Name: __________________________
Lab - Testing for Macromolecules
PART I: Establishing Positive Controls
INTRODUCTION
The structures of cells are made up of many different chemical molecules. Most of the common molecules
found in living things belong to four classes of carbon-containing molecules: carbohydrates, lipids, proteins, and
nucleic acids. Biological macromolecules are large molecules composed of smaller subunits. These subunits
link together to make up the complex structure that gives the macromolecules their diverse functions. Complex
carbohydrates such as starch, cellulose, and glycogen are made of simple sugars called monosaccharides.
Lipids, or fats, are made of long saturated or unsaturated chains of carbons attached to a glycerol backbone.
Together, this forms a triglyceride. Proteins are the only three-dimensional macromolecules that exist. They
are made of long chains of amino acids, linked together by peptide bonds, which then fold into their 3-D shape.
Water is the most versatile polar solvent. A polar molecule has charges that are unevenly distributed around
the atoms, when compared to a nonpolar molecule. Polar molecules chemically attract other polar molecules.
This explains why water can dissolve salt, but not oil.
MATERIALS
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vegetable oil
95% ethanol
test tubes (4)
labels
brown paper or newspaper
glucose solution
Biuret reagent
test-tube holder
droppers
test-tube stoppers (2)
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soluble starch solution
Benedict's solution
test-tube brush
hot plate
safety goggles
iodine solution
test-tube rack
500 mL beaker
tap water
protein solution
PROCEDURE:
Part A: Iodine Test for Starch
A blue-black color indicates the presence of starch.
1. Label four test tubes 1, 2, 3 and 4. Place them in a test-tube rack.
2. Using a separate dropper for each solution, add 10 drops of soluble starch solution to test tube 1, 10
drops of glucose solution to test tube 2, 10 drops of protein to test tube 3, and 10 drops of water to test
tube 4. Record the color of each test tube’s contents in Table 1.
3. Add 3 drops of iodine solution to each test tube. CAUTION: if iodine is spilled, rinse with water
immediately.
4. Record in Table 1 the color of each test tube’s contents after addition of the iodine.
5. Discard the contents of the test tubes in the appropriate waste beaker. Gently use a test-tube brush and
soapy water to clean the test tubes and rinse with clean water.
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Part B: Benedict’s Test for Reducing Sugars
Benedict’s solution tests for the presence of simple reducing sugars. This means you can detect
monosaccharides and some disaccharides, but not polysaccharides. Thus, a color change may or may not
occur when Benedict’s solution is added to a carbohydrate and heated. A change from blue to green, yellow,
orange, or red occurs if a monosaccharide or some disaccharides are present. The most drastic changes
occur with high concentrations of monosaccharides. The original blue color will remain after heating if a
polysaccharide or some disaccharides are present.
1. For a water bath, heat 250 ml of water to boiling using med-high heat on the hot plate.
2. Label four test tubes 1, 2, 3 and 4.
3. Using a separate dropper for each solution, add 10 drops of soluble starch solution to test tube 1, 10
drops of glucose solution to test tube 2, 10 drops of protein to test tube 3, and 10 drops of water to test
tube 4.
4. Record the color of each test tube’s contents in Table 2.
5. Add 20 drops of Benedict’s solution to each tube and place them in a boiling water bath for 3 minutes.
6. Remove the test tubes from the water bath using a test-tube holder and place them in a test-tube rack
to cool. CAUTION: Be careful not to burn yourself. If Benedict’s solution is spilled, rinse with water
immediately.
6. Record the color of each tube’s contents in Table 2.
7. Discard the contents of the test tubes in the appropriate waste beaker. Gently use a test-tube brush and
soapy water to clean the test tubes and rinse with clean water.
Part C: Spot Test for Lipids
If a semitransparent spot is evident, the sample contains lipids.
1.
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Place a drop of water on a piece of brown paper or newspaper.
In a separate spot on your brown paper, place a drop of oil.
Allow the paper to dry for a few minutes. You may need to use a paper towel to dab off excess.
Hold the piece of paper up to the light. Record the appearance of the two spots in Table 3.
Part D: Solubility Test for Lipids
Lipids are soluble only in nonpolar solvents because lipids themselves are nonpolar. If the mixture looks a little
cloudy and does not separate after a minute, then it is soluble.
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Make sure the test tubes are completely dry before you begin.
Label two test tubes 1 and 2.
Using separate droppers, add 20 drops of oil to test tube 1 and 20 drops of 95% ethanol to test tube 2.
Add 10 drops of water to test tubes 1 and 2 and stopper each tube.
Shake each tube gently and let it settle, and record in Table 4 whether water dissolves either solute.
Dispose of the contents of the test tubes in the appropriate waste beaker. Gently use a test-tube brush
and soapy water to clean the two test tubes and rinse with clean water.
Part E: Biuret Test for Proteins
When Biuret reagent is mixed with a protein, it will produce a lavender to violet color. The greater the colour
change, the more protein present in the sample.
1. Label three test tubes 1, 2, and 3.
2. Using separate droppers, add 30 drops of glucose solution to test tube 1, 30 drops of protein solution to
test tube 2, and 30 drops of water to test tube 3. Record the color of each tube’s contents in Table 5.
