CHEM 5007 – CHEMICAL BIOLOGY (BIOMOLECULES) (LABORATORY)

EXPERIMENT NO. 4 – CARBOHYDRATES: Part 4-1 – Qualitative Tests for

Carbohydrates

I. INTRODUCTION

Carbohydrates are a class of natural compounds that contain either an aldehyde or a

ketone group and many hydroxyl groups – they are often called polyhydroxy aldehydes or
ketones. Carbohydrates are used for energy. The carbohydrates that we eat are broken down in
our bodies and eventually form water and carbon dioxide. The energy obtained in this process is
used for other reactions that must occur in the body. Excess carbohydrates that we eat can be
stored in the liver as glycogen or can be converted to fats. Plants create carbohydrates in the
process of photosynthesis, where energy from the sun is used to build carbohydrates from water
and carbon dioxide.

A monosaccharide consists of a single carbohydrate molecule, containing between 3 and

7 carbons. The simplest group of carbohydrates and often called simple sugars since they cannot
be further hydrolyzed. Examples of monosaccharides are glucose and fructose. A disaccharide is
a carbohydrate made up of two monosaccharides by a glycosidic bond. Sucrose and lactose are
disaccharides. A polysaccharide consists of many monosaccharides linked together. They are
also called “glycans”. They yield more than 10 molecules of monosaccharides on hydrolysis.
Starch, pectin, glycogen, and cellulose are examples of polysaccharides.

Ketose and aldose are monosaccharides which can be differentiated based on the group
they contain. An aldose is defined as a monosaccharide whose carbon skeleton has an aldehyde
group. They are primarily found in plants. Examples of aldoses include glyceraldehyde,
erythrose, ribose, glucose and galactose. Ketose is a monosaccharide whose carbon skeleton has
a ketone group. Only in the presence of reducing sugar, can they isomerize to aldose. They are
used in processed food. Examples of ketose are ribulose, fructose, etc. Ketoses and aldoses can
be chemically differentiated through Seliwanoff's test.

Reducing sugars are carbohydrates that can act as reducing agents due to the presence of
free aldehyde groups or free ketone groups. These are sugars because this group of compounds
has a sweet taste as other sugars. All monosaccharides and some disaccharides are reducing
sugars. Non-reducing sugars on the other hand are carbohydrates that cannot act as reducing
agents due to the absence of free aldehyde groups or free ketone groups. Some disaccharides and
all polysaccharides are reducing sugars. In basic aqueous media, nonreducing sugars do not
generate any compounds containing an aldehyde group.

Pentose and hexose are common types of monosaccharides. Pentose is a five-carbon

monosaccharide such as ribose and deoxyribose, whereas hexose contains six carbon atoms. The
most common hexoses are glucose, galactose, mannose, and fructose. Small quantities of pentose
sugars such as arabinose, xylose, and ribose are naturally present in must and wine
 The qualitative analysis of carbohydrates is the significant test for detecting and
classifying carbohydrates, depending on the color change followed by chemical reactions. The
chemical tests are the following with their relative results.

Molisch’s test is a chemical test which is used to check for the presence of carbohydrates
in a given analyte. The formation of a purple or a purplish-red ring at the point of contact
between the H2SO4 and the analyte + Molisch’s reagent mixture confirms the presence of
carbohydrates in the analyte. Fehling’s test is used for the estimation or detection of reducing
sugars and non-reducing sugars. The presence of reducing sugars is indicated by the appearance
of brick-red precipitate in test tubes. Barfoed’s test is similar to Fehling’s test, except that in
Barfoed’s test, different types of sugars react at different rates. It is a chemical test that can be
used to check also for the presence of reducing sugars in a given analyte and also results in the
formation of a brick-red precipitate. Benedict’s Test is used to test for simple carbohydrates.
Benedict's test identifies reducing sugars (monosaccharides and some disaccharides), which have
free ketone or aldehyde functional groups. A positive test with Benedict's reagent is shown by a
color change from clear blue to brick-red with a precipitate. Seliwanoff’s test is used to
differentiate between aldose and ketose, i.e., it helps to detect the presence of aldo sugar and keto
sugar. Seliwanoff's reagent is a mixture of resorcinol and concentrated hydrochloric acid. Ketose
sugars react with the Seliwanoff's reagent to give immediately a deep cherry red color, while
Aldose sugars react with the Seliwanoff's reagent to give a faint pink color which intensifies with
time. Mucic acid test is a test that is highly specific and is used for the detection of the presence
of galactose and lactose. Crystals will be produced below the tube if the sample contains
galactose or its derivatives. The Iodine Test for Starch is used to determine the presence of
starch in biological materials. A positive result for the iodine test (starch is present) was a color
change ranging from violet to black; a negative result (no starch) was the yellow color of the
iodine solution.

h. Fermentation

II. OBJECTIVES

1. The students must develop a deeper understanding on what carbohydrates are.

2. The students ought to be able to identify the different types of carbohydrates.
3. The students must be able to observe and recognize how different carbohydrates react to
different chemical tests.

