Name ________________________
Biology 211 Laboratory Fall 2001
CHEMICAL COMPOSITION OF CELLS: WEEKS 1 AND 2
OBJECTIVES:
You will learn
 How to use pipets to measure volumes of liquid solutions
 That proteins are polymer chains made up of amino acids
 How to use a biuret test for the presence of proteins and peptides
 The function of enzymes
 That starch is a sugar polymer made up of glucose units
 How to use Benedict's test for reducing sugars
 That fats are made of glycerol and fatty acids
 How an emulsifier works
BACKGROUND READING: Read Campbell 5/e Chapter 5.
USE OF PIPETS: Pipets are devices to precisely measure volumes of liquids. To get
good results in this lab, you will need to learn to accurately use pipets. For today's lab,
you will mainly use two kinds of pipets.
For measuring 1 ml or less you can often use the plastic dropper pipets. To
use these, place the tip in the liquid, then squeeze the top to draw in liquid. Squeeze the
bulb again to release the liquid from the dropper. The dropper pipets can also be used to
measure drops of a solution to be added.
For measuring larger volumes or to measure a precise fraction of a ml (such
as 0.2 ml), you will use regular pipets. These require a device (called a pipet aid or
pipet pump) to assist in drawing up and expelling the fluids. Use the blue pipet aids for
the small pipets (1 ml or 2 ml) and the green pipet aids for the larger pipets (5 ml and
10 ml). Attach the blunt end of the pipet into the fitting on the pipet aid until it is held
snugly. Place the pipet tip into the liquid and use the roller ball to draw the liquid up to
the desired volume. The roller ball also is used to release the liquid from the pipet.
LAB SAFETY: For your safety in the lab, you should wear safety goggles when you
handle the testing chemicals and solutions that are hot or being heated. Laboratory
aprons will also be available to protect your clothing from spills.
CLEAN-UP: After performing tests, pour all solutions down the drain, rinse the test
tubes and place in the dishpans of water provided. Disposable dropper pipets and plastic
1 ml pipets should be placed in the orange bags provided on the benches. The glass
pipets you use (all sizes) should be placed with tips up in the white pipet holders in the
sink areas.
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ASSIGNMENT: As you carry out the lab over the next two weeks, fill in the blanks and
answer the questions. The completed handout is due at the end of the second lab period.
INTRODUCTION:
Several classes of organic molecules have biological significance. In this
laboratory, you will be studying proteins, carbohydrates (monosaccharides, disaccharides,
polysaccharides) and fats. As you shall see, amino acids are joined by peptide bonds
within a protein. Many glucose (monosaccharide) molecules are joined to form a
polysaccharide, such as starch. A fat contains one glycerol and three fatty acids.
Various chemicals will be used in this laboratory to test for the presence of these
molecules. Most often, you will be looking for a particular color change. If the change is
observed, the test is said to be positive because it indicates that a particular molecule is
present. If the color change is not observed, the test is said to be negative because it
indicates that a particular molecule is not present.
In all of the procedures, you will need to include a distilled water sample as a
control. Usually, a control goes through all the steps of the experiment but lacks one
essential factor (the experimental variable). This missing factor allows you to observe
the difference between a positive result and a negative result. If the control sample tests
positive, you know your test is invalid.
Proteins
Proteins have numerous functions in cells. Many are enzymes, organic catalysts
that speed up metabolic reactions. Pepsin is a digestive enzyme active in the stomach.
Other proteins have structural roles or act as carriers. Albumin is the major protein found
in blood.
Proteins are made up of amino acids. About twenty different amino acids are
found in cells. All amino acids have an acidic group (-COOH) and an amino group
(H2N-). They differ by the R group attached to the carbon atom. The R groups have
varying sizes, shapes and chemical activities.
A chain of several amino acids joined together is called a peptide, and a long
chain of amino acids is called a polypeptide. The amino acids in a peptide are held
together by peptide bonds. The function of a protein is determined by its three
dimensional shape. The shape is produced by the particular chemical interactions of the
R groups in the protein.
Test for Proteins
Biuret reagent (blue color) contains a strong solution of sodium or potassium
hydroxide (NaOH or KOH) and a very small amount of dilute copper sulfate (CuSO4)
2
solution. The reagent changes color in the presence of proteins or peptides because the
amino group (H2N-) of the protein or peptide chemically combines with the copper ions
in biuret reagent. In the presence of protein, the reagent turns violet; in the presence of
peptides the reagent is pink.
Experimental Procedure: Test for Proteins
Label four clean test tubes with numbers 1-4.
Table 1: Biuret test setup
Add:
2 ml distilled water
2 ml albumin solution
2 ml pepsin solution
2 ml starch solution
Tube #
1
2
3
4
Biuret reagent to add
3 ml
3 ml
3 ml
3 ml
Mix tube contents and record the final color in Table 2.
Table 2:Results of Biuret test
Tube # Contents Final Color
Conclusions
1
Water
2
Albumin
3
Pepsin
4
Starch
Conclusions

From your test results, conclude what kind of chemical is present and why the
results occurred. Enter your conclusions in Table 2.
 If your results are not as expected, offer an explanation._____________________
_____________________________________________________________________
Then inform your instructor, who will advise you how to proceed.

