Experiment 3: CARBOHYDRATES

OBJECTIVES:

 To identify common carbohydrates in nature

 To classify the different carbohydrates
 To perform simple qualitative tests for carbohydrates

PROCEDURE:

A. General Tests

Molisch test for all Carbohydrates

1. In eight (8) labelled test tubes, pipette 1mL of the following:

Test tube No. Sugar solutions

1 5% fructose

2 5% galactose

3 5% ribose

4 5% glucose

5 5% sucrose

6 5% lactose

7 5% maltose

8 5% starch

2. Add three drops of Molisch reagent and mix thoroughly

3. Incline each test tube and add 1mL of concentrated sulfuric acid down the side of the tubes

4. Do not shake to avoid mixing layers. Observe for the presence of a violet ring at the interface
of the two liquids.
Bial’s [Orcinol] Test for Pentoses

1. In four labelled test tubes, place 1mL of the following solutions:

Test tube No. Sugar solutions

1 5% arabinose

2 5% glucose

3 5% maltose

4 Water

2. Add 2mL of the Orcinol reagent. Mix well and transfer to a boiling water bath.

3. Observe for the appearance of blue-colored solution after 15 minutes of heating.

B. Reduction Tests

Benedict’s test

1. In nine labelled test tubes, place 2mL of Benedict’s reagent and 1mL of the following sugar
solution:

Test Tube No. Sugar Solution

1 5% fructose

2 5% galactose

3 5% arabinose

4 5% glucose

5 5% maltose

6 5% sucrose

7 5% lactose

8 5% starch

9 Water (control)
2. Place these test tubes with the carbohydrates in a boiling water bath and note the time
elapsed before the appearance of a brick red precipitate is observed. If there is no brick red
precipitate that appears after 30 minutes, discontinue heating.

C. Specific test for some Carbohydrates

Galactaric Acid Test

1. Place 1mL of the following solutions on four labelled test tubes:

Test tube No. Sugar solutions

1 5% lactose

2 5% glucose

3 5% maltose

4 5% galactose

2. Add approximately 10 drops of concentrated nitric acid.

3. Place in a boiling water for at least 90 minutes.

4. Remove the tubes from the water bath and allow them to stand overnight.

5. Observe for any crystals that will be formed.

Seliwanoff’s test

1. In nine labelled test tubes, place 2mL of Seliwanoff’s reagent and 1mL of the following sugar
solution:

Test tube No. Sugar solutions

1 5% fructose

2 5% galactose

3 5% arabinose

4 5% glucose

5 5% maltose
 6 5% sucrose

7 5% lactose

8 5% starch

9 Water (control)

2. Place these test tubes with the carbohydrates in a boiling water bath for 10 minutes and note
the time elapsed before a cherry red solution is observed.

3. Compare the results of each sugar solutions

Fermentration Tests

1. Fill seven (7) labelled test tubes with 10 mL of the following sugar solutions:

Test Tube No. Sugar solutions

1 5% fructose

2 5% galactose

3 5% arabinose

4 5% glucose

5 5% maltose

6 5% sucrose

7 5% lactose

2. Add 2mL of the yeast suspension and mix well. Incubate the mixture at 37oC .

3. Note the time required to produce a noticeable bubbling or gas as a result of fermentation.

4. If no gas has been formed after 3 hours, the test is considered negative.

Iodine test

1. In an evaporating dish, pipette 1mL of 1.0mL of I2/KI solution.

2. Add 2-3 drops of starch suspension and observe for a change in color from reddish brown to
blue. Repeat the test using the other carbohydrate solutions.
Experiment 3: CARBOHYDRATES

Score:

Advincula, Maria Yssabella B.

Aguilar, Allyzha Mae A.
Aguilar, Ariane S.
Aquino, Daniel Paolo C.
Alamon, Regine C.
Co, Jelena Amor A.
Dela Cruz, Nona Mae G.

