidize the aldehyde group to a carboxylic
acid. Cupric ions are reduced to cuprous oxide, which forms a red precipitate
RCHO + 2Cu2+ + 4OH- ----> RCOOH + Cu2O + 2H2O
•
The color of the precipitate varies from green to gold to red depending on
•
the concentration of the reducing sugar.
Procedure
1. Place 1mL of the following 1% carbohydrate solutions in separate, labeled test
tubes: glucose, fructose, sucrose, lactose, maltose, and starch.
2. Also place 1 ml of distilled water in another tube to serve as a control.
3. To each tube, add 1 ml of Benedict's reagent and heat the tubes in a boiling water
bath for 5 minutes.
4. Remove the tubes from water bath. Note and record the results.
 In the presence of a reducing sugar a precipitate which may be red, yellow or green
will form.
Picric Acid Test
(for reducing sugars)
•
Principle
Picric acid (2,4,6-trinitrophenol) or TNP reacts with reducing sugars to give a red colored
picramic acid C6H2.OH.NH2(NO2)2
Procedure
1. Into a test tube add 1 ml of maltose solution, into the second tube, 1ml of sucrose
solution.
2. Add into each tube 1 ml of a saturated solution of picric acid, and then add into each tube
0.5 ml of sodium hydroxide solution.
3. Heat both samples in a boiling water bath.

In the presence of reducing sugars, the solution stains red; a sodium salt of
picric acid is formed.
Bial's (Orcinol) Test for pentoses
( for the detection of pentoses)
Principle
Bial's reagent (0.1 % orcinol in concentrated HCl containing 0.1 % FeCl3.6H2O).
The action of concentrated acids causes the dehydration of sugars.
Bial’s test is used to distinguish between pentoses and hexoses. They react with Bial’s
reagent and are converted to furfural. Orcinol and furfural condense in the presence of ferric
ion to form a colored product. Appearance of a blue green colour or precipitate indicates the
presence of pentoses and formation of muddy brown precipitate shows the presence of
hexoses.
Procedure
1. Add about 1 ml of 1% xylose, glucose, fructose, maltose, arabinose, and xylose solution to
their respective labeled test tubes.
2. Add 1.5 ml of Bial's reagent to each tube and mix well.
3. Carefully heat each tube (with some agitation) directly over the burner flame. Hold the
tube at a diagonal and heat along the sides of the tube rather than at the bottom to prevent
eruption of the liquid from the tube. Move the tube diagonally in and out of the flame, until
the mixture just begins to boil. Stop heating when the mixture begins to boil.
 A blue-green color indicates a positive result. Prolonged heating of some
hexoses yields hydroxymethyl furfural which also reacts with orcinol to give
colored complexes.
 Aldoses (e.g., glucose) have an aldehyde group at one end.
Ketoses (e.g., fructose) have a keto group, usually at C2.
Seliwanoff's (Resorcinol) Test
(used for detection of Ketoses)
Principle
•
Seliwanoff’s test is used to distinguish between hexoses with a ketone group and
hexoses that are aldehydes.
•
With ketoses, a deep red color is formed rapidly.
•
Aldoses give a light pink color that takes a longer time to develop.
•
The test is most sensitive for fructose, which is a ketose.
Ketohexoses (such as fructose) and disaccharides containing a ketohexose (such as
sucrose) form a cherry-red condensation product.
Other sugars (e.g. aldose) may produce yellow to faint pink colors.
•
Seliwanoff's reagent (0.5 % resorcinol in 3N HCl).
•
It is a color reaction specific for ketoses.
•
When conce: HCl is added. ketoses undergo dehydration to yield furfural derivatives
more rapidly than aldoses.
•
These derivatives form complexes with resorcinol to yield deep red color.
•
The test reagent causes the dehydration of ketohexoses to form 5hydroxymethylfurfural. 5-hydroxymethylfurfural reacts with resorcinol present in
the test reagent to produce a red product within two minutes (reaction not shown).
•
Aldohexoses reacts so more slowly to form the same product.
Procedure
1. Add about 3 ml of Seliwanoff's reagent to each labeled test tube.
2. Add 1 drop of the respective sugar solution to the appropriate test tubes, and mix well.
3. Place all the test tubes in the boiling water bath at the same time and heat for 3 min after
the water begins to boil again. Record your observations.

A positive result is indicated by the formation of a red color with or without
the separation of a brown-red precipitate.
Barfoed's Test
(Used to distinguish between mono- & di-saccharides)
•
Principle
Barfoed's reagent reacts with mono-saccharides to produce cuprous oxide at a
faster rate than disaccharides do:
•
RCHO + 2Cu2+ + 2H2O -----> RCOOH + Cu2O + 4H+
Procedure
1. Place 1 mL of the following 1% carbohydrate solutions in separate, labeled test tubes:
glucose, fructose, sucrose, lactose, and maltose.
2. To each tube, add 1 ml of Barfoed's reagent, and heat in a boiling water bath for 10
minutes.
3. Remove the tubes from water bath. Note and record your observations.

A red precipitate will form if the test is positive.
 Starch : a carbohydrate consisting of a large number of glucose units joined by
glycosidic bonds. This polysaccharide is produced by most green plants as an energy
store
 starch generally contains amylose and amylopectin .
 glucogen, the glucose store of animals, is a more branched version of amylopectin.
Amylose is a glucose polymer with a(14) linkages.
Amylopectin is a glucose polymer with mainly a(14) linkages, but it also
has branches formed by a(16) linkages.
glycogen has more a(16) branches.
The highly branched structure permits rapid glucose release from glycogen stores, e.g., in
muscle during exercise.
•
Cellulose, a major constituent of plant cell walls, consists of long linear chains of
glucose with b(1®4) linkages.
Iodine test
This test is used for the detection of starch in the solution. The blue-black colour is due to
the formation of starch-iodine complex.
•
Starch contain polymer of α-amylose and amylopectin which forms a complex with
iodine to give the blue black colour.
•
When iodine (I2) is added to amylose, the helical shape of the unbranched
polysaccharide traps iodine molecules, producing a deep blue-black complex.
Amylopectin, cellulose, and glycogen react with iodine to give red to brown colors. Glycogen
produces a reddish-purple color. Monosaccharides disaccharides are too small to trap iodine
molecules and do not form dark colors with iodine.
Procedure:
Add 2 drops of iodine solution to about 2 mL of the carbohydrate containing test solution.
A blue-black colour is observed which is indicative of presence of polysaccharides.