EXPERIMENT 5 - CARBOHYDRATES.pptx

1. Qualitative tests
of Carbohydrate

2. LEARNING OUTCOMES
To perform chemical tests that identify the
type of carbohydrate
To use chemical tests in identifying an
unknown carbohydrate

3. Introduction
• Carbohydrates are biochemical compounds
made of carbon, hydrogen, and oxygen.
• Carbohydrates are the key source of energy used by
living things.
• 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)
• In human body, the D-glucose is used.
• Simple sugars ends with –ose
H O
C
H C OH
HO C H
H C OH
H C OH
CH2OH
D-glucose

5. Types of Carbohydrates
• Monosaccharides are the simplest carbohydrates, they form building blocks of
disaccharides and polysaccharides
- Also called “simple sugars”, cannot be hydrolyzed to simpler carbohydrates
- Examples: glucose, fructose, galactose, ribose
all of which are structural isomers with the molecular formula C6H12O6.
• Disaccharides are two monosaccharides bonded together by a glycosidic bond.
- Examples: sucrose (table sugar, which consists of a glucose and a fructose unit
joined together by a glycosidic bond), lactose (milk sugar).
• Oligosaccharides - a few monosaccharides covalently linked.
• Polysaccharides are polymers (many units) of monosaccharides
- Can be split into many monosaccharides with acid or enzymes
- Examples: starch, cellulose, glycogen

6. Glycosidic bond
Glycosidic bond is a condensation reaction
between two sugar units, where the H-
group from one sugar interacts with the -
OH group on another to release water and
link the sugar units together to form a
polysaccharide.
A glycosidic bond or glycosidic linkage is a type of
ether bond that joins a carbohydrate molecule to
another group, which may or may not be another
carbohydrate

7. Monosaccharide are classified as ketoses or
aldoses depending on the functional
group.
• A ketose contains a carbonyl group (ketone) attached to two R
groups having one or more hydroxyl groups.
• An aldose contains terminal aldehyde group in addition to R
group containing -OH.
D -
esoculg
H O
C
H C OH
HO C H
H C OH
H C OH
CH2OH
aldose
 trioses (C-3)
 tetroses (C-4)
 pentoses (C-5)
 hexoses (C-6)
 heptoses (C-7)
 They can be classified by the number of carbon atoms
C H
HO
C OH
H
C OH
H
CH2OH
CH2OH
C O
D-fructose
ketose

8. Reducingandnonreducing sugars
ALDOSES are called REDUCING SUGARS because the aldehyde group reacts with
oxidizing agents to form a carboxylic acid.
KETOSES are called NONREDUCING SUGARS because Ketones do not react with
oxidizing agents
• 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.
Anomeric
carbon
v
v
reducing
Non-
reducing

9. 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
All monosaccharides are reducing sugars because all monosaccharides have
an aldehyde group (if they are aldoses) or can tautomerize in solution to form
an aldehyde group (if they are ketoses). This includes common
monosaccharides like galactose, glucose, glyceraldehyde, fructose, ribose,

10. NON-
Reducing
Sugars.
is not oxidized by
mild oxidizing agents.
Sucrose
All polysaccharides

11. Chemical
Reactivity
Tests

12. Benedict'stest
• 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.

13. Benedict's Test for Reducing
Sugars
• It is an oxidation-reduction reaction during which the
aldehyde group of the carbohydrate is oxidized and
the cu2+ ions in Benedict's reagent solution are
reduced.
• The result of the reaction is a change in the color of
the reaction solution from blue, which is due to the
presence of Cu2+, to a range of possible colors such
as gold, green, orange, or red.
• The ketone group in ketoses cannot be oxidized to a
carboxylic acid. Yet, fructose, which is a ketose,
gives a positive test with Benedict's reagent. This is
because it undergoes a structure rearrangement in a
basic medium and changes into a glucose molecule.
Sugars that react with Benedict's test are called
reducing sugars.

14. Benedict'stest
• 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 oxide.
reddish precipitate of copper
lactose
sucrose
glucose

15. Seliwanoff'sT
est
• This test is used to distinguish between aldoses (like glucose)
and ketoses (like fructose).
• Objective: To distinguish between aldose and ketone sucrose.
Ketoses react faster than aldoses with the test reagent. A change in the color of
the solution from colorless to deep red indicates that a reaction has taken
place. A positive test is marked by the deep red color and it indicates the
presence of a ketose.
-Ve
(pale yellow)
+ Ve
(orange color)

16. Iodine Test for Polysaccharides
•The large structure of a polysaccharide traps iodine (I2) and reacts with it to
form a complex with a deep blue color, as in the case of starch, or a red-
brown color, as in the case of amylopectin and cellulose.
•The smaller structures of monosaccharides and disaccharides do not trap
iodine, and therefore, they do not form the same deep-colored solutions.
a negative test (left) and a positive test (right)
+Ve
(deep orange)
Dextrin
+ Ve
(blue)
starch

17. Solubility
• Monosaccharide and disaccharide can be
dissolved freely in water because water is a polar
substance, while polysaccharide cannot be
dissolved easily in water, because, it has high
molecular weight , which give colloidal solutions
in water soluble.

