Names: Joshua Carvajal Date: March 2, 2020

Clint Danniel M. Guarin

Ginafer S. Enomar

Activity 7: Qualitative Analysis of Carbohydrates

I. Introduction
One of the essential macromolecules is the carbohydrates, which are classified into
subtypes; monosaccharides, disaccharides, and polysaccharides. Monosaccharides are also
called simple sugars, made up of three to seven carbon chains, either linear or ring-shaped.
Glucose, galactose, and fructose are monosaccharides isomers. Glucose, the most common
monosaccharide, is used as an energy source, especially during cellular respiration.
Disaccharides are formed by two monosaccharides through dehydration reaction. They are
readily available energy stores. Examples are maltose (two glucose), sucrose (glucose and
fructose, and lactose (glucose and galactose). Polysaccharides are long chains of
monosaccharides linked by glyosidic bonds and may be branched or not. They are essentially
large sugar molecules, and can’t be digested directly. Starch, glycogen, cellulose, and chitin are
common examples of polysaccharides. Glycogen, in order to be used as energy, must be
converted into glucose.
The stoichiometric formula of carbohydrates is (CH2O)n, where n is the number of
carbons in the molecule. This is to say that the ratio of carbon to hydrogen to oxygen is 1:2:1,
which can be helpful when trying to identify a molecule as a carbohydrate. The experiment
below will explore the reactions of different substances to certain reagents that will ultimately
identify the presence of sugar molecules.
II. Objectives
At the end of the experiment, the students should be able to:
1. To study the properties of carbohydrates
2. To study some simple tests of carbohydrates
3. To characterized present in an unknown solution on the basis of various

III. Materials
Molisch reagent Test tube
Iodine solution Test tube rack
Seliwanoffs reagent Beaker
Benedict’s reagent Stirring rod
 Barfoeds reagent Stove
Osazone test
Bials reagent
Phenyl hydrazine reagent

IV. Procedures
A. General Test for Carbohydrates
Molisch Test
1. 1 ml of each sample (1% carbohydrate solution of sucrose, starch, maltose, lactose, saliva,
food sample, distilled water, and galactose) was placed separately in test tube.
2. 2 drops was put in each solution.
3. 2 ml of cons. H2SO4 was put in the test tube and slightly tilted.
4. The color of the interface between two layers was noted.
B. Iodine Test for Polysaccharides
1. 3 drops of each sample was put (1% solution of starch, milk and fruit juice separately on a
spot plate).
2. 1 drop of iodine solution was put.
3. The color obtained was noted.
C. Mucic Acid Test
1. 0.2 g fructose, 0.1 galactose was put separately in a test tube.
2. 2 ml of H2O was put and dissolve solids through heating.
3. 2 ml of concentrated HNO3 was put into boiling water bath for 1 hour in a hot plate.
4. The test tubes was taken and slowly allowed to cool after the heating period.
5. The test tubes was scratch with clean stirring rods to induced crystallization.
6. The test tubes was to cool at room temperature.
7. The test tube was put in an ice bath.
8. The test tubes were reserved until the next laboratory to complete the crystallization.
9. 2 ml of H2O was put to confirm the insolubility of the solid formed, shake the resulting
mixture.
D. Hydrolysis of Polysaccharides
1. 5 ml of each 1% solution (starch and sucrose) was put in each test tubes.
2. 5 drops of concentrated HCl was put.
3. It was cover with marble and boiled in a water for 30 minutes.
4. Hydrolysis was reserved for the next experiment.
E. Qualitative Test for Sugars
 E1. Benedict’s Barofoed’s, Seliwanoff’s Test
 1 ml of each solution with 2 ml of Benedict’s, Barofoed’s, Seliwanoff’s reagent was put
in each test tube.
 The solution was put in a boiling water bath until a positive responds.
 The changes of color was recorded.
E2. Bial’s (Orcinol) Test
 1 ml of each solution was put in each test tubes.
 1.5 ml of Bial’s reagent was put.
 The test tubes was heated carefully in a flame until misture begins to boil.
 The color produced was noted.
 2.5 ml of water and 0.5 ml pentanol was put and shaken.
E3. Osazone Test
 2 drops of test tube solution (fructose, sucrose, lactose, maltose, and galactose, and
hydrolysates of starch, and hydrolysates of sucrose) was put in each test tubes.
 4 drops of freshly prepared phenyl reagent was put.
 The test tubes was put in a boiling waster bath.
 Time was recorded when yellow crystals appeared.
V. Results and Discussion
Table A. General Test for Carbohydrates; Molisch Test
Samples Molisch Reagent After Adding Sulfuric Acid
Galactose Reddish violet solution with a
thin purple-colored ring
formed; reacted rapidly
Maltose Reddish violet solution with a
purple-colored ring formed;
reacted slowly
Lactose Light reddish violet solution
with a dense purple-colored
ring formed; reacted slowly
Sucrose Reddish violet solution with a
thin purple-colored ring
formed; reacted slowly
Milk Cloudy reddish violet solution
with a thin purple ring formed;
reacted slowly
Fruit Juice Cloud reddish violet solution
 with a thin purple ring formed;
reacted rather rapidly
Saliva Reddish violet solution with a
thick purple-colored ring
formed; some salivary bubble
remained; reacted slowly
Distilled Water Serves as the control ,so no
reaction
Starch Light reddish violet solution
with a purple-colored ring
formed: reacted slowly
Food Sample (Cheese Stick) Cloudy reddish violet solution
with a thick purple-colored
ring formed: reacted slowly

