I.

INTRODUCTION

Carbohydrates are one of the three principal classes of foods, and is

source of energy in our diets. Carbohydrates are compounds composed of
carbon, hydrogen and oxygen containing a ketone or aldehyde functional
group and alcohol group. These compounds can be characterized based
on the number of carbons (pentose, hexose), the functional group (ketone,
aldehyde), formation of ring (pyranose, furanose), isomers and the number
of monosaccharide units in the molecule (monosaccharide, disaccharide,
oligosaccharide and polysaccharide).

Monosaccharide is also known as simple sugars, the simplest form

of sugar and the most basic unit of carbohydrates. The general formula of
monosaccharide is CnH2nOn, and they cannot be further hydrolysed to
simpler chemical compounds. They are usually colourless, water-soluble
and crystalline solids (McMurry, J., 2008).
Disaccharides are formed from two monosaccharides and can be
classified as reducing sugar or non-reducing sugar. Reducing sugar is any
sugar that is capable of acting as a reducing agent because it has free
aldehyde or free ketone group, while non-reducing sugar has glycosidic
bonds between their anomeric carbons, thus cannot convert to an open-
chain form with an aldehyde group, they are stuck in the cyclic form (Pratt,
C.W., Cornely, K., 2013).
Oligosaccharide is a saccharide polymer containing a three to ten of
monosaccharides. This saccharide polymer can have many functions
including cell recognition and cell binding, they are normally present as
gylcans (Bruce, A., et. al, 2002).
The lastly, the polysaccharide, they are polymeric carbohydrate
molecules composed of long chains of monosaccharide units bound
together by glycosidic linkages, and on hydrolysis give the constituent
monosaccharides, they can be amorphous or even insoluble in water (Varki,
A., Cummings, R., et. al, 2000). Polysaccharide can be;
homopolysaccharide, composed of monosaccharide of the same type and
heteropolysaccharide are composed of different type of monosaccharides
(Matthews, C.E., et. al, 2001).

To verify the above concept, an experiment was conducted with the

following objectives: (1) to identify the reducing sugar from non-reducing
sugar; (2) to differentiate aldoses-containing to keotses; and (3) to know the
different test for carbohydrates.
II. Methodology

A. Materials and Apparatus

The chemicals used in the experiment are as follows: Molisch’s

reagent, Concentrated Sulfuric Acid (H2SO4), Dilute Iodine solution,
Concentrated HCl,, 0.5 mL of Bial’s reagent, Fehling’s reagent, Benedict’s
reagent, Barfoed’s reagent, Dilute Sodium Hydroxide, Seliwanoff’s reagent,
2% Ribose, 2% Maltose, 2% Sucrose, 2% Galactose, 2% Lactose, 2%
Fructose, 2% Glucose, 1% Starch. The apparatus included droppers, test
tube, test tube brush, test tube holder, 250 mL beaker, alcohol lamp, wire
gauze, iron stand and iron ring.

B. Procedure
I. Qualitative Test for Carbohydrates
1. Molisch’s Test

Three separate test tube was prepared and 2mL of

carbohydrates samples (glucose, maltose and starch) was added in
each test tube. Each test tube was added 2 drops of Molisch’s reagent
and mixed thoroughly. The test tube was slanted and the 2mL of conc.
H2SO4 was added, then test tube was erected slowly. Reddish violet ring
at the junction of two liquids was formed and this indicates the presence
of carbohydrates.
2. Iodine test
One or two drops of dilute iodine solution was added to 2-3mL of
dilute starch paste. The colour that develop was observed. Half of the
tube of starch was placed to another tube and heated it in boiling water
bath for 10 minutes. The two test tube was compared and the
observation was noted.
3. Bial’s Test for Pentose

One mL of sugar solution (glucose, fructose, maltose and

sucrose) was placed in a test tube then 3mL of concentrated HCl and
0.5mL of Bial’s reagent was added. The test tubes was heated in a
boiling water bath for one minute. The observations of different sugars
was observed.
 4. Seliwanoff’s Test

Two mL of the sugar solutions (glucose, fructose, maltose and

sucrose) was placed in separate test tubes, then 2mL of Seliwanoff’s
reagent was added. The test tubes was placed in a boiling water bath
for about 2 minutes. The colour change was observed.