18. Hydrolysis of Disaccharides and
Polysaccharides
The hydrolysis reaction of a disaccharide produces the two monosaccharide
units of which the disaccharide structure is formed.
Hydrolysis of polysaccharides produces smaller saccharides, which then
hydrolyze further into monosaccharides.
Hydrolysis of starch results in the formation of maltose in the first step. Then
maltose is hydrolyzed into glucose. Most of these hydrolysis reactions use an acid
as a catalyst to speed up the reaction.

19. 1- Molish ‘s Test :
(general test for carbohydrate)
(2ml) of suger
(0.5 ml) molish reagent
(2 drops) H2SO4 (con.) on wall
Purple ring
2- benedict ‘s Test :
(for reducing suger)
Starch
(-) blue
arabinose
(+) red
Sucrose
(-) green
(2ml) of suger
(1 ml) benedict reagent
heat for (3 min) in water bath
3- barfoed ‘s Test :
(for mono-suger)
Di suger
(-) blue
Mono suger
(+) red ppt
(1 ml) of suger
(1 ml) barfoed reagent
heat for (5 min) in water bath
4- bial ‘s Test :
(for pentose suger)
Glucose
(-) orange
Arabinose
(+) green-blue
(1ml) of suger
(1 ml) bial reagent
heat for (10 min) in water bath
5- seliwanoff ‘s Test :
(for ketose suger)
glucose
(-) yellow
fructose
(+) red
6- iodine ‘s Test :
(for poly suger)
Starch
(+) blue
Dextrin
(+) red
(1ml) of suger
(1 ml) seliwanoff reagent
heat for (5 min) in water bath
(1ml) of suger
(2 drops) iodine reagent

20. benedict ‘s Test :
(2ml) of suger
(1 ml) benedict reagent
heat for (3 min) in water bath
barfoed ‘s Test :
(1 ml) of suger
(1 ml) barfoed reagent
heat for (5 min) in water bath
bial ‘s Test :
(1ml) of suger
(1 ml) bial reagent
heat for (10 min) in water bath
seliwanoff ‘s Test :
(1ml) of suger
(1 ml) seliwanoff reagent
heat for (5 min) in water
bath
iodine ‘s Test :
(1ml) of suger
(2 drops) iodine reagent
benedict test
(+) Poly
(-) mono, di
Iodine test
(-) non-reducing (+) reducing
barfoed test
(-) di
(+) mono
seliwanoff test
bial test
(+) ketone
(+) pentose (-) hexose
(-) aldose

21. Molischtest
• This test is specific for all carbohydrates. Monosaccharide
gives a rapid positive test, Disaccharides and polysaccharides
react slower.
• Objective: To identify the carbohydrate from other
macromolecules lipids and proteins.

22. • 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
product.
α-naphthol Purpel color
furfural
α-naphthol
Purpel color
5- hydroxymethyl furfural

23. Method
Tube observation
1-glucose
2-ribose
3-sucrose
4-starch
1 Two ml of a sample solution is placed in a testtube.
2 Two drops of the Molisch reagent (which α-naptholin 95% ethanol) is
added.
3 The solution is then poured slowly into a tube containingtwo ml of
concentratedsulfuric acid so that two layers form, producing violet ring
appear as liaison between the surface separations.

24. Barfoed’sT
est
• This test is performed to distinguish between reducing
monosaccharides, reducing disaccharides and non reducing
disaccharides.
• Objective: 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.
• The nonreducing sugars give negative result.

25. • Barfoed’s reagent, cupric acetate in acetic acid ,
so in acidic medium , disacchride is a weaker
reducing agent than monosacchride, so mono
sacchride will reduce the copper in less time.

26. • 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).
Tube observation
1-glucose
2
- sucrose
3
- lactose

27. Bial’sT
est
• This test is used to distinguish between pentose and hexose
monosacharides.
• Objective: To distinguish between pentose monosaccharide
and hexose monosaccharide

28. • Principle: Bial’s test uses concentrated HCl as a dehydrating
acid and orcinol + traces of ferric chloride as condensation
reagent. The test reagent dehydrates pentoses to form
furfural. Furfural further reacts with orcinol and the iron ion
present in the test reagent to produce a bluish or green
product, while hexoses yield muddy-brown to grey
condensation product.

29. • Put 2 ml of a sample solution in a test tube.
• Add 2 ml of Bial's reagent (a solution of orcinol, HCl and ferric
chloride) 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.
Tube observation
1-glucose
2-ribose
3
- fructose

30. Test objective
Molisch test To identify the carbohydrate from other
macromolecules lipids and proteins
Benedict's test Benedict's reagent is used as a test for the
presence of reducing sugars.
Barfoed’s Test to distinguish between reducing
monosaccharides, reducing disaccharides
and non reducing disaccharides.
Bial’s Test To distinguish between pentose
monosaccharide and hexose
monosaccharide
Seliwanoff's Test To distinguish between aldose and keton
sugars
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