Islamic University_ Gaza
Faculty of Health Sciences.
Medical Technology Department
Lab 5: Qualitative Analysis Test
for Carbohydrates
Qualitative Analysis Test
• Is concerned with determining, the identity of
a substance.
• Enables us to detect the presence of things
which may be beyond the reach of our senses.
Types of Carbohydrates
• Most naturally occurring sugars are D isomers.
• Monosaccharides are the simplest carbohydrates
- Also called “simple sugars”, cannot be hydrolyzed to simpler carbohydrates
- Examples: glucose, fructose, galactose, ribose
• Disaccharides are two monosaccharides bonded together.
- Examples: sucrose (table sugar), lactose (milk sugar).
•
Oligosaccharides - a few monosaccharides covalently linked.
• Polysaccharides are polymers of monosaccharides
- Can be split into many monosaccharides with acid or enzymes
- Examples: starch, cellulose, glycogen
• The joining of two hexoses by the glycosidic
bond causes the formation of disaccharide.
• Longer
chains
composed
of
3-10
monosaccharide
units
are
called
oligosaccharides.
• Polysaccharides usually contain hundreds or
thousands of monosaccharide units.
Sugar Nomenclature
For sugars with more
than one chiral center,
D or L refers to the
asymmetric C farthest
from the aldehyde or
keto group.
Most naturally occurring
sugars are D isomers.
O
H
C
H – C – OH
HO – C – H
H – C – OH
H – C – OH
CH2OH
D-glucose
O
H
C
HO – C – H
H – C – OH
HO – C – H
HO – C – H
CH2OH
L-glucose
6 CH2OH
6 CH2OH
5
H
4
OH
O
H
OH
3
H
H
2
OH
a-D-glucose
H
1
OH
5
H
4
OH
H
OH
3
H
O
OH
H
1
2
H
OH
b-D-glucose
Cyclization of glucose produces a new asymmetric center
at C1. The 2 stereoisomers are called anomers, a & b.
Haworth projections represent the cyclic sugars as having
essentially planar rings, with the OH at the anomeric C1:
 a (OH below the ring)
 b (OH above the ring).
The active part in sugars is aldehyde or ketone group.
The presence of aldehyde or ketone groups and hydroxyl groups causes that
the sugars have typical reactions for aldehydes/ketones and alcohols.
Classification upon reducing end
1. Reducing Sugars
•
•
•
•
Oxidation: loss of electrons
Reduction: gain of electrons
Have aldehyde group
Sugars that can be oxidized by mild oxidizing
agents and the oxidizing agent is reduced in the
reaction.
( Can be oxidized to acid,Reduces another
compound)
• All monosaccharides
• Maltose, Lactose
• A reducing sugar is a carbohydrate possessing
either a free aldehyde or free ketone
functional group as part of its molecular
structure.
A functional groups are the regions of a
molecule that gives it particular properties.
A single molecule can have more than one
functional group as part of its structure.
• All of the monosaccharides and most of the
disaccharides can be oxidized.
• Sugars exist in solution as an equilibrium mixture
of open-chain and closed-ring (or cyclic)
structures.
• All monosaccharides have an open and closed
form structure, but oligosaccharides have only
closed structure.
• When the cyclic structure opens, the aldehyde
group is available for oxidation.
Opend and closed form of
monosaccarides
Lactose is a reducing sugar, why?
• ( one of the carbonyle groups are free)
• a carbonyl group is a functional group
composed of a carbon atom double-bonded
to an oxygen atom: C=O.
2. NON Reducing Sugars
• is not oxidized by mild oxidizing agents.
• Sucrose
• All polysaccharides
Sucrose is not a reducing sugar, why?
• Sucrose is not a reducing sugar because it cannot
revert to the open-chain form that would provide
the aldehyde group needed to reduce the cupric
ion. (the carbonyle groups are busy in the to
side)
• Common oxidizing agents used to test for the
presence of a reducing sugar are: Benedict's solution,
Fehling's solution and picric acid solution.