5. Reduction Tests for Sugars

A. Fehlings Test
One mL of sugar solutions (glucose, fructose, maltose and
sucrose) was added to 2mL of Fehling’s reagent solution (contain
equal volumes of Fehling’s A and Fehling’s B) and the mixture was
heated. the production of yellow or brownish red cuprous oxide
precipitate indicates the presence of reducing sugars.
B. Benedicts’ Test

Two mL of Benedicts’ reagent was added to 1mL of sugar

solution (glucose, fructose, maltose and sucrose) in a test tube, and
it has heated in a boiling water bath for 2 minutes. The appearance
of red precipitate indicates the presence of reducing sugars.
C. Barfoed’s Test

Two mL of Barford’s reagent was mixed with 1mL of sugar

solution (glucose, fructose, maltose and sucrose) in a test tube, and
it was heated in a boiling water bath for 3-5 minutes. The observation
was noted. The test tubes was continuously heated for 10 minutes,
and the observation was note. Red precipitate of cupric oxide (Cu2O)
indicates the presence of reducing sugar.

II. Hydrolysis
A. Hydrolysis of Sucrose
Two test tubes was prepared and labelled (TT1: Sucrose with
HCl and TT2: sucrose without HCl)

Three mL of 2% sucrose solution was place in each two

labelled test tubes. The first test tube was added with 3mL of water.
The second test tube was added with 3mL of water and 3 drops of
conc. HCl. The two test tubes was heated in a boiling water bath for
 about 5 minutes. Both solution was cooled in a room temperature.
The content of the test tube 2

B. Hydrolysis of Starch

Two mL of starch paste was added in a large test tube. Three

drops of conc. HCl was added and heated in a boiling water bath for
10 minutes. The solution was cooled and NaOH was added, the
solution was neutralized. The starch paste was divided into two
portions (approximately 1mL) and was place in two separate test
tubes for iodine test and Benedict’s test. The observation in each test
tubes was noted.

III. Results and Discussion

Carbohydrates can be identified or differentiated through different tests. One of these was
the use of condensation reagents which react with carbohydrates to produce highly
coloured products. The results shown below are the feedback of how carbohydrates
functions in our body. As for the observations, the positive results can be shown in
different samples.
Test 1. Qualitative Tests for Carbohydrates
Table 1.1 Molisch’s Test

Sample Observation Inference

Glucose Violet ring at the interphase of the two layers was Positive (+)
observed
Sucrose Violet ring at the interphase of the two layers was Positive (+)
observed
Starch Violet ring at the interphase of the two layers was Positive (+)
observed

Molisch’s test is a sensitive chemical test for all carbohydrates. When carbohydrates is
treated with concentrated sulfuric acid it undergo dehydration to produce an aldehyde
(either furfural or hydroxymethylfurfural), which then condenses with alpha naphtol to form
colored products. Pentoses yield furfural, while, hexoses yield 5-hydroxymethylfurfurals.
An appearance of purple coloured ring at the junction of two liquid is observed, this
indicates a positive result in molisch test. In table shows that all the solutions used in the
test give a positive result.

Table 1.2.Iodine Test

Sample Observation Inference

Glucose Yellow-brown color was observed Unbranched

Sucrose Yellow-brown color was observed Unbranched

Starch Blue-black/purple-ink color was observed Branched

Iodine test us used for the detection of starch in the solution. Because of the formation of
starch-iodine complex this result to the appearance of black colour. Starch contain
polymer of ɑ-amylose and amylopectin which form a complex with iodine and gives the
black colour. In solution, the amylase exists as a helically coiled structure, when the iodine
was added, the iodine molecules was trapped within the helical structure forming a
complex one. When heated, the black colour becomes colourless, due to unwinding, the
iodine molecules that trapped in the helical structure is released.

Test 1.3. Bial’s Test for Pentose

Sample Observation Inference

Glucose Light brown color precipitate was obseved Negative(-)

Fructose Dark brown Precipitate was observed Negative(-)

Arabinose Green color of precipitate was observed Positive (+)

Bial’s test is a test specific for pentoses, a carbohydrates containing 5 carbon atoms.
Bial’s reagent is contains orcinol solution, HCl and ferric chloride. The reagent dehydrates
pentoses to form furfural, and furfural reacts with orcinol and iron to produce bluish
product.

Table 1.4. Seliwanoff’s Test

Sample Observation Inference

Glucose It remained unaffected. Yellowish color was Negative(-)

observed
Fructose It gave off a deep cherry red color or a red Positive (+)
complex
Sucrose It gave off a deep cherry red color or a red Positive (+)
complex

Seliwanoff’s test is a test to distinguish a ketoses from aldoses. When heated, ketoses
are more rapidly dehydrated than aldoses. The reaction between a ketohexose and the
Seliwanoff reagent which contains resorcinol and dilute HCl, occurs rapidly in boiling
water bath within 1 minute, this results in the formation of cherry red complex. As you
can see in the table 1.4 only fructose and sucrose has a positive result in this test.