Table B. Iodine Test for Polysaccharides

Samples Iodine Test
Starch
Gum-Arabic
Agar-Agar2
Milk
Fruit Juice

Table C. Mucic Acid Test

Samples Mucic Acid Test: After Refrigeration
Fructose
Glucose
Galactose

Table D. Hydrolysis of Polysaccharides

Samples Color of Solution After 30 minutes of heating
Starch
Sucrose

Table E1. Qualitative Test for Sugars

Samples Benedict’s Test Barfoed’s Test After Heating
Galactose
Fructose
Maltose
Lactose
Sucrose
Milk
 Fruit Juice
Starch
Distilled Water
Saliva

Table E2. Bial’s Test

Samples Color after heating After adding with 2.5 ml
water and 0.5 butanol
Galactose
Fructose
Maltose
Lactose
Sucrose
Starch
Saliva
Milk
Fruit Juice
Distilled Water

Table E3. Osazone Test

Samples Osazone Test
Galactose
Fructose
Maltose
Lactose
Sucrose
Hydrolysates of Starch
Milk
Fruit Juic

VI. Questions with Answers

1. For the reaction of monosaccharides below, give the product responsible for its color?
For the reaction of monosaccharides below, give the product responsible for its color.
Depending on type of carbohydrate test conducted, the product produced by the reactions of the
monosaccharides experimented will result accordingly. The answer in question 5 breaks this
down in detail, and provides which monosaccharides yielded a positive reaction.

2. Suppose you saw no sign of a color change in the Benedict’s test and a dark red colored
solution with the Seliwanoffs test. Indicate which sugars you could have?

3. Suppose you saw a red ppt with Benedict’s test and a yellow solution after more than five
minutes with the Seliwanoff test. Indicate which sugar you could have?
4. Give the significance of the following test for carbohydrates.
Molisch’s Test – used to check for the presence of carbohydrates in a given analyte
Iodine Test for Polysaccharides – as the name suggests, this makes use of iodine to
distinguish starch from monosaccharides, disaccharides, and other polysaccharides
Mucic Acid Test – used to test for galactose; concentrated HNO 3 is heated along with an
aldose sugar to give a dicarboxylic acid called mucic acid that is formed from galactose,
and is insoluble in cold aqueous solutions
Benedict’s Test – used to test for reducing sugars (free or potentially free aldehyde or
ketone groups)
Barfoed’s Test – used to distinguish monosaccharides from di- and polysaccharides with
lower pH conditions and shorter incubation time
Seliwanoff’s Test – used to differentiate between ketoses and aldoses
Bial’s Test – used to detect the presence of furanoses (five-membered rings)
Osazone Test – one of the normal procedures for identifying sugars wherein such sugars
will reduce as a result of the reaction between the monosaccharide and phenyl hydrazine
(a crystalline compound), which will then form osazones. It involves two reactions:
1. Phenylhydrazine is involved in oxidizing the alpha carbon to a carbonyl group
2. Removal of one water moecule with the formyl group of the oxidized carbon
and the formation of a similar carbon-nitrogen bond.
5.What are the substances responsible for giving a positive color reaction with the different tests
for sugars?