III. RESULTS AND DISCUSSION

Part 4.1: Qualitative Tests for Carbohydrates

Table 1: Molisch test

 Sample Observations +/-

1. Glucose
There is a formation of purple ring between two layers of +
solution

2. Fructose
There is a formation of purple ring between two layers of +
solution

3. Galactose
There is a formation of purple ring between two layers of +
solution

4. Lactose
There is a formation of purple ring between two layers of +
solution

5. Maltose
There is a formation of purple ring between two layers of +
solution

6. Sucrose
There is a formation of purple ring between two layers of +
solution

7. Starch
There is a formation of purple ring between two layers of +
solution

8. Cellulose
There is a formation of purple ring between two layers of +
solution

Discussion:

1. Which samples are positive for the Molisch test?

Molisch test results are positive for all carbohydrates (monosaccharides, disaccharides,
and polysaccharides). All samples are carbohydrates thus showing a positive result in the
formation of purple rings.

2. Which samples are negative?

Since all samples are all carbohydrates, no negative results arose.

3. What do your results indicate about your samples?

In the chemical test of Molisch. The appearance of purple or violet rings confirms the
presence of carbohydrates. Carbohydrates reacting with mineral acids such as sulfuric acid get
dehydrated and form furfural derivatives. These compounds react with naphthol and get
condensed to form a purple-coloured ring product.

Table 2: Reduction test

Sample Observations (indicates whether + or -)

Fehling’s Test Benedict’s Test Barfoed’s Test

1. Glucose + + +

2. Fructose + + +

3. Galactose + + +

4. Lactose + + -

5. Maltose + + -

6. Sucrose - - -

7. Starch - - -

8. Cellulose - - -

Discussion:

1. What is your result for fructose on Fehling’s and Benedict’s test? Is fructose a reducing
sugar? Why?

Fructose gives positive results in both tests in which there is a formation of brick-red
precipitate. Fructose is a reducing sugar. Fructose is a type of sugar known as a monosaccharide.
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).

2. What is your result for sucrose in Fehling's and Benedict’s test? Is sucrose reducing
sugar? Why?

Sucrose gives negative results in both tests in which there is no formation of brick-red
precipitate. Sucrose is a non-reducing sugar. In sucrose, there are glycosidic bonds between their
anomeric carbons to retain the cyclic form of sucrose, avoiding its conversion into the form of an
open chain with an aldehyde group.

3. Maltose, sucrose and lactose are all disaccharides. Do they give the same results on
Fehling’s and Benedict’s test? Why or why not?

Sucrose shows different results compared to maltose and lactose which shows a negative
result in both tests. Though all of them are disaccharides, sucrose has a different chemical
structure. Sucrose (glucose + fructose) lacks a free aldehyde or ketone group and therefore is
non-reducing. In disaccharides, if the reducing groups of monosaccharides, i.e Aldehyde or
ketone groups are bonded, these are non-reducing sugars.

4. Starch, cellulose, agar-agar and gum Arabic are polysaccharides. What do your results
on Fehling’s and Benedict’s test reveal about polysaccharides?

Both Fehling’s and Benedict's test is used only for simple carbohydrates / reducing
sugars/ monosaccharides/ disaccharides. If such samples such as starch and cellulose will be
tested under the given tests, negative results will likely be observed since both samples are
polysaccharide and are not in the scope of test in Fehling’s and Benedict’s.

For Barfoed’s test:

1. How long did it take for the monosaccharide samples to give the positive result?

Barfoed's test is used for distinguishing monosaccharides from reducing disaccharides.

Monosaccharides usually react in about 1-5 minutes to show a brick-red precipitate. However, in
our case during experiment monosaccharides like glucose, galactose, and fructose, show a
positive result in the formation of brick-red precipitate from a long minutes of heating.

2. How about for the disaccharides?

In contrast to time of reaction in monosaccharides, disaccharides take 7-12 min to give

the colored product with Barfoed's test. However, during the experiment after a long minutes of
heating and waiting, no disaccharides gave positive results in Barfoed's test.

