Lab 3: Tests for Biological Molecules

Preparation
1. Read the entire exercise before lab and mark anything you will need to ask about. This will
prepare you to work more efficiently in lab because you might not then have to read
through all of the background information while you could be doing the activities. We will
be able to tell who is prepared and who did not read their lab material thoroughly.

Objectives
1. Know the names of the basic tests for carbohydrates and proteins.
2. Be able to accurately interpret the results of the tests for biological molecules
3. Use your knowledge of the basic tests for carbohydrates and proteins to determine
what is in an unknown solution.

Testsfor Biological Molecules

Before starting the following tests, your instructor will assign you an unknown mixture (A, B or
C). This sample contains a mixture of biological molecules. Determine the types of biological
molecules present in the solution as you do the tests of the molecules. Record the process of
your investigation, results, and conclusions. When you have finished, check with your TA to find
out if you’re right. Then go celebrate your success (or try to figure out what might have gone
wrong)!

Benedict’s test for Sugars

Benedict’s solution is composed of sodium carbonate, sodium citrate, and copper sulfate.
When Benedict’s solution is heated in the presence of a reducing substance, the blue cupric
ions (Cu++) are reduced to red, insoluble cuprous ions (Cu+).
The color varies from greenish to orange to red to brown, depending on the amount of
reducing substances present. Thus, Benedict’s solution allows us to test quantitatively for the
presence of reducing sugars.

Procedure:
1. Half fill the beaker with water and heat to a gentle boil.
2. Using a Sharpie, number the test tubes 1-8.
3. Add 1 ml (10 drops) of the correct test solution to each tube, as indicated in the table.
4. Add 2 ml (20 drops) of Benedict’s solution to each of the 8 test tubes.
5. Place the test tubes in a boiling water bath for approximately 3 minutes.
6. Allow the tubes to cool to room temperature and record your results below:

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 Tube Test Color/appearance Color Resulting Conclusion
# Solution before adding right after Color (Does the substance
Benedict’s adding after in the test tube
Benedict’s heating contain a reducing
sugar?)

1 Water

2 Glucose

3 Sucrose

4 Fructose

5 Lactose

6 Starch
solution

7 Egg
Albumin

8 Unknown____

General conclusions: Compare the Benedict’s test results obtained on the different solutions.

How can you explain these results? ________________________________________________

______________________________________________________________________________

Iodine Test for Starch

Iodine potassium iodide solution enters the amylase coils of the starch molecule. When this
happens, the color of the iodine potassium iodide solution changes from a yellowish orange to
a bluish black. This was seen previously when the starch in the parenchyma cells of the potato
was stained with iodine.

Procedure:
1. Obtain 4 clean test tubes and number them 1-5.
2. Add 1 mL (10 drops) of the correct test solution to each tube as indicated in the table below.
3. Add just 1 or 2 drops of Iodine Solution to each of the 5 tubes. Mix by gently shaking the
tube.
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*DO NOT HEAT tubes containing Iodine solution.
4. Record any color change for each tube in the table below.

Tube Test Solution Color/appearance Color after Conclusion

before adding adding (Does the substance in the test
# Iodine Iodine tube contain starch?)

1 Egg albumin

2 Starch
3 Glucose
4 Water
5 Unknown___

General conclusions: Compare the Iodine test results obtained on the different solutions.

How can you explain these results?_________________________________________________

______________________________________________________________________________

Biuret Test for Protein

The Biuret Test is one method of detecting the presence of proteins. The name biuret is derived
from the colored chemical complex formed between copper and proteins under alkaline
conditions. Whole proteins will give a violet color and if polypeptides are present rather than
whole proteins, a pink color results.
A number of complex substances besides proteins and polypeptides can respond with a violet
color to this test. Thus, a positive biuret test does not in itself conclusively prove the presence
of protein but since all proteins give a positive test even in high dilution, a negative biuret test
can be concluded as the near absence of protein.

Procedure:
1. Obtain 4 clean test tubes and number them 1-5.
2. Add 1 mL (10 drops) of the correct test solution to each tube as indicated in the table below.
3. Add 1 mL (10 drops) of Biuret Solution to each of the 5 tubes.
4. Wait 5 minutes. Record any color change for each tube in the following table.

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 Tube Test Solution Color Color right Resulting Conclusion (Does the
before after Color after substance in the test tube
# Adding adding contain protein?)
Biuret Biuret 5 minutes

1 Egg albumin

2 Starch

3 Glucose

4 Water

5 Unknown___

General Conclusions: Compare the Biuret test results obtained on the different solutions. How
can you explain these results?_____________________________________________________
_____________________________________________________________________________

What were the components of your unknown?

Clean up:

Washing test tubes:

1. Carefully pour the contents of the test tubes down the sink drain and rinse the test tubes
with tap water.

2. Fill the test tubes with warm soapy water. Then using a test tube brush, scrub the inside of
the test tube. Be sure to remove all of the red colored residue (from the sugar tests). Also,
remove your markings on the outside of the tubes.
3. Rinse well with warm tap water and return the tubes to the rack on your bench.

4. Invert the tubes so that they will drain.

5. Make sure the hot plate is turned off.

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Discussion Questions

1. Fill in the table by listing the 4 classes of large biological molecules, their building blocks and
their functions:

Large Building Examples Functions

Biological Block(s) or
Molecule Components

2. With regard to biological molecules, what does it mean to be hydrophilic? What does it
mean to be hydrophobic? Can a molecule be both at the same time? Provide examples.

3. What is a polyunsaturated fat? What is a monounsaturated fat?

4. Look at the food labels. Judging by the numbers on these labels, how many food calories are
there in a gram of fat? Protein? Carbohydrate?

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5. Which type of large biological molecule would be best to use for energy storage? Why?