Qualitative Analysis of Carbohydrates (Assignment)

BT 12022 – Cell and Biomolecules
Practical number – 05
Qualitative analysis of Carbohydrates
O.N.M. Perera
196099
Group C2

Date – 05/12/2020
Practical number – 05
Title – Qualitative analysis of carbohydrates.
Objective - To be familiarized with how to perform different chemical tests and classify different
carbohydrate materials.
• Introduction – Carbohydrates are biomolecules. They are mainly consist of Carbon(C),
Hydrogen(H) and Oxygen(O). Normally they have 2:1 ratio of Hydrogen and Oxygen.
Carbohydrates are polymer of monosaccharides. They are the most abundant organic
compound in the living organisms. Carbohydrates are central to nutrition and are found in a
wide variety of natural and processed foods. Carbohydrates are abundant in cereals and
processed food based on cereal flour. In addition, types of carbohydrates such as
polysaccharides can be found in the cell wall of all plants, is one of the main components of
insoluble dietary fiber.
• Physical, Chemical and Biological properties of carbohydrates are depend on their primary
structures and less frequently on their high order structures. They have (CH2O)n like empirical
formula. Carbohydrates organized in the form of aldehydes or ketones with multiple hydroxyl
groups coming off the carbon chain.
• Carbohydrates provide,
1. Energy and regulation of blood glucose.
2. Sparing the use of proteins for energy.
3. Breakdown of fatty acids and preventing ketosis.
4. Biological recognition processes.
5. Flavor and Sweeteners.
6. Dietary fiber.
• There are three main groups of carbohydrates are monosaccharides and polysaccharides. The
simplest carbohydrates are monosaccharides from which disaccharides and polysaccharides are
created. Monosaccharide’s are crystalline solids which are freely soluble in water and insoluble
in nonpolar solvents. There are two types of monosaccharide’s are aldoses and ketoses.
Monosaccharide’s except dihydroxyacetone have asymmetric centres since they contain one or
more asymmetric or chiral carbon atom and hence exist in optically active isomeric forms.
Simple monosaccharide’s are reducing agents and Disaccharides contain a Glycosidic bond.
Usually carbohydrates can be found in nature occur as polysaccharides.
1. Benedict's test: the reduction of sugars when heated in the presence of alkali is converted to a
potent reducing species known as enediols. When the reagent solution of Benedict and the
sugar reduction are heated together the solution changes its color to orange-red/brick red.

2. Oxidation: Monosaccharides reduce sugars if their carbonyl groups oxidize to create
carboxylic acids. In Benedict's test, D-glucose is oxidized to D-gluconic acid and thus glucose
is considered to be reducing sugar.
3. Reduction to alcohols: The C=O groups in open-chain forms of carbohydrates can be reduced
to alcohols by sodium borohydride, NaBH4, or catalytic hydrogenation (H2, Ni, EtOH/H2O).
The products are known as “alditols”.
Carbohydrates are divided into 4 groups, They are
1.Monosaccharides
2.Disaccharides.
3.Polysacchaides.
4. Oligosaccharides.
1. Monosaccharides
Monosaccharides are the simplest group of carbohydrates. They cannot be further hydrolyzed.
Therefore, they referred to as simple sugars. They are colorless, crystalline solids that are soluble in
water and insoluble in non-polar solvents. The general formula is Cn(H2O)n and CnH2nOn.
They can be categorized on the basis of the number of carbon atoms they contain and also on the basis
of the functional group present.
Monosaccharides are having 3,4,5,6,7... carbons are respectively called trioses, tetroses, pentoses,
hexoses, heptoses, etc., and even aldoses or ketoses, depending on whether they contain aldehyde or
ketone.
Trioses
It contains three carbon atoms. It has C3H6O3 general formula. There are only two trioses, an aldotriose
(glyceraldehyde) and a ketotriose (dihydroxyacetone). Trioses are important in respiration. Namely,
lactic acid and pyruvic acid are derived from aldotriose and ketotriose, respectively.
Tetroses
Tetrose are containing four carbon atoms. The general formula is C4H8O4
. Example is D- Erythrose-4-P is an intermediate in hexosemonophosphate shunt which is an alternative
of glucose oxidation.
Pentoses
Pentose are containing five carbon atoms. The general formula is C5H10O5.
Examples are -

