Prepa Anáhuac

Group #34

Laboratory Practice Date 22/sep/2023

Identification of Biomolecules
Team number:
Team members:
Fernanda Luque
Marisol Barrios
José Manuel Dorantes
Carlos Iván Vásquez

Objective: Identify the major types of organic compounds in some common foods, by
making the chemical reaction of each group with a specific indicator.

Pre Laboratory questions:

1. What is an indicator? How are indicators used in this experiment, explain the reaction for each
indicator that will be used in the experiment?
2. What is the purpose of using distilled water as one of your test substances?
3. What is the purpose of washing the test tubes thoroughly at the end of the practice?

Introduction: Starting with biomolecules, are chemical compounds found in living organisms that
means that they are everywhere and each one has their own purpose.
They are 4 major types of biomolecules and we are going to explain all:

Lipids: The Broad Pharm Web Site stipulates that, Lipids are a class of organic molecules that are
fatty acids or their derivatives. “Lipids are insoluble in water but soluble in organic solvents. The most
common lipids include fats, waxes, oils, certain vitamins, phospholipids, and steroids.” (Broad Pharm,
2022)
Carbohydrates are: “Compounds that contain mainly molecules of carbon, hydrogen, and oxygen,
although they can also contain nitrogen and phosphorus. They are the most abundant compounds and
are widely distributed in horticultural commodities” (Yahid, 2019, p. 09.1).
Proteins: Proteins are large, complex molecules that perform important functions in the body. They
perform many functions in cells and are necessary for the structure, function and organization of
muscles and organs. “Proteins are made up of hundreds or thousands of smaller units called amino
acids, which are attached to one another in long chains. There are 20 different types of amino acids
that can be combined to make a protein.” (MedlinePlus, 2021)
Nucleic Acids: “Nucleic acids are naturally occurring chemical compounds that serve as the
primary information-carrying molecules in cells. They play an especially important role in directing
protein synthesis. The two main classes of nucleic acids are deoxyribonucleic acid (DNA) and
ribonucleic acid (RNA).” (Roberts J, 2023)
Identifying biomolecules in a biological sample often involves the use of various chemical
indicators and their reactions. Biomolecules include carbohydrates, lipids, proteins, and nucleic acids
(DNA and RNA).
We also want to mention the implementation of Distilled Water to test the substance, which is used
because it does not contain impurities causing no interference with the results. Also it is important to
be in a place where all materials are clean and the surface also, we recommend to wash all the tubes
after using it so as not to contaminate and leave residues.

Hypothesis: When applying Sudan III, Iodine solution, Benedict’s solution and Biuret reagent
we’ll be able to see if biomolecules such as lipids, carbohydrates, proteins and nucleic acids are
present in certain substances. The first experiment is to test for lipids, if we apply Sudan III to the
substance given there’ll be a red stain caused by the presence of lipids, that happens because lipids are
nonpolar substances that are hydrophobic and that causes them to group together for the purpose of
getting away from surrounding water molecules; the second experiment is to test for carbohydrates,
we are using Iodine solution to see if starch is present, this’ll make a reaction because there is a charge
transfer that makes the color of the substance change to a black, yellow or brown color.

To detect if there is sugar we are using Benedict’s solution and also we’re putting the tests in a Maria
bath, this’ll make the Benedict’s solution blue color change to green, yellow or red depending on the
amount of sugars, this happens because the solution has copper (II) sulfate, when that is heated it
reduces the copper and makes the colors change; last experiment was testing for proteins, for this
experiment we’re using Biuret reagent to detect if there are proteins present, if there are proteins
present the color will change from yellow to violet or blue, this is the result of Biuret’s reagent copper
ions reaction to proteins’ peptide bonds.

Materials:
Read all the information on chemical safety from any Materials Safety Data Sheet that accompanies a
chemical.

6 test tubes paper towels

Test-tube rack 6 dropper pipettes
Test-tube holder 8 mL distilled water
Masking tape 8 mL simple sugar solution
Glass-marking pencil 8 mL egg white and water mixture
10-mL graduated cylinder 8 mL corn oil
Bunsen burner or hot plate 8 mL starch solution
600-mL beaker 2 mL unknown substance per group
Iodine solution
Sudan III stain
Biuret reagent
Benedict’s solution

Write on a little piece of Masking tape the name of the substance and the indicator that will
be used to identify the biomolecule.
 Test Tubes in rack

Methodology: (A flow chart might help you simplify the steps)

Part A. Testing for Lipids

1. Place 6 test tubes in a test-tube rack. Use masking tape to make labels for each test tube. Write the
name of a different food sample (listed in Materials) on each masking-tape label. Label the 5th test
tube as “distilled water” and the 6th one as “Unknown Substance”
2. Use a graduated cylinder to transfer 2 mL of distilled water into the test tube labeled “distilled
water.” Use a glass-marking pencil to mark the test tube at the level of the water. Mark the other test
tubes in the test-tube rack at the same level.
3. Use a separate dropper pipette to fill each of the other test tubes with 2 mL of the substance
indicated on the masking-tape label. Add 5 drops of Sudan III stain to each test tube. Sudan III stain
will dissolve in lipids and stain them red.
4. Gently shake the contents of each test tube. CAUTION: Use extreme care when handling Sudan III
to avoid staining hands or clothing.
5. In the Data Table, record any color changes and place a checkmark next to those substances testing
positive for lipids.
5. Wash the test tubes thoroughly but leave the labels on.

