This document describes several tests used to identify biological molecules including carbohydrates, lipids, and proteins in foods. Carbohydrates are tested using Benedict's test to detect reducing sugars and the iodine test to detect starch. Lipids are tested using the Sudan III stain and brown paper tests to detect hydrophobic regions. Proteins are tested using the Biuret test to detect peptide bonds. Students will perform each test on samples of water, syrup, potato, tofu, vegetable oil, oatmeal, avocado, white grape juice, lettuce, and chicken broth to identify which molecules are present in each food.

1. Identification of Biological Molecules
Introduction:
Molecules of a certain class have similar chemical properties because they have the same functional groups. A
chemical test that is sensitive to these groups can be used to identify molecules that are in that class. Practice the
following tests by using them to see what various foods contain. In each test, you will be including a substance that
does not react in the test to serve as the negative control. A substance known to give a reaction in the test would be
a positive control.
Carbohydrates:
These molecules contain the aldehyde and alcohol functional groups. Certain mono- and disaccharides can be
detected because of their aldehyde groups. Some polysaccharides can be detected because of their specific threedimensional structure. Carbohydrates, which are water soluble or hydrophilic substances, are not detected by their
hydroxyl groups.
Benedict’s Test: A reducing sugar test that depends on a free aldehyde group. Monosaccharides and some
disaccharides have a free aldehyde group and consequently will react to give a positive Benedict’s test. Such a
sugar acts as a reducing agent and is called a reducing sugar. The reaction proceeds as follows:
O
Benedict’s Reagent + R C
Heat
O
Benedict’s Reagent + R C
H
Oxidized Form
Reducing Sugar
O H
Reduced Form
Acid Sugar
This is a typical oxidation-reduction reaction in which the oxidation of the sugar occurs simultaneously with
the reduction of the Benedict’s reagent. Remember that
REDUCTION means removal of oxygen or addition of hydrogen.
OXIDATION means addition of oxygen or removal of hydrogen.
In this case, the sugar obviously gained an oxygen during the reaction and therefore the sugar underwent
oxidation.
Some disaccharides (e.g. sucrose) and all polysaccharides (e.g. starch) are not reducing sugars because they do
not have free aldehyde groups. You will use the Benedict’s test to compare the reducing sugar (e.g. glucose and
fructose) content of the foods listed on the data sheet.
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2. Identification of Biological Molecules
A. Prepare the water bath:
1. You should have a test tube rack with ten (10) test tubes. Number the tubes 1-10 to
correspond with the list of foods on your data sheet.
2. Prepare a boiling water bath for your test tubes by heating a half-full beaker of water on the
hot plate. Leave the heat setting on the middle setting and do not let the beaker boil dry.
B. Benedict’s Test:
1. Fill each test tube about 1cm full with the appropriate substance as listed on the data sheet
(i.e. sample #1 in tube #1). The actual amount in each tube is not critical as long as the same
amount of each substance is used in each tube. Be sure that you use only the specified dropper
for the solution, and return that dropper to the appropriate solution so that you avoid
contamination!
2. Add ten (10) drops of Benedict’s reagent to each tube and mix.
3. Heat the tubes for 4 minutes in the boiling water bath, then carefully remove the tubes using
your test tube holder.
4. A change in color from clear blue to red or orange indicates an abundance of reducing sugars.
A change to green indicates the presence of a smaller amount of reducing sugar.
5. Record your data then rinse the tubes out with water.
Iodine Test: Another test, the Iodine Test, can be used to distinguish starch from mono-, di-, and other
polysaccharides. Starch is a polymer of glucose in which the chains are coiled up in a particular way so that
they can interact with the iodine molecules to give a distinctive blue-black color. Other polymers, even those of
glucose, lack the precise coiled structure of starch and do not give the dark color. Thus the iodine test is a
specific test for starch that depends on the precise three-dimensional coiling of the glucose polymer so that it
can interact with iodine.
1. Fill each test tube about 1cm full with the appropriate substance as listed on the data sheet (i.e. sample
#1 in tube #1). The actual amount in each tube is not critical as long as the same amount of each
substance is used in each tube. Be sure that you use only the specified dropper for the solution, and
return that dropper to the appropriate solution so that you avoid contamination!
2. Add five (5) drops of iodine solution to each test tube.
3. A positive test will give a blue-black color. A yellow or brown result is negative.
4. Record your data then rinse the tubes out with water.
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3. Identification of Biological Molecules
Fats:
A fat is a hydrophobic molecule consisting of a glycerol
molecule joined to three fatty acids. The hydroxyl groups
of the glycerol react with the carboxyl groups of the fatty
acids in a condensation reaction, forming an ester linkage.
These functional groups are therefore no longer available
for a test reaction. Instead the Sudan III test depends on
the detection of the remaining hydrocarbon groups. The
colored dye, Sudan III, and the hydrocarbon groups are
non-polar and stick tightly together in their polar
surroundings. Simple hydrophobic interaction is the
basis for this rather inelegant test.
Sudan III Test:
1. Using a lead pencil and two (2) filter paper disks,
mark the disks with a small circle evenly spaced,
and number the circles to correspond
appropriately with the substance as listed on the
data table.
2. Add your initials somewhere on the filter paper
disk.
3. Add a SMALL drop from the appropriate
substance to the appropriate circled spot on the
filter paper disk. Be sure that you use only the specified dropper for the solution, and return that dropper to
the appropriate solution so that you avoid contamination!