Which of the four tubes is the control sample?______ What is a control sample?
__________________________________________________________________
Many Proteins are Enzymes
The enzyme pepsin can be found in the acidic environment of the stomach. In the
experiment that follows, you test the ability of this enzyme to speed up the following
reaction:
pepsin (enzyme)
protein + water
peptides
hydrochloric acid (HCl)
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Peptides can be detected in a biuret test. In the presence of peptides, the reagent
turns pink, while in the presence of proteins, the reagent is purple.
Experimental Procedure: Enzyme Function of Proteins
Label tubes 1 and 2.
 Tube 1: 1. Add 2 ml albumin solution, 2 ml water and 2 ml hydrochloric acid
(0.2% HCl).
2. Shake and incubate at 37C for 30 min.
3. After incubation, add 3 ml biuret reagent. Note any color change, and
record your results in Table 3.
 Tube 2: 1. Add 2 ml albumin solution, 2 ml pepsin solution and 2 ml
hydrochloric acid (0.2% HCl).
2. Shake and incubate at 37 C for 30 min.
3. After incubation, add 3 ml biuret reagent. Note any color change, and
record your results in Table 3.
Tube
1
2
Contents
Albumin
Water
HCl
Albumin
Pepsin
HCl
Table 3: Action of Pepsin
Final Color Conclusions
Conclusions

Why was incubation at 37C (body temperature)?__________________________
_________________________________________________________________

From your test results, conclude what kind of chemical is present and why the
results occurred. Enter your conclusions in Table 3. Enzymes are specific and
speed up only one type of chemical reaction. Considering this, do you predict that
pepsin will break down starch? Why or why not?__________________________
__________________________________________________________________

If your results are not as expected, offer an explanation. ____________________
__________________________________________________________________
Then inform your instructor, who will advise you how to proceed.
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Carbohydrates
Carbohydrates include sugars and molecules that are chains of sugars. Glucose,
which has only one sugar unit is a monosaccharide. Maltose, which has two sugar units,
is a disaccharide. Starch is a polysaccharide, a chain of glucose units. Sugars are an
immediate energy source in cells. In plant cells, glucose (a primary energy molecule) is
often stored in the form of starch.
Test for Starch
In the presence of starch, iodine solution (iodine-potassium iodide) turns from a
brownish color to blue-black.
Experimental Procedure: Test for Starch
Label three clean test tubes, tubes 1, tube 2 and tube 3.
 Tube 1: 1. Add 1 ml of starch suspension (1%--shake well before using), and add
five drops of iodine solution.
2. Note the final color change and record your results in Table 4.
 Tube 2: 1. Add 1 ml of potato solution and add five drops of iodine solution.
2. Note the final color change and record your results in Table 4.
 Tube 3: 1. Add 1 ml of water, and add five drops of iodine solution.
2. Note the final color change, and record your results in Table 4.
Sample
Tube 1: Starch
Table 4: Iodine Tests for Starch
Color Change
Conclusions
Tube 2: Potato solution
Tube 3: Water
Conclusions