DVM 1-2 February 24, 2022 February 25, 2022

Course/Year Date performed Date

submitted

W_F 7:00-10:00 AM Ms. Sarah Joy R. Del Rosario

Laboratory class schedule Laboratory Instructor/Professor

POST LAB ACTIVITY

RESULTS

Table 1. Reaction of different sugars with Molisch and Bial’s reagent

Sugar solution Observations

Molisch test Bial’s Test

5% fructose A purple ring is formed at the interface
of the two liquids.

5% galactose A purple ring is formed at the interface

of the two liquids.

5% arabinose A purple ring is formed at the interface Blue-colored solution is formed.

of the two liquids.
 5% glucose A purple ring is formed at the interface Brown-colored solution is
of the two liquids. formed.
5% maltose A purple ring is formed at the interface Yellow-orange solution is
of the two liquids. formed.

5% sucrose A purple ring is formed at the interface

of the two liquids.

5% lactose A purple ring is formed at the interface

of the two liquids.

5% starch A purple ring is formed at the interface

of the two liquids.

water Yellow-orange solution is

formed.

Exploratory Question/s:

1. Which of the sugar solutions gave a negative test/ positive test with  reagent? Why? What is
the purpose of this test? Why do many proteins give a positive test with Molisch reagent? What
is the reaction involved in this test?

All the sugar solutions showed positive in the test with Molisch regeant because they are
all carbohydrates. Molisch Test was positive when a purple ring appeared at the interface of the
two liquids. The purpose of this test is to determine whether a sample contains carbohydrates.
Because some proteins contain carbohydrates in the form of glycoproteins, a positive test with
Molisch reagent is expected. The reaction starts with the synthesis of
hydroxymethylfurfuraldehyde from concentrated sulfuric acid, which is then followed by the
condensation of this carbonyl molecule with an alcohol (α-napthol) to produce a violet colored
substance.

2. Which of the sugar solution gave a negative/ positive test with Orcinol reagent? Why? What is
the purpose of this test? How are carbohydrates differentiated by this test? What is the reaction
involved in this test?
 The sugar solution that gave a positive test is the 5% arabinose and the rest are
negative. Arabinose is aldopentose monosaccharide under reducing sugar. In Bial’s test the
formation of blue-colored solution indicates positive in the test. The purpose of this is to
evaluate general test for the presence of pentose. The reducing sugar monosaccharide sugar
solutions should be resulted positive to obtain the presence of pentose. The test reagent
dehydrates pentoses to form furfural. Furfural further reacts with orcinol and the iron ion present
in the test reagent to produce a bluish product.The reaction in this test proceeds similarly to the
Molisch test by forming a furfuraldehyde through the action of concentrated sulfuric acid. After
that, the aldehyde derivative will condense with orcinol (5-methyl-1,3-dihydroxybenzene).

Table 2. Reaction of different sugars with an Oxidizing agent

Sugar solution Observations

Benedict’s test Barfoed’s test

5% fructose Positive,  brick red precipitate is Positive,  brick red precipitate is

obtained. obtained.

5% galactose Positive,  brick red precipitate is Positive,  brick red precipitate is

obtained. obtained.

5% arabinose Positive,  brick red precipitate is Positive,  brick red precipitate is

obtained. obtained.

5% glucose Positive,  brick red precipitate is Positive,  brick red precipitate is

obtained. obtained.

5% maltose Positive,  brick red precipitate is Negative, shows no formation of

obtained. brick red precipitate.

5% sucrose Negative, shows no formation of Negative, shows no formation of

brick red precipitate. brick red precipitate.

5% lactose Positive,  brick red precipitate is Negative, shows no formation of

obtained. brick red precipitate.
 5% starch Negative, shows no formation of Negative, shows no formation of
brick red precipitate. brick red precipitate.
water Negative, shows no formation of Negative, shows no formation of
brick red precipitate. brick red precipitate.

