Qualitative Analysis of
Carbohydrates
Introduction
• Carbohydrates are the key source of energy used by living things.
• Also serve as extracellular structural elements as in cell wall of
bacteria and plant.
• Carbohydrates are defined as the polyhydroxy aldehydes or
polyhydroxy ketones.
• Most , but not all carbohydrate have a formula
• (CH2O)n (hence the name hydrate of carbon)
• Simple sugars ends with –ose
Classification
1-Simple sugar: (one unit)
Monosaccharides contain one monosaccharide unit.
2-Complex sugar (more than one):
Disaccharides contain two monosaccharide units.
- Oligosaccharides contain 3-9 monosaccharide units.
- Polysaccharides can contain more than 9 monosaccharide units.
- Complex carbohydrates can be broken down into smaller sugar units through
a process known as hydrolysis.
Monosaccharide
They can be classified by the number of carbon atoms
▪ trioses (C-3)
▪ tetroses (C-4)
▪ pentoses (C-5)
▪ hexoses (C-6)
▪ heptoses (C-7)
• also be classified as ketoses or aldoses.
• A ketose contains a carbonyl group attached to
two R groups having one or more hydroxyl groups.
• An aldose contains terminal aldehyde group in addition to R group
containing -OH.
Reducing and Non-reducing sugars
•Reducing and non reducing sugar: If the oxygen on the
anomeric carbon of a sugar is not attached to any other
structure, that sugar can act as a reducing agent and is
termed a reducing sugar.
Molisch test
•This test is designed to detect the presence of
carbohydrate molecules in a given test sample.
Monosaccharides give a rapid positive test,
Disaccharides and polysaccharides react slower.
•Objective: To identify the carbohydrate from other
macromolecules lipids and proteins.
•Principle: The test reagent (H2SO4) dehydrates pentose to
form furfural and dehydrates hexoses to form 5-
hydroxymethyl furfural.
•A positive result is indicated by the formation of purple ring
or product.
Method
1 - Two ml of a sample solution is placed in a test tube.
2 -Two drops of the Molisch reagent is added.
3 - The solution is then poured slowly into a tube containing two
mL of concentrated sulfuric acid so that two layers form, producing
violet ring appear as liaison between the surface separations.
Sample observation
1-glucose
2-ribose
3-sucrose
4-starch
Benedict's test
• Benedict's reagent is used as a test for the presence of reducing
sugars.
• All monosaccharides are reducing sugars; they all have a free
reactive carbonyl group.
• Some disaccharides have exposed carbonyl groups and are also
reducing sugars. Other disaccharides such as sucrose are
non-reducing sugars and will not react with Benedict's solution.
• Large polymers of glucose, such as starch, are not reducing sugars
Objective: To distinguish between the reducing and
non-reducing sugars.
Benedict’s Test
sucrose
lactose
glucose
• The copper sulfate (CuSO4) present in Benedict's
solution reacts with electrons from the aldehyde
or ketone group of the reducing sugar.
• Reducing sugars are oxidized by the copper ion in
solution to form a carboxylic acid and a reddish
precipitate of copper oxide.
• One ml of a sample solution is placed in a test tube.
• Two mL of Benedict's reagent is added.
• The solution is then heated in a boiling water bath for five
minutes.
• A positive test is indicated by: The formation of a reddish
precipitate.
Sample observation
1-glucose
2-sucrose
3-lactose
Barfoed’s Test
• This test is performed to distinguish between reducing monosaccharides,
reducing disaccharides and non reducing disaccharides.
• Objective: To distinguish between mono- , di- and poly saccharides.
• Reducing monosaccharides are oxidized by the copper ion in solution to
form a carboxylic acid and a reddish precipitate within three minutes.
Reducing disaccharides undergo the same reaction, but do so at a slower
rate.
• The nonreducing sugars give negative result.
•A disaccharide is a weaker reducing agent
than a monosacchride, so monosacchride
will reduce the copper in less time.
Procedure
• Place one ml of a sample solution in a test tube.
• Add 3 ml of Barfoed's reagent (a solution of cupric acetate and
acetic acid.
• Heat the solution in a boiling water bath for 6 minutes(after the 3
min check the tubes).
Sample Observation
1-glucose
2-sucrose
3-lactose
Bial’s Test
•This test is used to distinguish between pentose
and hexose monosacharides.
•Objective: To distinguish between pentose
monosaccharide and hexose monosaccharide
•Pentoses produce a bluish or green product, while
hexoses yield muddy-brown to grey condensation
product.
•Put 2 ml of a sample solution in a test tube.
•Add 2 ml of Bial's reagent to each tube.
•Heat the tubes gently in hot water bath.
•If the color is not obvious, more water can be added to
the tube.
Sample Observation
1-glucose
2-ribose
3-fructose
Seliwanoff's Test
• This test is used to distinguish between aldoses (like
glucose) and ketoses (like fructose).
•Objective: To distinguish between aldose and ketone
sucrose.
•Ketohexoses produce a cherry red
product within two minutes. Aldohexoses
react to form the same product, but do so
more slowly giving yellow to faint pink
color.
•One half mL of a sample solution is placed in a test tube.
•Two ml of Seliwanoff's reagent is added.
•The solution is then heated in a boiling water bath for two
minutes.
Sample Observation
1-glucose
2-fructose
Fehling’s Test
•Fehling’s test consists of a solution that is usually
prepared fresh in laboratories. Initially, the solution exists
in the form of two separate solutions which are labelled
as Fehling’s A and Fehling’s B.
•Fehling’s A is a solution containing copper(II) sulphate,
which is blue.
•Fehling’s B is a clear liquid consisting of potassium sodium
tartrate (Rochelle salt) and a strong alkali, usually sodium
hydroxide.
SAMPLE OBSERVATION
Glucose
Ribose
Fructose
Lactose
Tollen’s Test
•Tollens Test is a very useful
method to distinguish between
aldehydes and ketones.
• This qualitative lab test is also
referred to as the silver mirror
test.
1. Place 5 drops of the samples in separate test tubes.
2. Add 2 mL of Tollens reagent into each tube.
3. Boil for about 5 minutes. Note and record your
observations.
SAMPLE OBSERVATION
Glucose
Fructose
