Laboratory
3
Chemical Composition of Cells
(LM pages 29–42)
Time Estimate for Entire Lab: 2.0 hours
To reduce experimental time and preparation time, the following procedures can be set up as demonstrations: Enzyme Function of Proteins (LM page 32), Starch Composition (LM page 37), and Emulsification of
Lipids (LM page 39). In any case, to allow enough time for digestion, you may wish to do Enzyme Function
of Proteins first, and Starch Composition immediately thereafter.
Special Requirements
1. Incubation. Incubation is required for Enzyme Function of Proteins and for Starch Composition.
2. Fresh material. The potato and onion for Carbohydrates (LM page 34) and the materials for Testing
the Chemical Composition of Everyday Materials and an Unknown (LM page 41) can be obtained
locally, close to time of use.
Seventh Edition Changes
This was Lab 2 in the previous edition.
New or revised figures: 3.1 Peptide bond; 3.3 Glucose; 3.4 Maltose; 3.5 Starch; 3.6 Synthesis of a fat;
3.7 Emulsification
Notes
1. Test tubes. The exercises in this laboratory require students to add solutions to test tubes. As an
expedient, the students are asked to mark off the tubes at various centimeter levels with a ruler and
then to fill to these marks. You may prefer to have students use a standard method of measuring
volume, such as with a graduated cylinder or a pipette. Most experiments use the standard size test
tube. A few experiments require the large size test tube. Mini test tubes can be substituted for most
laboratory exercises as long as the total volume in a given tube does not exceed 9 cm. This will
reduce the volume of reagents used by approximately one-third.
Test tube sizes/volumes are as follows:
Mini
13 100 mm (Carolina 73-0008)
1 cm = 1.0 ml
Standard
16 150 mm (Carolina 73-0014)
1 cm = 1.5 ml
Medium large
20 150 mm (Carolina 73-0019)
1 cm = 2.4 ml
Large
25 150 mm (Carolina 73-0025)
1 cm = 4.0 ml
2. Students’ results. If students’ results are not as expected, you may wish to test the reagents.
3. Chemical solutions. All chemicals for this laboratory can be purchased from Carolina Biological
Supply. Information in the “Materials and Preparations” section may be helpful to those who wish
to prepare their own.
4. An incubator is recommended for some tests. However, a hot water bath (hot plate, beaker of
water, Celsius thermometer, test-tube clamp, beaker clamps, boiling chips) will also work.
MATERIALS AND PREPARATIONS1
All Exercises
_____
_____
_____
_____
_____
wax pencils
rulers, plastic millimeter
test tubes and racks
water, distilled
boiling chips, pumice (Carolina 84-8278, -8280)
13
3.1
Proteins (LM pages 30–33)
_____
biuret reagent (Carolina 84-8213)
_____
1% albumin solution (Carolina 84-2250, -2252)
_____
1–2% pepsin solution (Carolina 87-9378, -9380)
_____
1–2% starch solution (Carolina 89-2530)
_____
0.2% hydrochloric acid (HCl) (Carolina 86-7790, -7791)
_____
incubator set at 37°C or water bath
Biuret reagent. 30 ml per student group should be sufficient (using standard test tubes for all procedures).
If you buy prepared biuret, use only ten to fifteen drops; otherwise, the solution will be too dark, or dilute
to a 10% solution (10 ml biuret with 90 ml distilled water). To prepare your own biuret reagent, maintain
separate stock solutions of 3% copper sulfate—3 g of copper sulfate (cupric sulfate, Carolina 85-6550) per 100
ml of distilled water and 10% potassium hydroxide or sodium hydroxide—100 g of potassium hydroxide
(Carolina 88-3488) or sodium hydroxide pellets (Carolina 88-9470) per 1,000 ml of distilled water). Adding
five drops of copper sulfate solution and ten drops of potassium hydroxide solution to each experimental
tube produces more consistent results. Biuret reagent should be prepared fresh for each lab.
1% albumin solution. 20 ml per student group should be sufficient (using standard test tubes for all procedures). Prepare pH 7 buffer solution (Carolina 84-9380) as per directions on the vial, and dissolve 1 g of albumin per 100 ml of buffer. Allow time for precipitation to occur, and then decant. Also, check pH with indicator paper, and adjust to pH 7 with dilute acid or base. Swirl the stock, before distributing it to students.