Part B. Testing for Carbohydrates

Sugars and starches are two common types of carbohydrates.
1. To test for starch, use the same dropper pipettes to refill each cleaned test tube with 2 mL of the
substance indicated on the masking-tape label. Add 5 drops of iodine solution to each test tube. Iodine
will change color from yellow-brown to blue-black in the presence of starch.
2. Gently shake the contents of each test tube. CAUTION: Use extreme caution when using iodine as
it is poisonous and can also stain hands and clothing. In the Data Table, record any color changes and
place a checkmark next to those substances testing positive for starch.
3. Wash the test tubes thoroughly but leave the labels on.
4. For a sugar test, set up a hot-water bath (María bath) as shown in the next Figure. Half fill the
beaker with tap water. Heat the water to a gentle boil. CAUTION: Use extreme care when working
with hot water. Do not let the water splash onto your hands.
5. While the watert bath is heating, fill each cleaned test tube with 2 mL of the substance indicated on
the masking-tape label. Add 5 drops of Benedict’s solution to each test tube. When heated, Benedict’s
solution will change color from blue to green, yellow, orange, or red in the presence of a simple sugar,
or monosaccharide.
6. Gently shake the contents of each test tube. CAUTION: Use extreme caution when using
Benedict’s solution to avoid staining hands or clothing.
7. Place the test tubes in the hot-water bath. Heat the test tubes for 3 to 5 minutes. With the test-tube
holder, remove the test tubes from the hot-water bath and place them back in the test-tube rack.
CAUTION: Never touch hot test tubes with your bare hands. Always use a test-tube holder to handle
hot test tubes. In the Data Table, record any color changes and place a checkmark next to any
substances that test positive for a simple sugar.
Benedict’s solution will not react with sucrose. Avoid materials that contain sucrose.
8. After they have cooled, wash the test tubes thoroughly but leave the labels on.

Part C. Testing for Proteins:

1. Put 2 mL of the appropriate substance in each labeled test tube. Add 5 drops of biuret reagent to
each test tube. CAUTION: Biuret reagent contains sodium hydroxide, a strong base. If you splash any
reagent on yourself, wash it off immediately with water. Call your teacher for assistance.
2. Gently shake the contents of each test tube. Biuret reagent changes color from yellow to blue-violet
in the presence of protein. In the Data Table, record any changes in color and place a checkmark next
to any substances that test positively for protein.
3. Wash test tubes thoroughly but leave the labels on.

Part D. Recognizing the Unknown solution:

Look at your Data Table and observe where the 6th test tube was positive to the indicator reaction, so
you can tell what biomolecule was the one given to your team.
Results:

Sugars test 1: test 2: test 3: test 4: test 5: test 6:

stayed turned stayed turned stayed stayed
blue from green violet from clear and blue
to green to with a blue
yellowish intense dot
brown yellow/ora
nge

Lipids test 1: test 2: test 3: has test 4: has test 5: has test 6: has
has a red looks like a red color a red color an a dark red
color but it has a red orange/re color
at the line at the d color
very end upper part
of the of the tube
tube it
has a
transpar
ent color

Protein test 1: no test 2: has test 3: has test 4: has test 5: no test 6: no
s color a violet only a little a beige color color
(clear) color in bit of violet color (clear) (clear)
the upper color
part of the
tube

Starch test 1: is test 2: the test 3: has test 4: has test 5: has test 6: has
black upper part a brown a red color a brown a brown
of the tube color but at the color color
has a dark end of the
brown tube it has
color but a dark
the rest is brown
beige color
 Data Table

Substance Sudan III Iodine Benedict Biuret

(Lipids) (Starch) (simple sugars) (Proteins)

Water

Sugar

Starch

Corn Oil

Egg white

Unknown
Solution

Result’s Analysis

To test for lipids, we add Sudan III solution to the tested substance and observe any color
changes. To test for carbohydrates, we use iodine solution to test for starch and a hot-water
bath and Benedict's solution to test for reducing sugars.

