St.

Paul College of Ilocos Sur

(Member, St. Paul University System)
St. Paul Avenue 2727, Bantay, Ilocos Sur

COLLEGE DEPARMENT

DEPARTMENT OF NURSING

Fabie, Jemmalyn D. September 25, 2023

BSN 2-C

LABORATORY REPORT # 2
TESTS FOR CARBOHYDRATES

Introduction

Carbohydrates are important class of organic compounds that are essential in the
biological processes of all living organisms. These constitute carbon, hydrogen, and
oxygen atoms, which are primarily responsible for providing energy and serving as
structural components in cells. They involve a diverse range of molecules, from simple
sugars like glucose and fructose to complex polysaccharides like cellulose and
glycogen. The identification and characterization of carbohydrates are of great
importance in various fields, including biochemistry, nutrition, and clinical diagnostics.
In this laboratory experiment, we had the chance to dive in to the different tests in
identifying and studying carbohydrates, which are the Molisch test, Iodine test,
Benedict’s test, and Mucic test. These tests rely on explicit reactions that carbohydrates
undergo, generating observable changes in color or other measurable properties. In
conducting these tests, we were to uncover the complexity of the carbohydrates, and
gain insights into the presence, type, and structure of carbohydrates in a given sample.

Objectives
 To be able to provide a brief overview of carbohydrates and their different types.
 To be able to describe the different tests that can be used to identify
carbohydrates.
 To be able to explain the principles behind the tests for carbohydrates.
 To be able to underscore the importance of carbohydrate analysis in various
scientific disciplines.

Materials
 Analytes
- Distilled water  Test tubes
- Blue water  Beakers
- Milk  Test tube rack
- Sprite  Stirring rod
- Crackers (Sky Flakes)  Pipette
- Urine sample  Dropper
- Orange juice  Alcohol lamp
- Sugar  Tripod
 Reagents  Thermometer
- Molisch reagent
 Wire gauze
- Sulfuric acid
 Slides
- Iodine solution
- Benedict’s reagent  Microscope
- Concentrated nitric acid  Masking Tape
 Marker
Procedure

A. Molisch Test
1. Prepare 4 test tubes and label them as follows: distilled water, blue water,
milk, and sprite.
2. Put 2 ml of each analyte on each test tube.
3. Add two drops of the Molisch reagent into each test tube and mix thoroughly.
4. Using a dropper, slowly add 1 ml (15 drops) of concentrated sulfuric acid
alongside each tube to create an interface with the mixture.
5. Note what is formed in the interface. Record your observations.

B. Iodine Test
1. Prepare 3 test tubes and label them as follows: distilled water, crackers (sky
flakes), and sprite.
2. Put 2 ml of each analyte on each test tube.
3. Add a drop of iodine solution to each of the test tube and mix thoroughly.
4. Note the color of the resulting mixture.
5. Warm the three test tubes in water bath (60%) until a change is observed in
one of the test tubes.
6. Quickly remove all the tubes from the bath. Cool at room temperature.
7. Note the color of each mixture and record the results.

C. Benedict’s Test
1. Prepare 4 test tubes. Label each test tube as follows: distilled water, urine
sample, orange juice, and milk.
2. Into each test tube, place 1 ml (15 drops) of Benedict’s reagent.
3. Into each test tube, add 1 ml (15 drops) each of the analyte.
4. Note any changes. When no changes occur, place the tube in a war, water
bath (60%).
5. Remove all the tubes once a change is observed in some of the samples.
Allow to cool at room temperature.
6. Note the changes and record your observations.
Report Color of Solution Glucose Concentration
- Blue 100 mg/dl
+ Green 150 mg/dl
++ Yellow 1000 mg/dl
+++ Orange 2000 mg/dl
++++ Brick red +2000 mg/dl
D. Mucic Acid Test
1. Prepare 4 test tubes and label as follows: distilled water, milk, orange juice,
and sugar.
2. Add 1 ml (15drops) of each of the analyte.
3. Carefully place 1ml (15 drops) of concentrated nitric acid into each test tube
and mix thoroughly for a minute.
4. Hold a slide with the wooden test tube holder along one end. Transfer some
drops of the mixture onto the slide and heat over an alcohol lamp until they
are almost dry. DO NOT SCORCH! Continue doing this until 1ml or half of the
sample has been transferred.
5. Cool the slide to room temperature.
6. Examine the crystals formed under the microscope.
7. Draw and describe the crystals (color and shape) formed under LPO. Write
NONE if no crystals are formed with the given sugar.

Results/Discussions
A. Molisch Test

Molisch's test is a chemical test used to detect the presence of carbohydrates in

a sample. It is a general test for carbohydrates, meaning that it will give a positive
result for all types of carbohydrates, including monosaccharides, disaccharides,
and polysaccharides.
When a sample containing carbohydrates is heated with concentrated sulfuric
acid, the carbohydrates are dehydrated to form furfural or its derivatives. Furfural
and its derivatives are then able to react with α-naphthol to form a purple-colored
compound.

Analytes Analytes + Molisch Reagent Analytes + Sulfuric Acid

As seen on the pictures above, a purple ring formed at the interface between
the two layers of the liquids after the Sulfuric acid was added to the analytes;
giving the Molisch test positive results for all the analytes above. These results
indicate the presence of carbohydrates on the samples.
In Molisch test, the carbohydrate undergoes dehydration upon the introduction
of concentrated hydrochloric or sulfuric acid, resulting in the formation of an
aldehyde. This aldehyde undergoes condensation along with two phenol-type
molecules (such as α-naphthol, resorcinol, and thymol), resulting in the formation
of a purple or reddish-purple colored complex.