1–2% pepsin solution. 20 ml per student group should be sufficient (using standard test tubes for all procedures). To make a 1% solution, dissolve 1 g of pepsin in 100 ml of distilled water. Stir to dissolve.
1–2% starch solution. 20 ml per student group should be sufficient (using standard test tubes for all procedures). Care must be taken in preparing this solution. To make a 1% solution, dissolve 1 g of starch in a small
amount of cold water to form a pourable paste. Add this to 100 ml of boiling distilled water, while stirring,
and mix a few minutes. Cool. Add a pinch of sodium chloride (NaCl). If refrigerated, this solution will last
for several weeks; otherwise, a fresh supply should be prepared each day.
0.2% hydrochloric acid (HCl). 20 ml per student group should be sufficient (using standard test tubes for
all procedures). Add 2.3 ml of concentrated HCl to 100 ml of distilled water.
3.2 Carbohydrates (LM pages 34–37)
_____
1% starch solution (Carolina 89-2530), see prep notes above
_____
iodine-potassium-iodide (IKI) solution, premade (Carolina 86-9051, -9053, -9055),
or see Laboratory 2 for preparation instructions from
potassium iodide (KI) and iodine (I) crystals
_____
dropper bottles, one per student group (Carolina 71-6525)
_____
razor blades, single-edged
_____
cutting board for potato, onion
_____
potato, fresh
_____
microscopes, compound light
_____
lens paper
_____
slides
_____
coverslips
_____
onion, fresh
_____
paper towels
_____
boiling water bath:
_____ hot plate
_____ large beakers
_____ boiling chips, pumice (Carolina 84-8278, -82800
_____ beaker clamps
_____ thermometer, organic based, 50–150°C range (optional)
_____
1% glucose (dextrose) solution (Carolina 85-7430)
_____
onion juice from fresh onion
1
Note: “Materials and Preparations” instructions are grouped by exercise. Some materials may be used in more than one exercise.
14
_____
_____
_____
_____
_____
_____
_____
potato juice from fresh potato
mortar and pestle
Benedict’s reagent (Carolina 84-7091, 84-7111)
1% pepsin solution (Carolina 87-9378, -9380)
0.2% hydrochloric acid (HCl) (Carolina 86-7790, -7791)
1% amylase solution (Carolina 20-2350)
incubator at 37°C or hot water bath
Iodine (IKI) solution. Use one dropper bottle per student group. For ease of comparison, the same amount
should be used each time. Premade iodine-potassium-iodide solution can be purchased, or the ingredients
can be purchased separately as potassium iodide (KI) (Carolina 88-3790,-3792) and iodine (I) (Carolina 868970,-8972). These dry ingredients have a long shelf life and can be mixed as needed, according to the
instructions in Laboratory 2.
Boiling water bath. Place a large beaker of water on a hot plate. Adjust the dial on the hot plate so that the
water is maintained at a gentle rolling boil during the experiment. Thermometers are optional, since students should know that boiling water is 100°C.
1% glucose solution. This makes enough for all procedures for 20 student groups. Place 1 g of glucose in 50
ml of distilled water. Stir to dissolve, and bring the volume up to 100 ml.
Onion and potato juice. Onion may be mashed the day before, then stored in a refrigerator overnight.
Potato juice should be extracted as close to time of use as possible.
Benedict’s reagent. 50 ml per student group is sufficient. Benedict’s reagent can be purchased as a powder
to make 1 liter. Or to make 1 liter, mix 173 g of sodium citrate (Carolina 88-9060) and 100 g of sodium carbonate, anhydrous (Na2CO3) (Carolina 88-8770) with 800 ml of distilled water. Warm this mixture to dissolve; then cool and filter it. Add distilled water to make 850 ml. Then dissolve 17.3 g of copper sulfate
(cupric sulfate, Carolina 85-6550) in 100 ml of distilled water, and stir slowly into the first solution. Add distilled water to make 1 liter. When testing, Benedict’s reagent should be boiled approximately 5 minutes or
longer.