- Sugars: Tests 1, 3, 5, and 6 did not change color, while tests 2 and 4 changed from green to
yellowish brown and from green to intense yellow/orange, respectively. Tests 1, 3, 5, and 6: In
these tests, there was no noticeable change in color when we added Benedict's solution and heated
the samples. This lack of color change suggests that these substances contained minimal or no
reducing sugars. Reducing sugars are a type of sugar that can react with Benedict's solution to
produce color changes. 2 and 4: In contrast, when we added Benedict's solution and heated these
samples, the solution changed from green to yellowish brown (test 2) and from green to an intense
yellow/orange (test 4). This indicates the presence of sugars in these substances. The deeper color
change suggests a higher concentration of sugars. This suggests that the tested substances
contained different amounts of sugars, with tests 2 and 4 having higher sugar content than the
other tests.

- Lipids: Test 1 had a red color but at the very end of the tube it had a transparent color, while
test 2 looked like it had a red line at the upper part of the tube. This suggests that the tested
substances contained lipids, with test 1 having more lipids than test 2.Tests 1, 5, and 6: When
Biuret reagent was added to these tests, there was no visible color change. This suggests that these
substances did not contain proteins or had an exceptionally low protein content that was not
detectable by the Biuret reagent.Tests 2, 3, and 4: In these tests, the addition of Biuret reagent
resulted in a noticeable color change, ranging from violet to blue-violet. This color change indicates
the presence of proteins in these substances, with the intensity of the color change potentially
correlated with the concentration of proteins.
-Starch:. The results show that tests 1-6 all contained starch, as indicated by the color changes
observed after adding iodine solution.The results for starch were consistent across all tests (1-6).
When iodine solution was added, there was a color change observed, indicating the presence of
starch. The color change ranged from black (test 1) to various shades of brown, indicating that all
tested substances contained starch, with test 1 having a greater quantity.

-Proteins: The results show that tests 1, 5, and 6 did not contain proteins, while tests 2, 3, and
4 contained proteins, as indicated by the color changes observed after adding Biuret
solution.Tests 1, 5, and 6: When Biuret reagent was added to these tests, there was no visible color
change. This suggests that these substances did not contain proteins or had an exceptionally low
protein content that was not detectable by the Biuret reagent.Tests 2, 3, and 4: In these tests, the
addition of Biuret reagent resulted in a noticeable color change, ranging from violet to blue-violet. This
color change indicates the presence of proteins in these substances, with the intensity of the color
change potentially correlated with the concentration of proteins.

Conclusions: Based in the experiment we can prove our hypothesis because it states
that by applying Sudan III, Iodine solution, Benedict’s solution, and Biuret reagent, we will be
able to see if biomolecules such as lipids, carbohydrates, proteins, and nucleic acids are
present in certain substances. We were able to successfully identify the presence or absence of
lipids, carbohydrates (specifically starch), and proteins in the tested substances using the
specific indicators mentioned in the hypothesis. The laboratory practice was successful in
identifying the major types of organic compounds in common foods using specific indicators.

1. Lipids: In the examined compounds, the presence of lipids was confirmed in varied degrees
of intensity. Lipids' hydrophobicity and propensity to agglomerate in the presence of nonpolar
solvents were both highlighted by Sudan III staining.

2. Carbohydrates (Starch and Sugars): The color change with iodine solution revealed that
starch, a type of carbohydrate, was uniformly present in all compounds examined. On the other
hand, different amounts of simple sugars were found in several substances using Benedict's
solution.

3. Proteins: The existence of proteins was proven in some substances, but it was not apparent
in others when tested with the Biuret reagent. This suggests that the tested samples' protein
contents varied.
The outcomes are consistent with how we understand chemical interactions
We were also able to identify the different reactions to the indicators. This laboratory practice
provides a useful method for identifying the major types of organic compounds in common
foods.

Bibliography

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Online. https://www.biologyonline.com/dictionary/biuret-test

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3. Davidson, E. A. (2023, July 4). carbohydrates. Encyclopedia Britannica.

https://www.britannica.com/science/carbohydrate

4. No title. (n.d.). Study.com. Retrieved September 22, 2023, from

https://homework.study.com/explanation/what-is-the-basis-for-the-sudan-iii-test-why-does-it-i
dentify-non-polar-substances-such-as-lipids.html

5. Roberts, R. J. (2023, June 27). nucleic acid. Encyclopedia Britannica.

https://www.britannica.com/science/nucleic-acid

6. Roberts, R. J. (2023, June 27). nucleic acid. Encyclopedia Britannica.

https://www.britannica.com/science/nucleic-acid

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https://www.abdn.ac.uk/rowett/documents/Sourcing_Sugars_Teacher_BENEDICTS_2.pdf

8. Xucheng Hou, Tal Zaks, Robert Langer, Yizhou Dong, "Nature Review Materials", (2021)
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