Lab Activity 3
Carbohydrates Reactions
Alaa S Baraka
Islamic university of Gaza
Feb 2013
Content
Formation of osazons
 Test for individual carbohydrates:
1.
2.
3.
Bial's (Orcinol) Test for pentoses
Aniline Acetate Test for Pentoses
Seliwanoff's (Resorcinol) Test
Formation of osazones
Osazones are formed when the
sugars(monosaccharides) react with a
compound known as phenylhydrazine
These sugars are reducing ones which
have either a free aldehyde or a ketone
group to react with the phenylhydrazine.
Introduction
Phenylhydrazine is the chemical
compound with the formula C6H5NHNH2
General Reaction of osazons
formation
This reaction is complete in 3 step and
consume 3 moles of phenylhydrazine
During reaction with monosaccharides,
additional phenyl hydrazine is consumed in
oxidizing the adjacent OH-group to carbonyl
group which then forms a second phenyl
hydrazone.
Such bisphenyl hydrazones are called
osazones.
Osazone formation involves hydrazone
formation at C-1 of an aldose (or C-2 of a
ketose) and oxidation of C-2 (or C-1) of an
alcohol group to a ketone (or an
aldehyde). The new carbonyl group is also
converted to a hydrazone.
General Reaction of osazons
formation
osazones
a crystalline compound with a sharp
melting point will be obtained
Since only C1 & C2 of a saccharide are involved in
osazones, sugars with the same configuration at
the remaining carbon atom gives the same
osazone.
D-fructose and D-mannose give the same
osazone as D-glucose
seldom used for identification; we now use
HPLC or mass spectrometry
osazones
Depending on the time required to form the
insoluble yellow osazone, different sugars can be
classified into the following:
Mannose: 1-5 min
Fructose: 2 min
Glucose:5 min
Xylose: 7 min
Arabinose: 10 min
Galactose: 20 min
Maltose osazone soluble in hot water
Application
For identifying sugars esp. Reducing
sugars.
Osazones are used as dyes
Experiment 1: Formation of Osazones
(important in determining sugar structure)
Reagents:
1% solutions of glucose, fructose, maltose,
mannose, and xylose
Phenyl hydrazine mixture (2 parts phenyl
hydrazine hydrochloride are mixed with 3
parts sodium acetate).
Procedure
To 300 mg of phenyl hydrazine mixture add
5 ml of the tested solution,
 Shake well, and heat on a boiling water
bath for 30 – 45 min.
 Allow the tubes to cool slowly (not under
tap) and examine the crystals
microscopically, draw the shapes of the
crystalls
Results:
Viewed under the microscope:
Glucosazone
Viewed under the microscope:
Fructosazone
Viewed under the microscope:
Galactosazone
Xylosazone: Viewed under the
microscope
Tests for Individual Carbohydrates
1. Bial's (Orcinol) Test for pentoses
( for the detection of pentoses)
Principle
Pentoses are converted to furfural by this
reagent, which forms a blue green color
with orcinol
Reagents
Set up 1 % solutions of:
xylose,
arabinose,
glucose,
fructose,
maltose.
Bial's reagent (0.1 % orcinol in concentrated HCl
containing 0.1 % FeCl3.6H2O).
Procedure
– 1. Add about 2 ml of 1% xylose, glucose, fructose, maltose, in test
tubes.
– 2. Add 3 ml of Bial's reagent to each tube and mix well.
– 3. Carefully heat in boiling water bath
– 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.
Aniline Acetate Test
(for Pentoses)
The furfural produced by the reaction of
HCl on pentoses forms a bright red color
with aniline acetate in a test paper held
over the mouth of the reaction flask.
Reagents
1% solution of glucose, xylose
Aniline solution is prepared as follows:
Shake 5 ml of aniline with 5 ml of water
and add 5 ml of glacial acetic acid to
adjust clear the emulsion.
Procedure
Place 2.5 ml of solution to be tested and
10 ml water in 250 ml Erlenmeyer flask.
Add 10 ml of conc. HCl and boil gently for
about 1 min.
Cease heating, hold a filter paper
moistened with a few drops of aniline
acetate over the mouth of the flask.
Bright red color on a filter paper indicates
a positive result.
Seliwanoff's (Resorcinol) Test
(used for detection of Ketoses)
Principle
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.
Reagents
Set up 1 % solution of:
glucose,
sucrose,
fructose,
lactose,
maltose.
Seliwanoff's reagent (0.5 % resorcinol in
3N HCl).
Procedure
1.
2.
3.
4.
5.
Add about 2 ml of Seliwanoff's reagent to each
labeled test tube.
Add 1 ml of sugar solution to the test tubes and
mix well.
Place all the test tubes in the boiling water bath at
and heat for 3 min after the water begins to boil
again.
A positive result is indicated by the formation of
A red color with or without the separation of a
brown-red precipitate.