Exploratory Question/s:

3. Which of the sugar solutions gave a negative/positive test with Benedict’s reagent? Why?
What is the purpose of this test? How are the carbohydrates differentiated by this test? What is
the reaction involved in this test?

          The sugar solution that gave a negative test result with Benedict’s reagent are sucrose,
starch and water which indicates they are all under non-reducing sugar. While the fructose,
galactose, arabinose, lactose and maltose resulted positive which means they are all under
reducing sugar. The purpose of this test is to differentiate reducing from non-reducing sugar.
The reaction is based on the ability of sugar reduce Cu from +2 to +1 forming Cu2O which is
seen as a brick red precipitate. The Cu is provided by CuSO4, and by the presence of
Na2CO33 the medium is made slightly alkaline. 

4. Which of the sugar solutions gave a negative test/positive test with Barfoed’s reagent? Why?
What is the purpose of this test? How are carbohydrates differentiated by this test? Of what use
is Barfoed’s reagent in identifying an unknown sugar? What is the reaction involved in this test?

       The sugar solutions that gave negative test with Barfoed’s reagent are maltose, sucrose,
lactose, starch and water which shows they are under reducing disaccharide or non-reducing
sugar.  While fructose, galactose, arabinose and glucose resulted positive which means they
are reducing sugar monosaccharide. The purpose of this test is to modify a reducing sugar
monosaccharide from a reducing disaccharide. In order to classify a reducing sugar
monosaccharides, reducing sugar solutions should obtain positive result and negative reaction
for reducing sugar disaccharides. Barfoed’s test can be use in identifying the presence of
monosaccharide or disaccharide in an unknown sugar. Monosaccharide solutions make a
reddish precipitate, while disaccharide and polysaccharide solutions produce no precipitate

Table 3. (a) Reaction of Sugars with Seliwanoff’s reagent; and (b) Sugar Fermentability

Sugar solution Observations

Seliwanoffs’s test Fermentation test

5% fructose Cherry red colored solution is Gas bubbles were present when
formed. yeast is added.
 5% galactose There is no cherry red colored No gas bubbles were present when
solution formed. yeast is added.
5% arabinose There is no cherry red colored Gas bubbles were present when
solution formed. yeast is added.

5% glucose There is no cherry red colored Gas bubbles were present when
solution formed. yeast is added.
5% maltose There is no cherry red colored Gas bubbles were present when
solution formed. yeast is added.

5% sucrose Cherry red colored solution is Gas bubbles were present when
formed. yeast is added.

5% lactose There is no cherry red colored No gas bubbles were present when
solution formed. yeast is added.

5% starch There is no cherry red colored

solution formed.
water There is no cherry red colored
solution formed.

5. Which of the sugar solutions gave a negative test/positive test with Barfoed’s reagent? Why?
What is the purpose of this test? How are the carbohydrates differentiated by this test? How?
What's the reaction involved in this test?

All of the sugar solution, except for 5% fructose and 5% sucrose, tested negative in the
test. All of them with the exception of the two sugar solutions, did not result in a cherry red
solution. On the other hand, the sucrose and fructose solution tested positive– giving cherry red
colored solution.This is for that reason that the reaction of Seliwanoff reagent and a ketose
should result in a red colored precipitate. The purpose of this test was to distinguish a ketose
from an aldose and disaccharides. Because ketoses rapidly dehydrate more than aldoses when
heated, the results tend to be the formation of red colored solution. It can be concluded that the
fructose and sucrose are ketosis and the rest are aldoses. 

The reaction involved in this test uses a Seliwanoff  reagent that contains resorcinol and
dilute HCL that reacts with the ketose rapidly in boiling water. This will then result to the
formation of a red colored precipitate indicating that the solution now contains ketohexose and
ketopentose.

