University of Ninevah

College of Medicine
Department of Chemistry and
Biochemistry
First Stage

Practical Biochemistry
Carbohydrates

MSc. Sarah Abdulelah Younus

2024-2025
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Experiment of Carbohydrates MI
PRE-LAB ASSIGNMENT EI
This pre-lab assignment is worth 4 point for this experiment and
should be completed on a separate page and turned in at the beginning
of lab.
1) Read the experiment carefully.
2) Safety Considerations: list the health and safety hazards for
∆ Copper (ll) sulfate
∆ Concentrated HCL
∆ Concentrated H2SO4
3) Procedure: Summarize the procedure using a bullet list.
4) Pre-lab Questions
a. Draw the cyclic structures of glucose , fructose ,and galactose
b. What is reducing sugar?
c. Give example of a carbohydrate that would have a negative
result when test with the Benedicts reagent.
d. What is the main structural difference between starch and
cellulose?
OBJECTIVES:
At the end of this lecture we will understand the following points:
• Definition of carbohydrates
• Sources of carbohydrates
• Classification of carbohydrates
• Study the various qualitative tests of carbohydrates
• Identify an unknown carbohydrate.

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 Carbohydrates
(Structure, Function, Classification and Reaction)
Carbohydrates:
J Is
They are defined as poly hydroxyl compounds - aldehyde or
ketone functional group , they have most , but not all carbohydrate
have a formula (CH2O)n where n>4. (hence the name hydrate of
carbon).

Functions of Carbohydrates: -
MCQ
1. Carbohydrates are the main source of energy in the body.
2. Storage form of energy (starch and glycogen)
3. Excess carbohydrate is converted to fat.
4. Glycoproteins and glycolipids are components of cell
membranes and receptors.
5. They form structural basis of many organisms

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 CLASSIFICATION

1) Monosaccharide:
It consists of one sugar unit and cannot be hydrolyzed into smaller
units ( also called as simple sugars ) , the most important
monosaccharides are:

Hexes pentose

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Also monosaccharides are classified according to the nature of
carbonyl group into :-
a- Aldose:- such as Glucose
b- Ketose:- such as Fructose

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 Q:- Classify the following sugars ( Glucose , Fructose ,
Galactose , Ribose , Ribulose , Mannose) ?
2) Disaccharide:
which consist of two sugar units linked to each other by C-O-C
(glycoside bond ), The most abundant disaccharide sugars are:

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Maltose (malt sugar):- consist of ( Glucose + Glucose )
Sucrose (table sugar):- consist of ( Glucose + Fructose )
Lactose (milk sugar):- consist of ( Glucose + Galactose )

3) Oligosaccharides:
These sugars contain (2-10) sugar units that are linked to each
other by glycoside bond, such as the disaccharides.

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4) Polysaccharides:
Polysaccharides are carbohydrates having more than ten
monosaccharide units. such as starch, glycogen and cellulose.
They are also called complex carbohydrates.
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.

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Chemical Tests of Carbohydrate:
The various tests for carbohydrates are given below:
1. Molisch test: specific test for carbohydrates.
[Link]'s Test: used to detect pentose [5C] monosaccharides.
3. Benedict’s test: specific test for reducing sugars.
4. Barfoed’s test: specific test for monosaccharides.
5. Seliwanoff’s test: specific test for ketohexoses.
6. Iodine test: specific test for polysaccharides.

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1) Molisch’s Test:
It is a general test for all carbohydrates, 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.
•The furfural and 5- hydroxymethyl furfural further react with α-
naphthol present in the test reagent to produce a purple ring.

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Note: Reagent Preparation: Prepare α-naphthol solution by
dissolving 1 g of α-naphthol in 100 ml of ethanol.

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Procedure:
1- 2 ml of a sample solution is placed in a test tube.
2- 2 drops of the Molisch reagent (which α-napthol in 95%
ethanol) is added.
3-The solution is then poured slowly into a tube containing 2 ml
of concentrated sulfuric acid (H2SO4) so that two layers form,
producing violet ring appear as liaison between the surface
separations.

