Laboratory Experiment 3 Test For Carbohydrates

Activity 3
TEST FOR CARBOHYDRATES
INTRODUCTION: Carbohydrates include polyhydroxy aldehydes or ketones and their by-products. They
make up most of our diet and serve as the major source of energy. They play an important part in metabolic
processes by furnishing the carbon chain for compound synthesis by living organisms. The chemical tests used
to detect the presence of carbohydrates are based on the ability to (a) form furfural and its derivatives; and (b)
reduce and form characteristic compounds with reagents.
OBJECTIVE:
To be able to identify the different types of carbohydrates using the different specific chemical tests.
MATERIALS:
PROCEDURE/ RESULTS AND DISCUSSION
I. Test Based on the formation of furfural and its derivatives

1. Molisch’s Test
a. Mix 4 mL of distilled water and 2 drops of the Molisch’s reagent in a test tube. This tube will serve
as the control.
b. Place 4 mL of 3% solution of glucose in the second tube. Add 2 drops of the Molisch reagent and
mix the contents by gently shaking the test tube.
c. Incline the test tube and cautiously add about 5 mL conc. H 2SO4, allowing the acid to run down the
side of the tube. Sulfuric acid is denser than water and will form a lower layer. Note the color of the
ring formed at the junction of the two liquids.
d. In the same manner of adding acid, add sulfuric acid to the control tube. What do you observe?
e. Repeat the above test with 3% sample solutions of xylose, lactose and starch.
f. Record all results.
Substance Tested Description of visible results
Control Hot with a clear and transparent solution
Glucose There was a visible two-layered dark brown
substance on the bottom with a yellowish
substance on the top of the first layer.
Xylose The color of the solution became dark black.
Lactose The solution turned to a two-layered dark black
and light yellowish color visible on the test tube.
The dark black was formed on the bottom while
the light-yellowish substance was formed at the
top of the first layer.
Distinctively, the top layer of the lactose has a
lighter yellow color than the glucose.
Starch It turned to a dark black colored substance with
a light yellowish margin formed in the center
of the transparent and clear solution.
Based on the results, which carbohydrate gave positive result with Molisch reagent?
Based on the results, all of the substances tested, glucose, xylose, lactose, and starch, gave a
positive result with the Molisch reagent. In fact, almost all carbohydrates except tetrose and trioses
will give a positive result for Molisch reagent since they tend to undergo hydrolysis when exposed to
strong mineral acids and form monosaccharides. However, it should be noted that monosaccharide
will give a positive faster while disaccharide and polysaccharide will react slowly with the Molisch
reagent and give positive test in delay.
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2. Bial’s Orcinol Test
a. Place 1 mL each of 3% solution of xylose, glucose, and starch in separately-labeled test tubes.
b. Add 3 mL of Bial’s reagent to each test tube.
c. Carefully heat each tube over the flame until the solution starts to boil. Add 1-2 drops of 10% FeCl3
solution.
d. Note the color of the product formed.
e. Record your results.
Substance Tested Results
Glucose The substance turned to a light yellowish
color with a pinch of black precipitate on
the bottom. (Negative Result)
Xylose The substance became darker compared
to glucose with a pinch of black
precipitate on the bottom. (Negative
Result)
Starch The starch was initially cloudy when
mixed with 3mL Bial’s reagent. But after
being heated with two drops of 10% FeCl3
solution, the substance turned to a light
yellowish color with presence of
precipitate on the bottom. (Negative
Result)
3. Seliwanoff’s Test
a. Place 1 mL each of 3% xylose, glucose, fructose and sucrose in separately-labeled test tubes.
b. Add 4 mL of Seliwanoff’s reagent to each test tube.
c. Place the tubes in a water bath filled with boiling water and allow them to stay there for exactly
1 minute.
d. Note the changes and record which tube gives a positive result in the shortest time.
e. Continue heating and observe the color changes at 1-minute intervals.
f. Record the time required for a positive test for each sample.
Substance Tested Time Result Explanation
Xylose 2:36 Clear bluish-green in Xylose gave a
color negative Seliwanoff’s
test since it did not
change to cherry-red
color within the first 2-
minutes of heating.
This indicates that the
sample does not
contain ketoses.
Glucose 3:26 No change in color Negative Seliwanoff’s
test since the solution
did not change to
cherry-red color within
the first 2- minutes of
heating.
This indicates that the
sample does not
contain ketoses.
Fructose 1:34 Cherry-red color Fructose reacted faster
than glucose and the
solution changed to
cherry red-color
within the first 2
minutes of heating,
which means that it
contains ketoses.
Positive Seliwanoff’s
test
Sucrose 2:16 Reddish orange Sucrose gave positive
Seliwanoff’s test since
it is a disaccharide
consisting of fructose
and glucose. Meaning,
the sample contains
ketoses.
II. Tests based on the reducing property of sugars.