3. How about for the polysaccharides?

 Like Fehling’s and Benedict's test, Barfoed’s is done to distinguish between
monosaccharides and reducing disaccharides only. Thus, even after hours of heating of
polysaccharide samples no positive results will be seen.

Table 3: Test for Specific Carbohydrates

Sample Observations (indicates whether + or -)

Seliwanoff’s test Mucic Acid test Iodine test

1. Glucose - - -

2. Fructose + - -

3. Galactose - + -

4. Lactose - + -

5. Maltose - - -

6. Sucrose + - -

7. Starch - - +

8. Cellulose - - -

Discussion:

1. Which of the samples gave a positive result for Seliwanoff’s test? Why?

Fructose and sucrose gave a positive result of red-orange coloration which means they
are classified as ketose sugars. Ketoses (e.g. fructose) are more readily dehydrated by HCl than
the aldoses to form hydroxymethyl furfural, which then condenses with resorcinol of
Seliwanoff's reagent to form a red colored complex.

2. Which of the samples gave a positive result for the Mucic Acid test? Why?
The carbohydrates in the sample that gave a positive result for the mucic acid test are fructose
and lactose. Monosaccharides, such as galactose contain sugars, such as lactose have a positive
response to this chemical reaction. In the presence of acidic substances such as nitric acid create
saccharic acids. The saccharic acid produced is insoluble and forms clear crystals.

3. Which of the samples gave a positive result for the Iodine test? Why?

Starch is the carbohydrate in the sample that gave a positive iodine test result. The basis for this
test is that starch is a coiled polymer of glucose — iodine interacts with these coiled molecules
and becomes bluish black. Iodine does not react with other carbohydrates that are not coiled, and
remains yellowish brown.

Table 4: Fermentation

Sample Observations +/-

1. Glucose
There were a lot of bubbles on the sample after the +
fermentation.

2. Fructose
There were tiny bubbles appeared in the sample after the +
fermentation.

3. Galactose
There were no bubbles on the sample. -

4. Lactose
There were no bubbles on the sample. -

5. Maltose
After the fermentation, there were small bubbles on the +
sample.

6. Sucrose
After the fermentation, there were some slightly large +
bubbles on the sample.

7. Starch
There were no bubbles on the sample. -

8. Cellulose
There were no bubbles on the sample. -

Discussion:
1. What is fermentation?

By the metabolic process of fermentation, which is enzyme-catalyzed, organisms turn sugar or

starch into alcohol or an acid anaerobically, generating energy. It is a metabolic process that
releases energy from sugar or other organic substrates. Hence, it is called an Anaerobic
biochemical process.

2. Which among your samples are fermentable sugars?

Among the 8 carbohydrates that we have tested, results show that the fermentable sugars in our
samples are the following; Glucose, Fructose, Maltose, and Sucrose which produces a formation
of bubbles.

IV.CONCLUSION

Although both starch and sugar are complex carbohydrates, which have large molecule weights
and significantly reduce their affinity for water, the hydroxyl groups that exist in the monomers
themselves actually contribute to their insignificant solubility. However, the solubility will
increase when these complex carbohydrates are broken down into their monomers, where the
hydroxyl groups can form hydrogen bonds with other water molecules easily due to the reduced
molecular weight and size that affect the affinity for water. It was concluded that carbohydrates
can be qualitatively analyzed using different tests.

The positive result for the Molisch test is the presence of a purple liquid layer. All carbohydrates
in our experiment gave a positive result for the Molisch test. The Fehling’s, Benedict's, and
Barfoed tests are the common tests that are used to determine the presence of reduced sugar.
Glucose, galactose, lactose, and maltose are the carbohydrates in the sample that give a brick-red
precipitate after the solutions are heated for both Fehling’s and Benedict's tests, while for
Barfoed’s test, only the three carbohydrates, which are galactose, glucose, and fructose, give a
positive result.

The formation of the pink complex indicates a positive result, which means that the given sample
in the experiment, like fructose and sucrose, contains aldohexoses. The absence of such color or
the appearance of the color after a prolonged period of time indicates a negative result, which
means that the test sample doesn't have aldohexoses. For the mucic acid test, crystals will be
produced below the tube if the sample contains galactose or lactose. The iodine test is used to
test for the presence of starch. Starch is a type of polysaccharide carbohydrate that is made up of
amylose and amylopectin. It is one of the main sources of carbohydrate and is present naturally
in plants. Amylose is starch from a dark blue complex with iodine. Lastly, for fermentation, in
the sample that contains starch, it appeared that the glucose, fructose, maltose, and sucrose were
the only sugars in which bubbles appeared due to the carbon dioxide, as they were made by
yeast, as they grow using sugars as a source of energy.

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