• D- ribose is a constituent of RNA and many co-enzymes e.g. FAD, NAD.
• D-2 deoxy is a constituent of DNA component of DNA.
• D-Lyxose is a constituent of lyxoflavin found in the human heart.
• D- arabinose is a constituent of plant cell wall
• Phosphate esters of D- Ribulose and D- xylose occurs as an intermediate in the HMP pathway.
Hexoses
Hexoses are containing six carbon atoms. The general formula is C6H12O6.
I. D – Galactose
They are very rarely found to be free in nature as a constituent of milk sugar lactose and in tissues such
as galactolipids and glycoproteins.
II. D – Mannose
D- Mannose used to stamp proteins via the glucosylation process. It can’t be found in free in nature but
is commonly distributed in combination as polysaccharide mannan, e.g. ivory nut. It is also considered
to be a constituent of glycoproteins
III. D- Fructose
it is a ketohexose and is commonly called the fruit sugar, as it occurs in fruit. It is a sweet sugar sweeter
than glucose and sucrose. It is found in honey as laevulose. In the seminal fluid of man fructose is the
chief source of energy for sperms.
2. Disaccharides
Disaccharides are glycosides formed from two monosaccharides that may be aldoses or ketoses. Each
of the—OR groups is given by the original cycle for the hemiacetal or hemiketal distribution. The
second—OR group is derived from an aglycone; it is the second monosaccharide that supplies the
alcohol to the glycosid bond functional group. Maltose, Sucrose, lactose are common disaccharides.
Glucose + Fructose Sucrose

Galactose + Glucose Lactose
Glucose + Glucose Maltose
3. Polysaccharides
Polysaccharides contain more than 2 monosaccharide units. Polysaccharides differ from each other in
the identity of their recurring monosaccharide units, in the length of their chains, in the types of bond
linking units and in the degree of branching. They are further classified depending on the type of
molecules produced as a result of hydrolysis. They are homopolysaccharidese, containing
monosaccharides of the same type orheteropolysaccharides. Examples of Homopolysaccharides
are starch, glycogen, cellulose, pectin. Heteropolysaccharides are Hyaluronic acid, Chondroitin.
Polysaccharides can divide further,
1. Based on their function
Storage- Starch, Glycogen, Inulin
Structural- Cellulose, Hemicellulose, Pectin, Chitin
2. Based on their architecture
Linear forms- Cellulose, Amylose

Branched forms- Glycogen, Amylopectin, Hemicellulose
3. Based on the homogeneity of the monomer
Homopolysaccharides (homoglycans)
Starch – amylose and amylopectin are the two types of starch Monomer of both is glucose
Glycogen – glucose
Cellulose – glucose
4. Heteropolysaccharides (heteroglycans)
Glycosaminoglycans (composed of an acidic sugar and amino sugar) – heparin, hyaluronic acid,
proteoglycans
5. Based on the oxidoreductive properties
Reducing sugars – contain hemiacetal or hemiketal group – glucose, galactose, fructose, maltose,
lactose
Non-reducing sugars – contain no hemiacetal groups – sucrose and all polysaccharides
4. Oligosaccharide
Oligosaccharide is a saccharide polymer containing a small amount of monosaccharides.
Oligosaccharides can have many roles, including cell recognition and cell binding. Oligosaccharides
are formed when two or more monosaccharides join together by O-glycosidic bonds. Examples
include sucrose, lactose and maltose. Specific enzymes are used to catalyze the glycosidic bonds in
olgosaccharides and each sugar must be specific to each enzyme used for each new glycosidic bond
Procedure:
1. Iodine test
1. 2 ml of each sample was added in separate test tubes.
2. Four drops of I2/KI solution was added. (0.01 M iodine in 0.12 M KI).
3. The solution was shaken.
2. Barfoed’s Test for Reducing Monosaccharides
1. 1 ml of each sample was added to separate test tubes.
2. 2 ml of Barfoed’s Reagent was added.
3.Tubes were mixed and placed in boiling water bath for 5 min.
4. the mixture was cold for few minutes.
If the reaction mixture was not positive for the test, Mixture was boiled for other 5 minutes.
3. Benedict’s Test for Reducing Carbohydrates
1. 5 drops of each sample was added to separate test tubes.

2. 2 ml of Benedict’s reagent was added.
3. Tubes were mixed and placed in boiling water bath for 5 min.
4. Fehling’s test
1. 2 ml of given sample was added to the boiling tube.
2. 1 ml of Fehling’s A solution was added to the tube containing the sample.
3. 1 ml of Fehling’s B solution was added to the same tube.
4. Tube was mixed well and heated over a Bunsen flame.
Discussion:
1. 1. Other tests that can be used to identify carbohydrates and Chemistry of each test
performed?
There are more tests for identify Carbohydrates. Iodine test, Barfoed’s test, Benadict test and
Fehling’s tests are the most used tests. Except those tests, there are few another tests to identify
Carbohydrates such as Molisch’s test, Tollen’s test….
1. Molisch’s test
• This test is given by almost all of the carbohydrates. In this test concentrated HCl acid converts the
given carbohydrate into furfural or its derivatives, which react with α-naphthol to form a purple
coloured product. The appearance of purple or violet ring confirms the presence of
carbohydrate.
.
• Procedure -
1. Take 2ml of the given sample solution in a clean test tube.
2. Add 2-3 drops of Molisch reagent slowly.
3. Now add concentrated sulfuric acid along the sides of the test tube.
4. The acid layer forms a layer at the bottom.

