Identification of Biological Molecules
All living things are essentially composed of the same basic groups of molecules. The four
types of carbon-based molecules you will perform chemical tests for in this laboratory exercise
are carbohydrates (sugars and starch), lipids, amino acids, and proteins.
These molecules come from what you eat, so many of the samples you test will be food
products.
Objectives
When you have finished this lab, you will be able to
1. list the four groups of biological molecules.
2. explain the necessity of using controls in experiments.
3. define all boldface terms.
4. distinguish positive from negative test results.
Note: Goggles must be worn for all parts of this exercise.
Procedures:
Molecules that are built the same way can be expected to behave nearly the same in a
chemical reaction. In this lab you will perform tests to detect the presence of several classes of
molecules and macromolecules (large molecules). When performing chemical tests, first a set
of standards or controls, is done against which you may compare your results. A negative
control uses a substance that will not react with the chemicals. This will show you what a
negative result looks like. A positive control is a substance that will react with the chemicals. It
will show you a positive result.
Be sure you follow directions and carefully measure out the proper amount of each material.
Carbohydrates:
Carbohydrates like sugars and starches are important sources of energy in your diet. Foods
like pasta, bread and potatoes are examples of the complex carbohydrates dieticians suggest we
eat more of while lessening our intake of refined “quick-energy” sugars like sucrose (table sugar)
and high fructose corn syrup, a component of many sweetened foods and drinks.
Spring 2006
1
Sugar Test
Simple sugars can be detected using Benedict’s test. Benedict’s reagent changes color (after
heating) in the presence of these molecules. However, some other sugars and starches are
structurally different enough that they do not give a positive test (a color change). Perform the
following tests:
1. Number 7 test tubes and add your initials.
2. Add equal amounts of Benedict’s reagent and the proper sample (see Table 1).
3. Heat in the boiling water bath for 5 to 10 minutes. Remove the hot tubes with test tube
holders and place them in your test tube rack.
4. Record your results and interpretations (what those results mean; e.g., does the sample
contain sugar?) in Table 1. The color you get also indicates how much simple sugar is
present. For instance, blue-green = small amount, yellow = medium, and red-orange = a
large amount.
Tube #
1
2
3
4
5
6
7
Table 1. Results of Benedict’s test for sugars
Sample Material
Final Color
Interpretation of Results
Water
Clear blue
No simple sugars present
Glucose
Onion juice*
Potato juice*
Sucrose
Mashed green banana*
Mashed ripe banana*
*To make a sample of these items, cut a small cube of the material, add a small amount of water
and mash it with a mortar and pestle.
Answer the following questions about this test:
What is the negative control in this test? _____________________________________________
What is the positive control in this test?______________________________________________
Which banana (ripe or green) has more sugar?________________________________________
Does the onion or potato contain more sugar?________________________________________
Spring 2006
2
Starch Test
Starch turns bluish-black in the presence of iodine.
Procedure:
1. Cut a very thin slice of onion and potato. Place each on a separate microscope slide.
2. Add one or two drops of iodine and a coverslip to each slide and observe them with the
compound microscope.
Describe what you see inside the potato cells (structures, which parts are stained, etc.):
______________________________________________________________________________
Describe what you see inside the onion cells (structures, which parts are stained, etc.):
______________________________________________________________________________
According to the results of the Benedict’s and starch tests, a potato plant stores most of its
carbohydrates in the form of (starch or sugar?) _________________________, while onion
plants store most of theirs in the form of (starch or sugar?) _____________________________.
3. Label two slides at one end with either the word “Green” or Ripe”.
4. Place a very small amount of mashed green banana and mashed ripe banana on the
appropriate slide. Spread the sample on the slide with a toothpick so that it is very thin.
5. Add a drop of iodine and a coverslip. Observe the slides microscopically.
Which banana has the most starch? ________________________________________________
Keeping in mind which banana has the most sugar and which has the most starch, describe the
chemical changes that take place in a banana as it ripens: ______________________________
_____________________________________________________________________________
Wild bananas have seeds. How does becoming sweeter aid in the dispersal of these seeds?
______________________________________________________________________________
Name two other fruits or vegetables that follow the same pattern: _________________________
Lipids:
Lipids are a diverse group of molecules that share one characteristic property: they do not
dissolve in water. Lipids are an efficient form of energy storage. One gram of lipid contains
twice as much energy as one gram of either carbohydrates or proteins. This form of condensed
energy storage is especially useful to small active animals, such as birds. Lipids also play a
Spring 2006
3
significant role in the structure of biological membranes. Cells, as we know them, would not
exist without lipids. Despite popular belief, lipids (fats), are an important part of a balanced diet
and can be found in oils, fried foods and nuts. Perform the following tests:
1. Add 10 mL of water to a test tube.
2. Add 10 drops of vegetable oil. Shake. Observe the dispersal of oil in the water followed by a
rapid separation into two distinct layers.
Which substance floats to the top? __________________________________________________
3. Add to this tube a few drops of an emulsifier. An emulsifier breaks oil into small droplets
and prevents them from separating into layers again. (This is how creamy salad dressings are
made.) Shake the tube again. This mixture of oil and water is an emulsion.
Describe the distribution of oil in the emulsion now compared to the previous test.
______________________________________________________________________________
4. Put a drop of the emulsion (not the emulsifier) on a slide. Add a small drop of Sudan IV stain,
mix, and add a coverslip. Examine microscopically.
Which substance (water or oil) is stained by the Sudan IV? ______________________________
Proteins:
Long chains of amino acids are joined together chemically by peptide bonds to form
proteins. Proteins are much more than meat or muscle. They determine the shape and structure
of living organisms and act as enzymes that promote the chemical reactions necessary for life.