1. Benedict’s Test
a. Place 1 mL each of 3% solutions of glucose, xylose, fructose, lactose and sucrose in
separately-labeled test tube. Add 5 mL of Benedict’s reagent in each tube.
b. Place all the tubes in boiling water bath for 2 to 3 minutes.
c. Observe the color of the solution and note whether a precipitate was formed. A change in
color of the solution is not considered a positive reaction.
d. Avoid prolonged heating
e. Record your results.
Substance Tested Result Explanation
Xylose Brick Red Xylose has more than 2%
reducing sugar which
resulted to a positive
Benedict’s test. This
reaction is caused by the
reducing property of
simple carbohydrates.
The copper (II) ions in
the Benedict’s solution
are reduced to Copper (I)
ions, which causes the
color change.
Glucose Reddish-brown Glucose tested positive
since there was a
formation of reddish
precipitate within
3minutes. So basically,
reducing sugars are aslo
present.
Fructose Orange Fructose is an alpha-
hydroxy-ketone, and
gives a positive test
because it is converted to
the aldoses glucose and
mannose by the base in
the reagent.
Sucrose Green Sucrose is a non-
reducing sugar and does
also not react with the
iodine
This study source was downloaded by 100000828797886 from CourseHero.com on 12-07-2022 01:10:35 GMT -06:00or with the
Benedict Reagent. Thus,
it resulted to a Negative
result.
Lactose Dark Brown Lactose tested positive
since it contains glucose
with a free reducing
aldehyde moiety, after
isomerization.
2. Barfoed’s Test
a. Place 1 mL each of 3% solutions of glucose, xylose, fructose, lactose and starch in
separately-labeled test tubes.
b. Add 3 mL Barfoed’s solution in each test tube
c. Prepare a control tube using distilled water instead of sugar solution.
d. Place all the tubes in a boiling water bath for 10 minutes. Record your observations
Control Light blue The control is the basis
of the observation.
Xylose Light bluish-green with Since xylose is a
presence of copper (||) oxide monosaccharide, it
precipitate oxide reacted after 3minues and
produced a red
precipitate. The red
precipitate indicate a
positive result for
Barfoed’ Test.
Glucose Light bluish-green with Glucose gave a positive
copper (||) oxide precipitate Barfoed’s test since it is a
monosaccharide
containing reducing
agent. Evidently, it has
also produced red
precipitate.
Fructose Light bluish-green with Fructose gave a positive
copper (||) oxide precipitate Barfoed’s test since it is a
monosaccharide
containing reducing
agent. It has also
produced red precipitate.
Starch The substance with the Starch tested negative
lightest blue color. There because it is a
was no change. polysaccharide with
weaker reducing agent.
Lactose Darker blue compared to In lactose, the reaction
starch was negative since it is a
disaccharide sugar which
means that it has weaker
reducing agent.
3. Tollen’s Test
a. Place 5 drops of 3% solutions of glucose, xylose and sucrose in separate test tubes.
b. Add 2 mL of Tollen’s reagent into each test tube
c. Boil for about 5 minutes. Note and record your observations
Xylose Presence of dark gray The formation of a dark
precipitate grey precipitate or silver
mirror on the bottom and
sides of the test tube
indicates a positive
result, which means that
the given sample
contains reducing sugars/
aldoses.
Glucose Absence of dark gray The absence of such
precipitate precipitate indicates a
negative result, which
means that the test
sample doesn’t have
reducing sugars/ aldoses/
α-hydroxy ketoses.
Sucrose Presence of silver mirror on The formation of a silver
the bottom of the test tube mirror on the bottom and
sides of the test tube
indicates a positive
result, which means that
the given sample
contains reducing sugars/
aldoses.
4. Phenylhydrazine
a. Place 5 mL each of glucose, lactose and maltose in separate test tubes. Add small amount of
solid Phenylhydrazine and 10 drops of sodium acetate.
b. Heat the tubes and note the formation of precipitate.
c. Let it stand for and reserve the products for the next laboratory period.
d. Observe crystals under the microscope.
e. Draw the osazone crystals as seen under the microscope.
RESEARCH QUESTIONS
1. Will disaccharides and polysaccharides give positive result for Molisch test?
Yes, both disaccharides and polysaccharides will produce a positive result because a Molisch's
test is a chemical test for the presence of carbohydrates that is based on the dehydration of the
carbohydrate by sulfuric or hydrochloric acid to produce an aldehyde, which condenses with two
molecules of a phenol, resulting in a violet ring. In this experiment, disaccharides such as lactose and
sucrose, as well as polysaccharides such as starch, demonstrated this feature.
2. Which of the different carbohydrate tests would give a positive result for maltose?
Molisch test, Benedict's Test, Tollen's Test, Seliwanoff's Test, Barfoed's Test
3. Can Seliwanoff’s test be used to distinguish sucrose from fructose? Explain.