Test 1.5. Reduction Test for Sugars

5A. Benedict’s test

Sample Observation Inference

 Glucose Brick-red precipitate was observed Positive (+)

Sucrose Aqua-blue precipitate was observed Negative(-)

Maltose Brick-red precipitate was observed Positive (+)

Fructose Brick-red precipitate was observed Positive (+)

Benedict’s test is a test for reducing sugars. Free aldegyde groups, ɑ-hydroxy ketones
and hemiacetals are capable of being reducing sugars. All monosaccharides are reducing
sugars. Some disaccharides and polysaccharides that are easily hydrolysed will give a
positive test. In this test, Cu+2 ions (blue in solution) are reduced to Cu+1 ions that form
Cu2O which is brick-red precipitate. As you can see in table 5B, only sucrose gives a
negative result, because the ring of the glucose cannot open due to the glycosidic bond.

5B. Barfoed’s Test

Sample Observation Inference

Glucose Red precipitate/definite red cloudiness was Positive (+)

observed
Fructose Red precipitate/definite red cloudiness was Positive (+)
observed
Maltose No red precipitate was observed Negative(-)

Lactose No red precipitate was observed Negative(-)

Barfoed’s test is a test to distinguish reducing monosaccharides from reducing

disaccharides. Monosaccharides react very fast whereas disaccharides react very slowly.
Table 5C shows that glucose, fructose and galactose are a reducing monosaccharides
while maltose and lactose are reducing disaccharides. A scanty brick-red precipitate is
observed in a positive reaction.
Table 6. The Inversion of Sucrose
Sample Observation Inference

Without HCl Blue color was developed and observed Clock reaction

With HCl Red color was observed. The hydrolytic product Confirmatory test
gave positive reaction with benedict’ Test for sucrose

Sucrose is a disaccharide, which means that it is a molecule that is derived from two simple sugars
(monosaccharides). In the case of sucrose, these simple sugars are glucose and fructose. Inverted sugar is
a mixture of glucose and fructose. It is obtained by splitting sucrose into these two components. The
splitting of sucrose is a hydrolysis reaction which can be induced simply by heating an aqueous solution
of sucrose. Acid also accelerates the conversion of sucrose to invert.

I. Discussion

.
 Hydrolysis of sucrose, table 1 shows that sucrose with no HCl added
gives a negative result in Fehling’s test and Seliwanoff, in the other hand,
sucrose with HCl gives a positive in Fehling’s Test and Seliwanoff’s Test, this
is because of HCl hydrolysed sucrose, in result sucrose break into glucose and
fructose and give a positive result.
In table 2 shows the result of hydrolysis of starch, in the two tests,
specifically Benedict’s test and Iodine test starch gives positive result, a
hydrolysis occurred due to the HCl.

II. Summary and Conclusion

In the experiment conducted, it has been shown that all monosaccharides
are reducing sugars together with some disaccharide and polysaccharide
namely maltose and maltose, while sucrose in the other hand is an example
of non-reducing sugar because the ring of glucose cannot open due to
glycosidic bond, but when sucrose undergo hydrolysis, sucrose will break into
glucose and fructose, and can give positive result in the test. Fructose and
ribose are an example of ketoses, while the remaining carbohydrates are
aldoses. Molisch’s Test is a general test for carbohydrates that gives a purple
ring that indicates a positive result, Iodine test is a test for starch which gives
a black colour for a positive result, Bial’s test is a test specific for pentoses,
carbohydrates that contains 5 carbon atoms, which gives a bluish for a positive
result, Seliwanoff’s test is a test to distinguish ketoses from aldoses, while
Fehling’s and Benedict’s Test are test to differentiate reducing sugar from non-
reducing sugar, lastly Barfoed’s test is a test to distinguish reducing
monosaccharides from reducing disaccharides.

V. References
Matthews, C.E., et. al. 1999. Biochemistry. 3rd Edition. Benjamin Cummings NY. ISBN 0-
8053 -3066-6
McMurry, J. 2008. Organic Chemistry. 7th Edition. Belmont, CA: Thompson Brooks/Cole.
Print
Pratt, C.W., Cornely, K. 2013. Essential Biochemistry 3rd Edition. Wiley.p. 626. ISBN 978-
111 8083505
Varki, A., Cummings, R., et. al. 1999. Essentials of glycobiology. Cold Spring Har J. Cold
SpringHarbor Laboratory Press. ISBN 0-87969-560-9.