For the Molisch’s test, the formation of a purple or a purplish-red ring at the point of contact
between the sulfuric acid and the analyte plus the Molisch’s reagent mixture confirms the
presence of carbohydrates in the analyte.
Substances positive for this reaction are: (itype diri ang mga positive)

For the iodine test, iodine interacts with the coiled molecules of glucose and becomes bluish-
black in color, indicating the presence of starch, while a yellow-ish brown solution is a negative
reaction.
Substances positive for this reaction are: (itype diri ang mga positive)

For the mucic acid test, the formation of insoluble precipitate is an indication of of the
presence of galactose.
Substances positive for this reaction are: (itype diri ang mga positive)

For the hydrolysis of polysaccharides, polysaccharides (and even disaccharides) will react
positively under acidic conditions (such as adding HCl into the solution). Since starch is a
polysaccharide and sucrose is a disaccharide, both can be hydrolyzed to monosaccharides.

For the Benedict’s test, copper is reduced and the product forms a red precipitate.
Substances positive for this reaction are: (itype diri ang mga positive)

For the Barfoed’s test, a rusty or brownish-red color will indicate monosaccharides; no color
change or weak change will indicate either a disaccharide or polysaccharide.
Substances positive for this reaction are:

For the Seliwanoff’s test, the concentrated acid (HCl) when heated along with a sugar will
produce furfural or hydroxymethylfurfural, which further reacts to give a red color. If the
solution turns red after < 1 min, this indicates the presence of a monosaccharide ketose. If the
solution turns red after ~ 1 min, this indicates the presence of a disaccharide ketose. If the
solution turns red after > 1 min, this indicates the presence of an aldose.
Substances positive for this reaction are:

For the Bial’s (Orcinol) test, the yellow color turns into greenish is the sugar is a furanose (has
a five membered ring-like furan), green/olive indicates a pentose-furanose, muddy brown
indicates a hexose (or higher)-furanose, and no color change indicates a pyranose.
Substances positive for this reaction are: (itype diri ang mga positive)

As for the Osazone test, the sugar present is indicated by the crystals formed: different petal-
shaped crystals indicates the presence of maltose; puff-shaped crystals indicates the presence
of lactose; and phombi-plate shaped crystals indicates the presenhce of galactose.

6. What factors may interfere with producing negative color reactions?

Negative color reactions are typically caused by conditions that are not met for the
reaction to have positive results. For example, if the polysaccharide tested is not starch, then the
iodine test will be negative, resulting to a yellow-ish brown solution. Temperature, time, and
volume ratios are also influential factors, and depending on the stringency of a particular
carbohydrate test procedure, these factors may have to be as exact as possible to yield accurate
results.

7. What is the physiological importance of knowing the specific classes of carbohydrates?

Carbohydrates are classified based on their glycosidic linkages — the more linkages they
have, the more complex the sugar molecule is. This also means that that more glycosidic
linkages means a bigger sugar molecule, which relates to how they are needed to be broken
down in the first place during the body’s ingestion, digestion, and absorption, or more
commonly known as metabolic processes. The mouth starts breaks down the starch, then by the
time that starch reaches the small intestine, it is turned into a disaccharide (maltose, sucrose, or
lactose) by enzymes. Finally, before it enters the glycolysis, the disaccharide is broken down into
glucose, a simple sugar, by intestinal organs (mucosal cells and lining). It is indeed necessary to
realize why the structures of these sugar molecules matter, especially since stereoisomers exist,
leading to the immense diversity of sugar structure and function.