3. Add 10 drops of Biuret reagent to each test tube. CAUTION: Biuret reagent is extremely caustic to the
skin and clothing. If Biuret reagent spills, rinse with water immediately.
4. Record in Table 5 the color of each tube’s contents after adding the Biuret reagent.
5. Discard the contents of the test tubes into the appropriate waste beaker. Gently use a test-tube brush
and soapy water to clean the test tubes and rinse with clean water.
6. Fill in the last column of all five tables with the correct interpretation of the test results.
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OBSERVATIONS
Table 1. Iodine Test for Starch
Test Tube
Substance
1
Starch
2
Glucose
3
Protein
4
Water
Colour Before
Colour After
Presence of Starch
(+/-)
(─) indicates a negative test result (i.e., the substance being tests for was not present)
(+) indicates a positive test result (i.e., the substance being tests for was present)
Table 2. Benedict’s Test for Reducing Sugars
Test Tube
Substance
1
Starch
2
Glucose
3
Protein
4
Water
Table 3. Spot Test for Lipids
Substance
Colour Pre-Boiling
Colour PostBoiling
Presence of Sugar
(+/-)
Table 4. Solubility Test for Lipids
Presence of
Lipids (+/-)
Translucent?
Substance
Water
Oil
Oil
Ethanol
Dissolves?
Presence of
Lipids (+/-)
Table 5. Biuret Test for Proteins
Test Tube
Substance
1
Glucose
2
Protein
3
Water
Colour Before
3
Colour After
Presence of
Proteins (+/-)
PART II: Testing for Unknowns
Using your data from Part I, you will now work to identify unknown macromolecules in various samples.
PROCEDURE
1. Obtain 20 mL of unknown sample #1. Make sure the solution has been stirred.
2. Place 3 mL of unknown sample #1 in each of four test tubes labeled 1a-1d.
3. Add 3 drops of iodine solution to test tube 1a. Using the positive controls you established in Part I
(refer to Table 1), determine whether this sample contains starch. Record your results for the presence
of starch in Table 6, indicating either a positive result (+) or a negative result (–).
4. Add 20 drops of Benedict’s solution to test tube 1b. Place this test tube in a hot water bath for 3
minutes. Remove the test tube from the water bath using a test-tube holder and place it in the test-tube
rack to cool. CAUTION: Be careful not to burn yourself. Using the positive controls from Part I (refer to
Table 2), determine whether this sample contains glucose. Record your results for the presence of
reducing sugars in Table 6.
5. In test tube 1c, carefully add 10 drops of water. Place a stopper on the test tube. Shake the test tube
gently, and let it settle. Using the positive controls from Part I (refer to Table 4), determine whether this
sample contains lipids. Record your results for the presence of lipids in Table 6.
6. Add 10 drops of Biuret reagent to test tube 1d. Using the positive controls you established in Part I
(refer to Table 5), determine whether this sample contains proteins. Record your results for the
presence of proteins in Table 6.
7. Dispose of the contents of test tubes 1a-1d as instructed by your teacher. Clean the test tubes using
soapy water, and thoroughly dry each test tube before proceeding.
8. Obtain 20 mL of unknown sample #2. Again, make sure the sample has been stirred. Label four clean
test tubes 2a-2d. Using the clean test tubes, repeat steps 2-7 for sample #2. Be sure to record all of
your results for sample #2 in the appropriate spaces in Table 6.
9. Obtain 20 mL of unknown sample #3. Again, make sure the sample has been stirred. Label four clean
test tubes 3a-3d. Using the clean test tubes, repeat steps 2-7 for sample #3. Be sure to record all of
your results for sample #3 in the appropriate spaces in Table 6.
10. Obtain 20 mL of unknown sample #4. Again, make sure the sample has been stirred. Label four clean
test tubes 4a-4d. Using the clean test tubes, repeat steps 2-7 for sample #4. Be sure to record all of
your results for sample #4 in the appropriate spaces in Table 6.
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OBSERVATIONS
Table 6. Data Table for Unknown Samples
Unknown Sample #
Tests
1
a
Starch
b
Glucose
c
Lipids
d
Proteins
2
3
4
– indicates a negative test result (i.e., the substance being tested for was not present)
+ indicates a positive test result (i.e., the substance being tested for was present)
ANALYSIS
1. Based on the results you recorded in Table 6, determine the macromolecule in each of the five unknown
samples. (4 marks)
Unknown Sample #1:
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Unknown Sample #2:
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Unknown Sample #3:
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Unknown Sample #4:
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2. Why was water tested in each of the tests for Day 1? (2 marks)
3. Biuret reagent will turn the skin and fingernails a brownish-purple colour. Explain why. (3 marks)
4. When greasy food is spilled, why is it difficult to clean with water alone? How should grease spills be
cleaned (other than using plain water)? Include a description of water’s properties that apply. (4 marks)
5. List 2 characteristics that carbohydrates, lipids, and proteins have in common. (4 marks)
6. Describe the reaction that forms macromolecules such as carbohydrates, lipids, and proteins. Are these
reactions anabolic or catabolic? (4 marks)
7. Explain why triglycerides are not considered polymers. (3 marks)
8. Differentiate between organic and inorganic molecules. (3 marks)
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