4. Allow the paper to dry completely.
5. Soak the paper for three (3) minutes in the Sudan III stain.
6. Use forceps to remove the paper from the Sudan III stain.
7. Rinse the paper in the water bath for one (1) minute.
8. Examine the intensity of the staining of the spots and rate the staining on your data sheet as follows:
0 = no staining, + = faint orange, ++ = definite orange color
Brown Paper Test:
1. Using a pencil, mark the strip of brown paper bag with small circles evenly spaced along the length of the
paper, and number the circles to correspond appropriately with the substance as listed on the data table.
2. Add your initials somewhere on the paper.
3. Add a SMALL drop from the appropriate substance to the appropriate circled spot on the brown paper. Be
sure that you use only the specified dropper for the solution, and return that dropper to the appropriate
solution so that you avoid contamination!
4. Proceed with the rest of the lab and check your paper at the end. A positive test for lipids is a wet spot on
the paper when viewed from the back. It may be helpful to hold the paper up in front of a bright light to
look for a “wet” semi-translucent area.
5. Record your data.
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4. Identification of Biological Molecules
Proteins:
Proteins are polymers of amino acids in which the carboxyl group of one subunit joins head-to-tail with the amino
group of its neighbor. In this case, the bound amino group is nevertheless sufficiently reactive to change the Biuret
test reagent from blue to violet. Thus the basis of the test is a subtle interaction between the copper ions in the Biuret
reagent and the amide bonds in the protein backbone. This test is specific for the amino group found in the peptide
bonds of proteins.
1.
2.
3.
4.
Fill each test tube about 1cm full with the appropriate substance as listed on the data sheet (i.e. sample #1
in tube #1). The actual amount in each tube is not critical as long as the same amount of each substance is
used in each tube. Be sure that you use only the specified dropper for the solution, and return that dropper
to the appropriate solution so that you avoid contamination!
Add ten (10) drops of Biuret reagent to each tube and mix. This test may take a few minutes to complete
the reaction.
After a few minutes observe the tubes and note the color change. A color change from blue to violet
indicates the presence of protein. Because the color change is more subtle for this test (blue and violet are
very similar) you must observe the tubes very carefully.
Record your data then rinse the tubes out with water.
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5. Identification of Biological Molecules
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6. Identification of Biological Molecules
Name
Partner
Section
Date
Data Sheet
Carbohydrates
Benedict’s
Iodine Test
Test
Tube #
Substance
1
2
3
4
5
6
7
8
9
10
Sudan III
Test
Lipids
Brown Paper
Test
Protein
Biuret Test
Distilled Water
Syrup
Potato
Tofu
Vegetable Oil
Oatmeal
Avocado
White Grape Juice
Lettuce
Chicken Broth
Questions:
1.
Carbohydrates, which are water soluble are not detected by their hydroxyl groups. Why not?
2.
Will ordinary table sugar give a positive test for Benedict’s solution? Why or why not?
3.
Distilled water is obviously not a food, so what is its purpose in each of the tests?
4.
If you get a positive test using the water; what does this mean?
5.
According to tradition, chicken soup is a good food for persons who are ill. Based upon the following
nutritional facts from a soup can, evaluate whether or not chicken soup is a good food.
Amount/serving
Total Fat 2g
Sat. Fat 1g
Cholest. 15mg
Sodium 980mg
%DV
3%
5%
5%
41%
Amount/serving
Total carb. 9g
Fiber 1g
Sugars 1g
Protein 3g
6
%DV
3%
4%

7. Identification of Biological Molecules
6.
A.
Using your data, identify the substance described by each of the following nutrition labels.
HINT: Total Carbohydrate = sugars (simple sugars) + dietary fiber (cellulose) + starch
Amount/serving
Total Fat 0g
Sat. Fat 0g
Cholest. 0mg
Sodium 20mg
%DV
0%
0%
0%
1%
Amount/serving
Total carb. 38g
Fiber 0g
Sugars 36g
Protein 0g
%DV
13%
0%
C.
Amount/serving
Total Fat 14g
Sat. Fat 1.5g
Cholest. 0mg
Sodium 0mg
%DV
5%
3%
0%
0%
Amount/serving
Total carb. 1g
Fiber 0g
Sugars 0g
Protein 7g
%DV
0%
4%
%DV
4%
2%
0%
0%
Amount/serving
Total carb. 27g
Fiber 4g
Sugars 0g
Protein 5g
%DV
9%
16%
13%
Identity:
Reason:
Identity:
Reason:
B.
Amount/serving
Total Fat 3.5g
Sat. Fat 0.5g
Cholest. 0mg
Sodium 0mg
%DV
21%
7%
0%
0%
Amount/serving
Total carb. 0g
Fiber 0g
Sugars 0g
Protein 0g
%DV
0%
0%
D.
Amount/serving
Total Fat 2.5g
Sat. Fat 0.5g
Cholest. 0mg
Sodium 0mg
Identity:
Reason:
Identity:
Reason:
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8. Identification of Biological Molecules
Setup
Stock solutions of
1. dH2O
2. Syrup
3. Potato
4. Tofu
5. Vegetable Oil
6. Oatmeal
7. Avocado
8. White Grape Juice
9. Lettuce
10. Chicken Broth
Student Stations – 5x
Benedict’s Solution – Wheaton Bottle
Iodine Solution – Wheaton Bottle
Biuret Solution – Wheaton Bottle
Paper Bag Strips (2)
Filter Paper Disks (4)
Sudan III Stain
Sudan III Rinse
Test Tube Rack
`
10 Test Tubes
Sharpie Marker
Pencil
10 Small Erlenmeyer Flasks for stock solutions (labeled)
10 pipettes (1 per stock solution)
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