6. Which of the sugar undergoes fermentation? Which does not? Why? What is the purpose of
this test? How are carbohydrates differentiated by this test? What is the reaction involved in this
test?
 All of the sugar solutions, except galactose and lactose, underwent fermentation. All the
other sugar solutions had a presence of bubbling in them while galactose and lactose did not
experience such. It is for this reason that lactose can not be fermented by yeast. Lactose can
only be digested in the presence of Lactase– a β-galactosidase enzyme that hydrolyzes lactose
into glucose and galactose. The purpose of this test is to indicate whether or not a sugar can be
broken down into smaller compositions by microorganisms including yeast. Because of this, one
can use this test to determine a monosaccharides from a lactose. 

Table 4. Reaction of Carbohydrates with (a) Nitric acid and ; (b) I /KI
2

Sugar solution OBSERVATION

(a) Galactaric acid test (b) Iodine test

5% galactose Crystal formation is observed. No change in color.

5% glucose No crystal formation is No change in color.

observed.
5% maltose No crystal formation is No change in color.
observed.
5% sucrose No crystal formation is No change in color.
observed.
5% lactose No crystal formation is No change in color.
observed.

5% starch No crystal formation is The color became a bluish black.

observed.

Exploratory Question/s:

7. Which of the sugar solutions gave a negative test/positive test with nitric acid? Why? What is
the purpose of this test? How are the carbohydrates differentiated by this test?

Only 5% of galactose was found to contain nitric acid or only galactose test positive with
nitric acid. The rest of sugar solutions test negative.This is because nitric acid only distinguishes
galactose from other carbohydrates that aren't or don't contain it. Because nitric acid is an
oxidizing agent, it breaks down the aldehyde and alcohol groups to generate the crystal.

8. Can this test be used to determine the presence of milk in milk-chocolate bar? How? 

Lactose is a sugar found in milk that is made up of glucose and galactose. Sugars are
catalyzed and oxidized by nitric acid, resulting in crystal formation. The hydrolysis of lactose to
glucose and galactose is catalyzed by nitric acid initially. Nitric acid then converts both of those
sugars to tetrahydroxy acid. Galactaric crystallizes, indicating that the test is positive.

9. Which of the sugar solutions gave a negative test/positive test with I /KI? Why? What is the
2

purpose of this test? How are the carbohydrates differentiated by this test? What is the reaction
involved in this test?

The only carbohydrate that tests positive in an iodine test is starch. The iodine test is
unique in that it is not necessarily based on the presence of a specific carbohydrate, but rather
on the structure of starch. Starch is a large polysaccharide with a helical structure, but the
hollow middle of amylose is where iodine enters to produce a straight triiodide structure that is
soluble and gives the color deep black blue.

REFERENCES:

Anupama Sapkota. (2020, November 18). Barfoed’s Test- Definition, Principle, Procedure,

Result, Uses. Retrieved February 25, 2022, from Microbe Notes website:

https://microbenotes.com/barfoeds-test/

‌Carbohydrates - Bial’s Test. (2022). Retrieved February 25, 2022, from Harpercollege.edu

website:http://dept.harpercollege.edu/chemistry/chm/100/dgodambe/thedisk/carbo/bial/

bials.htm

Carbohydrates - The Molisch Test. (2022). Retrieved February 25, 2022, from Harpercollege.edu

website:http://dept.harpercollege.edu/chemistry/chm/100/dgodambe/thedisk/carbo/

molisch/molisch.htm
Chhabra, N. (2014). Qualitative tests for carbohydrates. Retrieved February 25, 2022, from

Slideshare.net website: https://www.slideshare.net/namarta28/qualitative-tests-for-

carbohydrates-35884145

‌Hernandez, G. (2016, March 4). Mucic Acid Test. Retrieved February 25, 2022, from

Academia.edu website: https://www.academia.edu/22832264/Mucic_Acid_Test

https://www.facebook.com/sag.micro. (2015, October 29). Benedict’s Test- Principle,

Preparation, Procedure and Result Interpretation. Retrieved February 25, 2022, from
Microbiology Info.com website: https://microbiologyinfo.com/benedicts-test-principle-

composition-preparation-procedure-and-result-interpretation/