8. Characterize carbohydrates based on the following reactions:

a. Oxidation
Carbohydrates, by nature, contain many alcohol groups as well as a carbonyl group
which implies that under proper conditions, they can undergo oxidation reactions. For
instance, under mild aldehyde conditions, the aldehyde group of an acyclic sugar can be
transformed into carboxylic acid to form the molecule aldonic acid. Bromine water is a
common reagent for this type of reaction.
b. Reduction
Between their cyclic and acyclic forms, carbohydrates can exits in dynamic equilibrium,
but only the acyclic form can potentially undergo a reduction reaction in which the
aldehyde group is converted into alcohol in the presence of some type of reducing agent
such as sodium borohydride. Sugars that can form acyclic versions in which they
contain the aldehyde group and thus undergo reduction are known as reducing sugars.
c. Hydrolysis
Disaccharides and polysaccharides can be hydrolyzed under acidic conditions to
produce monosaccharides by breaking the glycosidic links (ether bonds) between
monomer units in the structure of the molecule. Through the help of necessary enzymes,
consumed food can be digested.
d. Reaction with strong NaOH
 e. Dehydration with strong HCl
Simple sugars undergoing dehydration reactions will be synthesized into their more
complex forms — a disaccharide. Common dehydrating reagents sulfuric acid and
alumina. Hydrolysis is the basically the reverse reaction of dehydration.

9. What are glycosides? Amino sugars? Explain the clinical significance of glycosides and amino
sugars.
Glycosides are compounds formed when the anomeric (hemiacetal or hemiketal)
hydroxyl group of a monosaccharide undergoes condensation with the hydroxyl group of a
second molecule, with the elimination of water. In living organisms like, like plants for example,
store chemicals in the form of inactive glycosides, which can be activated by enzyme
hydrolysis. This causes the sugar to be broken off, making the chemical available for use. Many
of these plant glycosides can then be used for medications. Amino sugars are formed by the
replacement of one of the hydroxyl groups of a sugar by an amino group or substituted amino
group. One of their roles is to the basic structures in most of the lubricating fluids in the body,
in the basement membrane which organizes cells into tissues and in other important
biomolecules.

10. What are stereoisomers? Do all isometric sugars have the same physical and chemical
properties?
Stereoisomers are molecules that have the same molecular formula and sequence of
bonded atoms, but differ in the three-dimensional orientations of their atoms in space. For
example, cis and trans isomers have different physical and chemical properties, since atoms may
be place on the same side of the double bond (cis) or on opposite sides of it (trans). The same is
true for isometric sugars. Glucose, for example, has 16 possible stereoisomers.

VII. Conclusion

VIII. Documentation
A. General Test for Carbohydrate: Molisch Test
 From left to right From left to right
1st Test tube: Galactose 6th Test tube: Fruit Juice
2nd Test tube: Maltose 7th Test tube: Saliva
3rd Test tube: Lactose 8th Test tube: Distilled water
4th Test tube: Sucrose 9th Test tube: Starch
5th Test tube: Milk 10th Test tube: Food sample
(cheese stick)

After Adding Acid: 1-5 After Adding Acid: 6-7

B. Iodine Test for Polysaccharides

From left to right

1st Test tube: Starch
2nd Test tube: Gum-arabic
3rd Test tube: Agar-agar2
4th Test tube: Milk
5th Test tube: Fruit Juice
C. Mucic Acid Test

1st Test tube: Fructose

2nd Test tube: Glucose

D. Hydrolysis of Polysaccharides

1st Test tube: Sucrose

2nd Test tube: Starch

E1. Qualitative Test for Sugars

 Benedict’s Test and Barfoed’s Test (left to right)
 Galactose, Fructose, Maltose, Lactose, and Sucrose has Saliwanoff solution on the
last test tube in each picture

Galactose Fructose
 Maltose Lactose

Sucrose Milk

Fruit Juice Starch

Distilled Water Saliva

E2. Bial’s Test

Galactose Fructose

Maltose Lactose

Sucrose Starch

Saliva Milk
 Fruit Juice Distilled Water

E3. Osazone Test

IX. References