B. Iodine Test

The Iodine Test is a chemical test used to detect the presence of starch in a
sample, which is a complex carbohydrate composed of glucose molecules linked
together. It is a specific test for starch, meaning that it will only give a positive
result for starch and not for other types of carbohydrates.
This test relies on the unique property of starch to form a deep blue-black
complex with iodine in the presence of water.

As
we

have +observed
Analytes on
Iodine Solution the Samples in Warm Bath Iodine test,After
Samples only theBath
Warm crackers
formed a deep blue-black color, and the sprite and distilled water samples did not
undergo any change – their deep brown color remained. This implies that only
the crackers sample is positive of starch among the three analytes.
The iodine test takes advantage of the fact that iodine molecules can enter the
helical structure of amylose molecules, forming a stable inclusion complex. This
complex results in a distinct color change from amber or yellow (in the absence
of starch) to a deep blue-black or purple color (in the presence of starch). This
test is commonly used in food quality control to determine the starch content in
various food products, including potatoes, grains, and sauces, as well as in
 biological and biochemical experiments to detect the presence of starch in plant
tissues or cell extracts.

C. Benedict’s Test

Benedict's test is a chemical test used to detect the presence of reducing

sugars in a sample. Reducing sugars are carbohydrates that can donate
electrons to other molecules resulting to a color change. Benedict's solution
contains copper (II) ions, which are blue in color. When reducing sugars are
added to Benedict's solution, they reduce the copper (II) ions to copper(I) ions.
Copper(I) ions form a red precipitate of cuprous oxide.

After+ Benedict’s
Analytes undergoing a warm
Solution Samples in Warm Bath Samples After Warm Bath
water bath, the only sample that had a significant color change is the orange
juice giving it a ++++ result as based on the given table. Basing on the table
given for the solution reports, the brick red color of the analyte denotes that there
is an approximately +2000 mg/dl concentration on the sample. As seen on the
photos above, we have also observed that the urine and milk samples have
formed green precipitates denoting a + result with an approximately 150 mg/dl
glucose concentration. While the distilled water did not show any change,
wherein the solution’s color is blue with a negative result with an approximately
100 mg/dl glucose concentration.
Benedict's test is a sensitive test for reducing sugars and can detect even small
amounts of reducing sugars in a sample. It is a simple and easy-to-perform test,
and it is widely used in laboratories and in industry to test for the presence of
reducing sugars in food, beverages, and other products.

D. Mucic Acid Test

The Mucic Acid Test is a chemical test used to detect the presence of galactose
or lactose in a sample. It is a specific test for these two carbohydrates, meaning
that it will only give a positive result for galactose or lactose and not for other
types of carbohydrates. When galactose or lactose is heated with nitric acid, it is
oxidized to form mucic acid. Mucic acid is a white, insoluble substance. When
mucic acid is heated with sulfuric acid, it forms a white precipitate giving a
positive result.
 Analytes + Concentrated Nitric Acid Drying Samples on Slide

Analytes Under the Microscope in LPO

Distilled Water Sugar Orange Juice

Milk

As we have observed under the microscope, we have observed the

following:
Analyte Crystallization
Distilled water NONE
Sugar NONE
Orange juice NONE
Milk Clusters of brown fine dots

As stated on the table, only the milk formed crystallizations on the mucic acid
test. This entails that among the three analytes, only the milk is positive in this
test meaning it is the only analyte that contains galactose or lactose. While the
other analytes may contain carbohydrates, but they do not have the presence of
galactose or lactose.
The mucic acid test is specific for galactose and lactose and is not suitable for
detecting other carbohydrates. Additionally, this test requires caution when
working with concentrated nitric acid, which should be handled with care due to
its hazardous properties. The mucic acid test is primarily used in carbohydrate
analysis to identify and distinguish galactose and lactose in various biological
and food samples.
Conclusion

In this laboratory experimentation, we have conducted a series of tests for

carbohydrates to identify their presence, type, and characteristics within various
analytes. These tests allowed us to acquire insights into the carbohydrate
content of the analytes and illustrate the diversity of carbohydrates present in
different materials. We have the Molisch Test, Iodine Test, Benedict’s Test, and
the Mucic Acid Test. These four tests are used as identifiers of the presence of
different carbohydrates in various analytes.
Discerning the presence and conformation of carbohydrates in different
analytes has extensive implications across several scientific disciplines. In food
analysis, these tests help in determining sugar content and starch presence,
which is essential for nutritional labeling and quality control. In clinical
diagnostics, detecting carbohydrates like glucose and lactose aids in diagnosing
conditions such as diabetes and lactose intolerance. Furthermore, carbohydrate
analysis is essential in biological and biochemical researches for studying
metabolic pathways, enzymatic reactions, and cellular processes.
 Overall, the carbohydrate tests conducted in this laboratory experiment have
provided valuable understandings into the composition of the tested analytes.
These tests serve as fundamental tools in various scientific applications, enabling
researchers and analysts to explore the complex world of carbohydrates and
their importance in the domains of biology, nutrition, and quality control. Further
investigations and advanced analytical techniques will continue to enhance our
understanding of carbohydrates and their roles in diverse contexts.

References

https://www.angelo.edu/faculty/kboudrea/index_2353/Chapter_07.pdf

https://byjus.com/chemistry/molischs-test/

https://byjus.com/chemistry/tests-of-carbohydrates/

https://byjus.com/chemistry/benedicts-test/#:~:text=Benedict's%20test%20is%20a
%20chemical,be%20identified%20with%20this%20test.

https://microbiologynote.com/mucic-acid-test/