5. Note the junction of the two layers.
6. If there is a formation of the violet ring then the presence of carbohydrate is confirmed.
2. Tollen’s test
• Carbohydrates reacts with Tollens reagent forms a silver mirror on the inner walls of the test tube.
This confirms the presence of reducing sugars. Silver ions are reduced to metallic silver.
• Procedure –
1. Take the given sample solution in a clean test tube.
2. Add 2-3ml of tollens reagent to it.
3. Keep the test tube in a boiling water bath for 10 minutes.
4. If there is the appearance of shiny silver mirror confirms the presence of reducing sugars.
3. Bial’s Test
• It is responsive only to pentoses. Any compound containing pentose sugar will give a positive
Bial test. Pentases form furfural compounds in the presence of concentrated acid. Furfural
compounds formed by pentoses are condensed with orcinol to form blue colored compounds.
Bial reagent is used to dissolve 300 mg of orcinol in 100 ml of concentrated HCL and 0.25 mL
of ferric chloride solution.. The test is positive for all the compounds that contain pentose sugar
like DNA, RNA, etc.
• Hexoses form green color with Bial’s reagent.
• Procedure -
1. Add 3 ml of Bial’s reagent in an empty test tube
2. Add 3 ml of test solution to the above test tube
3. Heat the test tube in boiling water bath
4.Allow the solution to cool at room temperature

4. Barfoed’s Test
• The Barfoed test is a test to differentiate between monosaccharides and disaccharides. It is also
focused on the reduced capacity of sugar. Monosaccharides give early positive tests, and
disaccharides give late positive results. Reducing sugar is tautomerized in a mildly acidic
medium to form enediols. These enediols convert cupric ions to cuprous ions that form cupric
hydroxide. This cuprous hydroxide has been converted to cuprous oxide for heating and
precipitation. Its principle is similar to Benedict’s test except the acidic environment.
• If there are monosaccharides, can be seen red precipitates after the initial first 5 minutes
indicates. If precipitates are formed after 15 minutes, a disaccharide is present in the test
solution.
• Procedure -
1. Take 2 ml of Barfoed’s reagent in a test tube
2. Add 2 ml of the test solution to the above test tube
3. Mix the solutions
4. Hold the test tube on flame and boil for minutes
5. Allow to cool at room temperature
6. Look for the precipitates
7. If no precipitates are formed, boil for an additional 10 minutes
8. Allow to cool and look for the precipitates
5. Seliwanoff’s Test
• This test is used for the identification of monosaccharides. It is commonly used to distinguish
fructose, keto sugar, glucose and galactose. This test includes the formation of furfural
derivatives with hydrochloric acid monosaccharides. Furfural derivatives formed by a sugar
with a ketone functional group of resorcinol condensate to form a chromogene with a cherry-
red color. Seliwanoff’s test is specific only for hexoses having a ketonic functional group.
Sucrose also gives a positive test because it is hydrolyzed to glucose and fructose. It is highly
sensitive for sucrose even at 1% concentration.
• Procedure -
1. Take 3 ml of Seliwanoff’s reagent in a test tube
2. Add 1 ml of test solution in the above test tube
3. Hold the test tube on flame and allow to boil for 30 seconds

4. Allow to cool at room temperature
6.Phloroglucinol test
• This test is specifically designed to detect galactose and lactose in a solution. This test also
includes the development of furfural derivatives in the presence of concentrated HCL. The
furfural derivatives formed by galactose are then condensed with phloroglucinol to form a red-
colored compound.
• As the solution is heated and cooled, it turns yellow to red. The change in color to red shows
the presence of galactose in the solution. Lactose also gives this test a positive effect as it is
hydrolyzed by acid to create glucose and galactose. To make a distinction between the two,
perform the Barfoed test. A similar red-colored compound with phloroglucinol also forms
furfural compounds formed by pentoses having a keto group.
• Procedure
1. Take 2 ml of test solution in a test tube
2. Add 2 ml of Tollen’s reagent to the above test tube
3. Mix the two solutions thoroughly
4. Hold the test tube on flame and boil for some time
5. Allow to cool at room temperature
7.Osazone Test
• This test is a confirmatory test for carbohydrates. It gives final inference about the type of
carbohydrate in the solution. Osazone derivative of carbohydrate forms specific crystals that
are characteristic of the carbohydrate. The shape of the crystal tells us about the present
existence of the carbohydrate. Phenyl hydrazine responds to the formation of phenylhydrazone
by reducing sugar in an acidic atmosphere at high temperatures.
• Procedure
1. Take 5 ml of the given solution in a test tube
2. Add 3 pinches of osazone mixture to the above test tube
3. Mic thoroughly
4. Hold the test on flame and boil for 5 minutes
5. Check for yellow crystals. If not formed boil further