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 [Link]'s test:
To detect 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, since the concentration of hemiacetal groups is very low.
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Principle :
•The copper sulfate (CuSO4) present in Benedict's solution reacts
with electrons from the aldehyde or ketone group of the reducing
sugar in alkaline medium.
•Reducing sugars are oxidized by the copper ion in solution to
form a carboxylic acid and a reddish precipitate of copper (I)
oxide.

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Procedure:

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1- 1ml of a sample solution is placed in a test tube.
2- 2 ml of Benedict's reagent is added.
3- The solution is then heated in a boiling water bath for 5
minutes.
4-A positive test is indicated by: The formation of a reddish
precipitate.

Note: All sugars give a positive results with a Benedict test except
sucrose because it is does a non-reducing sugar group.

Reagent Preparation:
In 1000 mL volumetric flask dissolve 173 g of sodium citrate with
100 g of sodium carbonate in 800 ml of distilled water, filter the
solution and add 17.3 g copper sulfate dissolved in 100 mL
distilled water, then fill of to 1000 mL.

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[Link]’s Test:
It is a chemical test used for detecting the presence of
monosaccharides. To distinguish between mono- , di- and poly
saccharides.
Principle :
Barfoed’s test used copper (II) ions in a slightly acidic medium.
Reducing monosaccharides are oxidized by the copper ion in
solution to form a carboxylic acid and a reddish precipitate of
copper (I) oxide within three minutes. Reducing disaccharides
undergo the same reaction, but do so at a slower rate.
Since the reduction in the weak acid medium occurs with
difficulty, so only monosaccharide can reduce the Cu+2 ions and
thus we can distinguish between the monosaccharide and
disaccharides (increasing the time boiling can give a false positive
result for disaccharides due to their hydrolysis in the acid
mediumto mono saccharide units). The general reaction is:

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Procedure: -
1-Place 0.5 ml of a sugar solution in a test tube.
1-Add 2 ml of Barfoed's reagent (a solution of cupric acetate and
acetic acid).
3-Heat the solution in a boiling water bath for 3 to 5 minutes (after
the 3 min check the tubes).

Reagent Preparation:
Dissolve 13.3 g copper acetate in 200 mL of distilled water,
filter the solution and add 1.8 ml of glacial acetic acid to the
filtrate.

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4. Bial’s Test:
Bial’s test is used to detect the presence of pentoses (five carbon
sugars) such as ribose .and hexose monosaccharide
Principle:
Bial’s test uses concentrated HCl In this test, the pentose is
dehydrated by concentrated HCl to form furfural that combines
with orcinol and the solution turns to blue-green. Hexoses form
a hydroxy methyl furfural with HCl , which then combines with
orcinol to form a brown complex.

Procedure:
Take (0.5) ml of the sugar solution in a test tube and add (2mL)
of the Bial's reagent solution. Shake the tube well and place in a
boiling water bath for 3-5 minutes. the blue-green precipitate
formed.

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Reagent Preparation:
Dissolve 1.5 g of orcinol in 500 ml of concentrated hydrochloric
acid and then add 1 ml of 10% ferric chloride solution.

5) Seliwanoff's Test:
Seliwanoff’s Test used to distinguish between aldoses (like
glucose) and ketoses (like fructose).

Principle: -
This test is based on the fact that, when heated, ketoses are more
rapidly dehydrated than aldoses. The ketose reacts with HCl (3M)
to give furfural derivative, which condenses with resorcinol to
give a cherry red complex, so attention to the heating time
should be given.
Aldohexoses react to form the same product, but do so more
slowly giving yellow to faint pink color.

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Procedure: -
1-0.5 ml of a sugar solution is placed in a test tube.
2-Add 2ml of Seliwanoff's reagent (a solution of resorcinol and
HCl) is added.
3-The solution is then heated in a boiling water bath for two
minutes. Notice red color develop with fructose (a ketohexoses ) and no
such color or yellow with ( glucose) an aldohexoses.

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Questions:
1- Name the complex formed by the addition of
concentrated sulfuric acid to sugar solution and
explain the reaction?

2- Why sucrose gives negative Benedict test?

3- Explain starch it gives negative Benedict test?

4- Why glucose (monosaccharide) and maltose

5- What is the difference between Benedict and

6- What are the carbohydrates’ that give

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