Twenty amino acids are used in the formation of proteins. Your body can manufacture only
twelve while the other eight, the essential amino acids, must be provided by your diet. Foods
high in the essential amino acids are fish, eggs, and beans.
Amino Acids
Most amino acids (except proline) will produce a violet color in the presence of the chemical
ninhydrin. Proline produces a yellow color.
Procedure:
1. Obtain a round piece of filter paper, being careful to handle it only by the edges. Label it as
shown below with a pencil.
Spring 2006
4
2. With a transfer pipette place a drop of the appropriate substance on the paper. Allow the spots
to dry. In the “Thumbprint” circle press your thumb very firmly, trying to leave a heavy
thumbprint. (This was one of the first methods used to try to reveal fingerprints.)
3. Spray these areas with 1% ninhydrin solution. Caution: Ninhydrin spray is to be used only in
the fume hood.
4. Place the papers on the hot plate to dry. The color should develop in 15-20 minutes.
Record your results in Table 2.
Table 2. Results of amino acid test
Sample
Final Color
Interpretation of Results
Water
Phenylalanine
Proline
Thumbprint
Why does your thumbprint give a positive result? ______________________________________
Spring 2006
5
Proteins
Biuret reagent changes color from 'sky blue' to pink or lavender in the presence of proteins,
but not amino acids.
Procedure:
1. Label four test tubes from 1-4.
2. With a graduated cylinder, add 10 mL Biuret reagent to each test tube. Caution: Biuret
reagent is caustic. Do not get it on your skin or clothes. Wash off with water
immediately.
3. Add an equal amount of the appropriate material listed in Table 3 to the appropriate test
tube.
4. Record your results in Table 3.
Table 3. Results of Biuret test for proteins
Tube #
Sample
1
Water
2
Egg white solution
3
Egg yolk solution
4
Phenylalanine
Spring 2006
Final Color
Interpretation
6
Testing Unknowns:
Each group will be provided with an unknown food. The letter tells you the group of possible
unknowns from which your sample came. Your food is one of the ones listed under that letter.
A
B
C
Soy flour
Corn starch
Ground coffee
Glucose
Honey
Instant coffee
Powdered milk
Table sugar
Enriched Flour
Egg white
D
E
Vermont Maid Syrup
Glucose
Maple syrup
Table salt
Potato starch
Gelatin
The compositions of these foods are given in Table 4*. Look on this table for simple sugar,
starch, fat, and protein compositions of these foods. Low levels of particular molecules will
probably not show up in these tests.
Procedure:
1. Place a small sample of the unknown in each of several clean test tubes. Add the proper
reagent for sugar to test tube #1. Which reagent will you use?______________________
2. Use tubes 2, 3, and 4 to test for the other substances [starch (iodine), lipids (Sudan IV),
and protein (Biuret)]. Do not add reagents to the entire original unknown. Look back to
earlier parts of this lab to recall these procedures. Test only a small portion of solid
unknowns and dissolve them in water when appropriate, such as for Benedict’s test.
3. Mix the reagent and unknown food thoroughly so that you don’t get a false negative
reaction.
4. After each test, decide which, if any, substance on your list of possible unknowns was
ruled out. For example, a positive test for simple sugar would rule out corn starch (starch
must give a negative test). A negative test for simple sugar would rule out honey
(glucose in honey must give a positive test).
5. Identify your unknown and enter it in Table 5.
Spring 2006
7
Table 4. Composition of Foods
Grams per 100 grams edible portion (or %). Amounts in parentheses cannot be detected
by your tests.
Corn Starch
Egg White
Egg Yolk
Enriched Flour
Gelatin (dry)
Glucose
Ground Coffee
Honey
Instant Coffee
Maple Syrup
Potato Starch
Powdered Skim Milk
Soy Flour
Table Salt
Table Sugar
Vermont Maid Syrup
Water
% Protein
% Fats
% Carbohydrate
12
87.6
51.1
12
13.0
9
4.1
17.2
2.6
21.5
7.6
4
8
0.2
0.5
25
(0.3)
10.9
16.0
(10.5)
85.6
(0)
(12.5)
(0.3)
(0)
(0)
(8.0)
35.8
41.2
(0)
(0)
(0)
(trace)
(trace)
30.6
(1.0)
(0.1)
(0)
15.4
(0)
(0)
(0)
(0.8)
(0.7)
12.1
(0)
(0)
(0)
87.6 (as starch)
(0.8)
(0.6)
76.1 (as starch)
(0)
91
28.5
82.3 (as glucose + fructose)
35.0
(77) (only sucrose)
79.9 (as starch)
51.6 (as starch)
33.3
(0)
(99.5)
74 (as glucose + fructose)
Table 5. Results of Testing Unknowns.
Letter of your unknown _____________.
Test Performed
Results
Substance (s) Eliminated by This Test
Your unknown substance is _______________________ Instructor’s Initials __________
Before you leave the lab . . . make sure that all the equipment you used is spotlessly clean for
the next class. Be sure your work area has been washed clean. Do not throw any garbage down
the sink. All solid wastes must go in the waste can.
Modified slightly from Experiments in Biology (1988) by Carolyn Eberhard, published by Saunders College Publishing.
**Watt, B. and A. Merrill, “Composition of Foods, Raw, Processed, Prepared.” Agricultural Handbook No. 8. Revised Dec. 1963. Sup. of
Documents, U.S. Govt. Printing Office, Washington, DC.
Spring 2006
8