6. Keep checking after every 5 minutes for yellow crystals
7. Once the crystals are formed, allow the solution to cool at room temperature
8. Take the crystals out and prepare slide
9. Observe the slide under the microscope
Carbohydrates are found in foods like breads, vegtables, fruits and dairy products. It is most abundant
biomolecule on the earth. It produced by the conversion of more than 100 billion metric tons of Carban
dioxides and Water in photosyenthesis.
Carbohydrates are source of energy, it provides energy for brain, kidneys, heart muscles, and central
nervous system and it prevents the breakdown of proteins for energy. It helps to keeps blood
cholesterol levels in check. The extra energy stored as fat it’s called fat metabolism. Carbohydrates
supply carbon for synthesis of another compounds.
Structural components of plants (cellulose). Central to materials of industrial products: paper, lumber,
fibers. Most absorbed carbohydrates such as glucose used to make energy, some glucose is converted
to ribose and deoxyribose, which are essential building blocks of important macromolecules, such as
RNA, DNA, and ATP.
Carbohydrates are an important component of many industries such as textiles, paper, lacquers and
breweries. To some degree, detoxification of physiological value is done with carbohydrate
derivatives. Agar is a polysaccharide used in culture media, laxatives and food.
They continue to make up the body mass by being present in all areas of the cells and tissues.
Carbohydrates are part of genetic material, such as DNA and RNA, in the form of deoxyribose and
ribose sugars.
They form components of bio-molecules that play a key role in blood clotting, immunity, fertilization,
etc. They form components of bio-molecules that play a key role in blood clotting, immunity,
fertilization, etc.
It provides sweetness to foods. Carbohydrates is basically the main fibre of the diet or provide the bulk
fibre for better digestion. Starch is the form the food is stored in plants. Carbohydrates help clear gut
and prevent constipation. Starch is the form the food is stored in plants.
It gives sweetness to the food. Pectine and hemiceliulose are structural carbohydrates in the walls of
plant cells. It plays a significant role in cell recognition processes. Chitin forms the cell wall of the
fungi and the outer insect schelitone. Murine is a structural carbohydrate in the wall of bacterial cells.
By using mentioned tests, can identify easily whether Carbohydrates are presents in the unknown
sample. If it does not contain a carbohydrate, further tests can be performed to classify and possibly to
identify it. In addition can identify types of Carbohydrates such as whether it is monosaccharide,
disaccharide or polysaccharide.

Simple carbohydrates containing a free ketone or aldehyde functional group can be identified with
Benedict’s test.
From Fehling's test can differentiate between water-soluble carbohydrate and ketone functional groups,
and as a test for reducing sugars and non-reducing sugars.
By doing Molisch's test can determine the presence of carbohydrates or sugars in the substance .
Bial's test is helps distinguishing pentoses sugar from hexoses sugars.
From Seliwanoff's test ican distinguishes aldose and ketose sugars.
By doing Osazone test allows to differentiation of different reducing sugars on the basis of the time of
appearance of the complex.
Exercise -

Conclusion – There are more types of Carbohydrates and there are various tests to identify them.
1. Iodine test is used to differentiate polysaccharides from the rest of carbohydrates and
differentiate between glycogen, starch, and cellulose.
2. Benedict’s test is used to determine reducing sugars.
3. Fehling’s test is used to identify reducing sugars (but is known to be NOT specific for
aldehydes)
4. Barfoed’s test is used to distinguish reducing monosaccharides from reducing
disaccharides
5. Phloroglucinol test is used to detect galactose and lactose in a solution
6. Tollen’s test confirms the presence of reducing sugars
7. Osazone test is used to detect reducing sugars.
8. Seliwanoff’s test is used for the identification of monosaccharides
9. Bial’s Test is responsive only to pentoses
10. Molisch’s test is given positive result for carbohydrates

O.N.M. Perera
196099
Group C