• An oxidizing agent (also called an oxidizer or oxidant) is
referred to as a chemical compound that readily
transfers oxygen atoms or a substance that gains
electrons in a redox chemical reaction.
• Aldose(glucose) ---- oxidation ----- carboxylate
• Ketose(fructose) ----- oxidation----- hydroxyl
carboxylate
Oxidizing Reagent
Benedict's Solution
Fehling's Solution
Tollen's Reagent
copper sulfate in
copper sulfate in alkaline
silver nitrate in aqueous
alkaline citrate
tartrate
ammonia
Color of Solution
deep blue
deep blue
colorless
Color After Reaction with a
brick red precipitate
brick red precipitate
silver mirror forms
Reducing Sugar
Cu2O(s)
Cu2O(s)
Ag(s)
Species Being Reduced
Cu2+
Cu2+
Ag+
(the oxidant)
Cu2+ + e ---> Cu+
Cu2+ + e ---> Cu+
Ag+ + e ---> Ag(s)
Species Being Oxidized
reducing sugar
reducing sugar
reducing sugar
(the reductant)
oxidized to carboxylate
oxidized to carboxylate
oxidized to carboxylate
Composition
1. 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.
a negative test (left) and a positive test (right)
5. 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.
3. 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.
Results
two negative tests (left, middle) and a positive
test (right)
Monosaccharides
Aldoses (e.g., glucose) have an
aldehyde group at one end.
H
Ketoses (e.g., fructose) have
a keto group, usually at C2.
O
CH2OH
C
C
O
HO
C
H
OH
H
C
OH
OH
H
C
OH
H
C
OH
HO
C
H
H
C
H
C
CH2OH
CH2OH
D-glucose
D-fructose
4. 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 cherryred 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 5-hydroxymethylfurfural. 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.
a negative test (left) and a positive test (right)
Deep coulr in short time in fructose compared to a light pink colour produced
by glucose
2. Barfoed's Test
(Used to distinguish between mono- & di-saccharides)
• Principle
Barfoed's reagent reacts with monosaccharides to produce cuprous oxide at a
faster rate than disaccharides do:
• RCHO + 2Cu2+ + 2H2O -----> RCOOH + Cu2O + 4H+
a negative test (left) and a positive test (right)
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.
Polysaccharide
CH2OH
H
O
H
OH
H
H
H 1
O
OH
6CH OH
2
5
O
H
4 OH
OH
H
H 1
O
H
OH
O
H H
H
OH
H
O
H H
H
H
OH
H
H
O
O
O
OH
2
3
H
H
CH2OH
CH2OH
CH2OH
H
H
OH
H
OH
H
OH
OH
amylose
Amylose is a glucose polymer with a(14)
linkages.
CH2OH
CH2OH
O
H
H
H
OH
H
H
OH
O
OH
CH2OH
H
H
OH
H
H
OH
H
H
OH
CH2OH
O
H
OH
O
H
OH
H
H
O
O
H
OH
H
H
OH
H
H
O
4
amylopectin
H
1
O
6 CH2
5
H
OH
3
H
CH2OH
O
H
H
H
1
2
OH
O
CH2OH
O
H
4 OH
H
H
H
O
OH
Amylopectin is a glucose polymer with mainly a(14) linkages, but it
also has branches formed by a(16) linkages.
H
O
H
OH
H
H
OH
H
OH
Glycogen
glycogen has more a(16) branches.
The highly branched structure permits rapid
glucose release from glycogen stores, e.g., in
muscle during exercise.
CH2OH
CH2OH
O
H
H
OH
H
H
OH
H
O
OH
CH2OH
H
H
OH
H
H
OH
H
H
OH
CH2OH
O
H
OH
O
H
OH
H
H
O
O
H
OH
H
H
OH
H
H
O
4
glycogen
H
1
O
6 CH2
5
H
OH
3
H
CH2OH
O
H
2
OH
H
H
1
O
CH2OH
O
H
4 OH
H
H
H
H
O
OH
O
H
OH
H
H
OH
H
OH
• 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 starchiodine 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.
a negative test (left) and a positive test (right)