Yes, fructose is a ketose, and Seliwanoff's test is particularly sensitive to it. Sucrose is neither a
ketose nor an aldose, but rather a combination of the two. It will react, but much more slowly, and will
produce a much milder pinkish color.
4. Inulin is a polysaccharide composed entirely of fructose units. Which test should be used to best identify
the presence of fructose?
Seliwanoff's test. The Seliwanoff's test is used to differentiate between ketoses and aldoses.
When ketoses, such as fructose, are treated with a concentrated acid, they dehydrate more quickly,
yielding furfural derivatives, and when combined with resorcinol, they yield cherry red complex.
CONCLUSION:
After performing the various experiments which include the test for the formation of furfural and its
derivatives (Molisch’ Test, Bial’s Orcinol Test, Seliwanoff’s Test) and the Test based on the reducing property of
sugars (Benedict’s Test, Barfoed’s Test, Tollen’s Test, Phenylhydrazine), I was able to differentiate if a substance
is composed of ketone or aldehyde through observing if it has either a positive or negative reaction to a certain
test. Generally, the experiment on carbohydrate has enlightened my understanding to the deep definition of the
positive or negative result and classification of monosaccharide, disaccharide, and polysaccharide substances
when they are mixed or heated to an alcohol lamp. Overall, the experiment has shed light to my pre- existing
knowledge on the basic concepts of carbohydrates to a whole new set of information and excitement.
REFERENCE/S:
Tollens’ Test- Definition, Principle, Procedure, Result, Uses. (2020, November 19). Microbe Notes.
https://microbenotes.com/tollens-test/#:~:text=The%20formation%20of%20a%20dark
Bial’s Test: Objective, Principle, Procedure, Reagent And Results Interpretation – BIOCHEMINSIDER. (n.d.).
Retrieved June 26, 2022, from https://biocheminsider.com/bials-test-objective-principle-procedure-
reagent-and-results-interpretation/#:~:text=Positive%20Test%3A%20The%20presence%20of
Sapkota, A., & Waiswa, J. (2020, November 19). Seliwanoff's test- definition, principle, procedure, result,
uses. Microbe Notes. Retrieved June 26, 2022, from https://microbenotes.com/seliwanoffs-test/
Aryal, S. (2019, August 15). Benedict’s Test- Principle, Composition, Preparation, Procedure and Result
Interpretation. Microbiology Info. https://microbiologyinfo.com/benedicts-test-principle-composition-
preparation-procedure-and-result-interpretation/
Byjus. 2022. Molisch’s Test - Principle, Procedure, Reaction, & Reagent, from
https://byjus.com/chemistry/molischs-test/
What are molischs test? Definition, Types and Importance - chemistry | AESL. (n.d.). Www.aakash.ac.in.
https://www.aakash.ac.in/important-concepts/chemistry/molischs-test
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Activity 3

TEST FOR CARBOHYDRATES

PROCEDURE/ RESULTS AND DISCUSSION

I. Test Based on the formation of furfural and its derivatives

II. Tests based on the reducing property of sugars.

3. Can Seliwanoff’s test be used to distinguish sucrose from fructose? Explain.

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