From your test results, draw conclusions about what organic compound is present
in each tube, and write these conclusions in table 4. If your results are not as
expected, offer an explanation. ________________________________________
_________________________________________________________________
Then inform your instructor, who will advise you how to proceed.
Test for Sugars
Sugars react with Benedict's reagent after being heated in a boiling water
bath. Increasing concentrations of sugar give a continuum of colored products
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(Table 5). This experiment tests for the presence (or absence) of varying amounts
of reducing sugars in a variety of materials and chemicals.
Chemical
Water
Glucose
Maltose
Starch
Table 5: Color Reactions with Benedict's Reagent
Chemical Category
Benedict's Reagent (After Heating)
Inorganic
Blue (no change)
Monosaccharide
Varies with concentration
Very low--green
Low--yellow
Moderate--yellow-orange
High--orange
Very high--orange-red
Disaccharide
Varies with concentration-see "glucose"
above
Polysaccharide
Blue (no change)
Experimental Procedure: Test for Sugars
Label 5 clean test tubes 1-5. Add sample (0.2 ml) and Benedict's reagent (1 ml)
to each tube according to Table 6 below.
Tube
1
2
3
4
5
Table 6: Sample Tubes for Benedict's Tests
Volume sample
Volume Benedict's reagent
0.2 ml distilled water
1 ml
0.2 ml 1 M glucose
1 ml
0.2 ml 0.1 M glucose
1 ml
0.2 ml 0.05 M glucose
1 ml
0.2 ml starch suspension
1 ml
Place the tubes in the boiling water bath and heat for 5-10 min. Note the color
change in Table 7. Be very careful handling the tubes in the water baths! Wear goggles
and use test tube clamps to remove the tubes from the baths! If you need to move the
water bath use insulated gloves.
Tube
1
Contents
Water
2
Glucose
1M
Glucose
0.1 M
Glucose
0.05 M
Starch
3
4
5
Table 7: Benedict's Reagent Test
Color (After Heating) Conclusions
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Conclusions
 From your test results, conclude what kind of chemical is present and why the
results occurred. Enter your conclusions in Table 7.
 If your results are not as expected, offer an explanation._____________________
__________________________________________________________________
Then inform your instructor, who will advise you how to proceed.
Lipids
Lipids are compounds that are insoluble in water and soluble in organic solvents,
such as alcohol and ether. Lipids include fats, oils, phospholipids, steroids, and
cholesterol. Typically, fats and oils are composed of three molecules of fatty acids
bonded to one molecule of glycerol.
Test for Lipids
Fats do not evaporate from brown paper; instead they leave an oily spot.
Experimental Procedure: Test for Lipids
1. Place a small drop of water on a square of brown paper. Describe the immediate
effect. __________________________________________________________________
2. Place a small drop of vegetable oil on a square of brown paper. Describe the
immediate effect.__________________________________________________________
3. Wait at least 5 minutes. Evaluate which substance penetrates the paper and which is
subject to evaporation. Record your results and conclusions in Table 8.
Sample
Water spot
Oil spot
Table 8: Test for Lipids
Results
Conclusions
Emulsification of Lipids
Some molecules are polar, meaning that they have charged groups of atoms, and
some are non-polar, meaning that they have no charged groups or atoms. A water
molecule is polar, and therefore, water is a good solvent for other polar molecules. When
the charged ends of water molecules interact with the charged groups of polar molecules,
these polar molecules disperse in water.
Water is not a good solvent for non-polar molecules, like fats. A fat has no polar
groups to interact with water molecules. An emulsifier, however, can cause a fat to
disperse in water. An emulsifier contains molecules with both polar and non-polar ends.
When the non-polar ends interact with the fat, and the polar ends interact with the water
molecules, the fat disperses in water.
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Bile salts (emulsifiers found in bile produced by the liver) are used in the
digestive tract. Detergents are commercially produced emulsifiers.
Experimental Procedure: Emulsification of Lipids
Label two test tubes 1 and 2.
Tube 1: 1. Add 3 ml water and 1 ml vegetable oil with dropper pipets. Cover and shake.
2. Observe for the initial dispersal of oil, followed by rapid separation into two
layers. Does vegetable oil appear to be soluble in water? Why or why not?
_________________________________________________________________
Tube 2: 1. Add 2 ml water, 1 ml vegetable oil and 1 ml of detergent. Shake.
2. Observe and describe how the distribution of oil in tube 2 compares with the
distribution in tube 1._____________________________________________
Testing Chemical Composition of Everyday Materials and Unknowns
Everyday materials, such as foods, are composed of organic compounds such as
carbohydrates, proteins, and lipids. It will be possible for you to determine if these
organic substances are present by using the tests you learned in this laboratory.
Experimental Procedure: Chemical Composition
1. Your instructor will provide you with several everyday materials. Use the tests for
carbohydrates (reducing sugars and starches), proteins, and lipids in this laboratory to
determine the macromolecules present in these materials.
2. Write the name of a known material on the container. Assign a letter to any unknowns
provided (Unknown A, Unknown B, Unknown C, Unknown D) and write it on the
container.
3. Record your result as positive or negative in Table 9.
Sample Name
Table 9: Results of Tests on Knowns and Unknowns
Reducing Sugar Starch (Iodine) Protein (Biuret) Lipid (Brown
(Benedict's)
Paper)
Slimfast
Wisdom drink
Apple juice
I can't believe
it's not butter
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Unknown A
Unknown B
Unknown C
Unknown D
Conclusions


Was there any material that tested positive for only one of the organic
compounds? Explain.________________________________________________
_________________________________________________________________
What types of foods would you expect to test positive for more than one of the
organic compounds studied in this laboratory? ____________________________
__________________________________________________________________
Laboratory Review
1. What molecules studied in this lab are present in cells? ________________________
_______________________________________________________________________
2. To build a protein, what building block would you use? ________________________
3. How would you test an unknown solution for each of the following:
a. Sugars________________________________________________________________
b. Fat__________________________________________________________________
c. Starch________________________________________________________________
d. Protein_______________________________________________________________
4. Why in many of these tests is water used instead of a sample substance?
_______________________________________________________________________
_______________________________________________________________________
5. A test tube contains albumin and pepsin. After 1 hour, the test for protein is positive,
but the test for peptides is negative. Explain. ___________________________________
_______________________________________________________________________
_______________________________________________________________________
Source of Laboratory:
Mader, S. (1998) Biology, A Laboratory Manual. WC Brown/McGraw-Hill. pp. 19-34.
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