Biological Macromolecules
Introduction
All organisms are composed of organic compounds. An understanding of some basic
concepts of chemistry is necessary for an understanding of life.
Four biochemical assays will be introduced in this exercise. These are qualitative
assays—assays that simply test for the presence or absence of a substance.
Objectives
Having completed this lab, you should be able to
1. Conduct and interpret the results of biological molecule assays to determine the
presence or absence of biological macromolecules.
2. Explain how and why positive and negative controls are used in biological
molecule assays.
Learning to Conduct and Interpret Results of the Biomolecule Assays
Introduction
In the study of living material, it is often necessary to determine if certain biological
macromolecules are present in a sample. To do this, certain standard tests may be
performed. The tests are qualitative not quantitative; however, the darkness of the
reaction or the amount of precipitate often gives some general idea of the amount
present.
In this exercise, you will learn how to perform the four biochemical assays described
above by analyzing the carbohydrate, protein and fat content of a series of six known
samples:

distilled water

glucose solution

Sucrose

starch solution

Albumin

Vegetable Oil
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using four of the assays (Benedict’s, Iodine, Biuret’s and Sudan III). This will
provide you with the tools you will need for addressing research questions and
designing experiments later in the course.
Note: Before collecting data, you should create a data-recording table in your lab
notebook to help you organize your thoughts and your efforts at the lab bench and to
record your results in an easily understandable way in your notebook. Your goal here
should be learning which assays are used to detect the various biological molecules
and learning to read positive and negative results. Additionally, you should understand
the purpose of using positive and negative controls in chemical assays.
Materials

6 test tubes

distilled water

glucose solution

Sucrose

starch solution

Albumin

Vegetable Oil

Iodine solution

Benedict’s solution

hot plate

400 mL beaker

A lab notebook to record your observations
Iodine Test
1. Set up six test tubes, mark each with a number, and add the following:
o
Tube 1: 5 mL of glucose solution
o
Tube 2: 5 mL of sucrose solution
o
Tube 3: 5 mL of starch solution
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o
Tube 4: 5 mL of albumin solution
o
Tube 5: 5 mL of vegetable oil
o
Tube 6: 5 mL of distilled water
2. Add 5 drops of Iodine solution to each test tube and observe. A positive result is
indicated by a color change from orange to a blue-black color.
3. In your lab notebook record 1) your color observations and 2) your interpretation
of results (i.e., Iodine test positive or negative).
4. Iodine solution gave a positive test for which solution(s)?
5. Deposit waste solutions in the collection jar for hazardous materials and clean
test tubes.
Benedict's Test
1. Make a boiling water bath by placing 150 mL of distilled water into a 400 mL
beaker and heat on a hot plate.
2. Set up three test tubes, mark each with a number, and add the following:
o
Tube 1: 5 mL of glucose solution
o
Tube 2: 5 mL of sucrose solution
o
Tube 3: 5 mL of starch solution
o
Tube 4: 5 mL of albumin solution
o
Tube 5: 5 mL of vegetable oil
o
Tube 6: 5 mL of distilled water
3. To each test tube add 5 drops of Benedict’s solution, then heat the tubes in a
boiling water bath for 2 minutes or until there is a color change. A positive result
is indicated by a color change in the solution from blue to: green, yellow, orange
or brown. The color is determined by the amount of glucose present.
4. In your lab notebook record 1) your color observations and 2) your interpretation
of results (i.e., Benedict's positive or negative).
5. Benedict’s solution gave a positive test for which solution(s)?
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Sudan III Test
1. Wearing latex or nitrile gloves to avoid fingerprints, get a disc of filter paper. Use
a pencil (not a pen) to draw 6 equally-spaced circles and label them with the six
known samples. Put your names on the filter paper.
2. Add one small drop (only) of the appropriate sample to its labeled circle. Make
sure you use the pipette labeled for that sample to avoid cross-contamination
between samples.
3. Put the paper in one half of a Petri dish and place under a lamp at your lab
bench, allowing the samples to dry.
4. Once dry, (Note: The vegetable oil spot my never look completely dry) soak the
paper in the Sudan III solution for 3 minutes.
5. Using forceps, carefully remove the paper from the stain and rinse the paper in a
large beaker filled with distilled water. Rinse the paper for a longer time if no
spots are visible (the paper retains excess dye).
6. In your lab notebook record 1) your color observations and 2) your interpretation
of results (i.e., Sudan positive or negative).
7. Sudan III gave a positive test for which solution(s)?
8. Dispose of all the liquid waste in the labeled liquid chemical waste jar.
Biuret Test
1. For each sample, transfer 2 ml of test solution into the appropriate test tube.
2. Add 5 drops of sodium hydroxide (NaOH) to each sample and mix thoroughly.
3. Add 5 drops of
thoroughly.
copper sulfate (CuSO 4) to each sample and again mix
4. In your lab notebook record 1) your color observations and 2) your interpretation
of results (i.e., Biuret positive or negative).
5. The Biuret test was positive for which solution(s)?
6. Clean up when finished. Discard the liquid chemical waste into the liquid
chemical waste jar. Then thoroughly clean the tubes at the sink.
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Determining the Content of an Unknown Sample
1. Once you have learned how to conduct and interpret each of the assays, get an
unknown sample from your instructor.
2. Record the letter of your unknown sample here and in your lab notebook.
3. Conduct the four assays to determine the contents of your unknown.
4. Record your results in your lab notebook and include an interpretation of your
results.
5. Which biological macromolecule(s) were in your unknown sample?
6. Why was distilled water included in each of the tests?
About this document ...
Biological Macromolecules
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