1% amylase solution. 20 ml per student group is sufficient. Mix 1 g alpha amylase with 100 ml distilled
water. Keep the alpha amylase powder refrigerated when not in use, and make the solution fresh when
needed.
3.3 Lipids (LM pages 38–40)
_____
paper, brown (squares 4 cm 4 cm)
_____
water
_____
vegetable oil
_____
test tubes
_____
Tween® (Carolina 20-7861, -7862) or bile salts (oxgall, Wards Biology 38W2179)
_____
medicine droppers, dropper bottles
_____
slides
_____
coverslips
_____
microscopes, compound light
_____
lens paper
_____
adipose tissue slide (Carolina 31-2698, -2728)
_____
everyday materials for unknowns; suggestions: bread crumbs, flour and water mixture,
squeeze bottle of margarine, skim milk, orange juice, syrup from canned fruit
1% Tween® or bile solution. Tween is a standard wetting agent. Measure 1 ml of Tween or 1 g of bile salts,
and dissolve in 100 ml of distilled water.
15
EXERCISE QUESTIONS
3.1 Proteins (LM page 30)
Test for Proteins (LM page 30)
Experimental Procedure: Test for Proteins (LM page 31)
Table 3.2 Biuret Test
Tube
Contents
Final Color
Conclusions
1
Distilled water
Blue
No protein is present.
2
Albumin
Purple
Protein is present.
3
Pepsin
Purple
Protein is present.
4
Starch
Blue
No protein is present.
Conclusions (LM page 32)
• If your results are not as expected, offer an explanation. Answers will vary.
• Which of the four tubes is the control sample? tube 1
What is a control sample? In a control sample, the sample does not contain the factor being tested.
Therefore, a control sample is expected to give negative results.
Why do experimental procedures include control samples? The control samples usually validate the
experiment. If control samples give positive results, the experiment is invalid—in this case, the reagents may be
contaminated or the procedure may need improvement.
Experimental Procedure: Enzyme Function of Proteins (LM page 32)
Table 3.3 Action of Pepsin
Tube
Contents
Final Color
Conclusions
1
Albumin
Water
HCl* (incubate)
Purple
The protein albumin is present.
Albumin
Pepsin
HCl (incubate)
Pinkish-purple to pink
Albumin is digested to peptides by
the enzyme pepsin.
2
*HCl may cause some breakdown.
Conclusions (LM page 33)
•
Why were the tubes incubated at 37°C rather than at another temperature? This is normal human
body temperature, the optimum for digestion.
•
Enzymes are specific and speed up only one type of chemical reaction. Considering this, do
you predict that pepsin will break down starch? no Why or why not? Starch does not contain peptide bonds, which are specifically broken by pepsin.
•
If your results are not as expected, offer an explanation. Answers will vary.
16
3.2 Carbohydrates (LM page 34)
Test for Starch (LM page 35)
Experimental Procedure: Test for Starch (LM page 35)
Potato (LM page 35)
3. What is the color of the small oval bodies? blue-black
What is the chemical composition of these oval bodies? starch
Onion (LM page 35)
Table 3.4 Iodine (IKI) Tests for Starch
Sample
Color Change
Conclusions
Tube 1: Starch
Blue-black
Starch is present.
Tube 2: Water
Orange-brown (no change)
No starch is present.
Potato
Blue-black
Starch is present.
Onion
Orange-brown (no change)
No starch is present.
Conclusions (LM page 35-36)
• Is glucose stored as starch in the potato? yes
Is glucose stored as starch in the onion? no
Test for Sugars (LM page 36)
Experimental Procedure: Test for Sugars (LM page 36)
Table 3.6 Benedict’s Reagent Test
Tube
Contents
Color (After Heating)
Conclusions
1
Water
Blue
No sugar is present.
2
Glucose solution
Orange
A very high amount of sugar is present.
3
Onion juice
Yellow to orange
A moderate amount of sugar (as per Table 3.5)
is present.
4
Potato juice
Yellow
Some sugar is present.
5
Starch suspension
Usually blue; may be yellow
No sugar to some sugar is present.
Conclusions (LM page 37)
• From your test results, conclude what kind of chemical is present and why the results occurred.
Enter your conclusions in Table 3.6. Which tube served as a control? tube 1
Starch Composition (LM page 37)
Experimental Procedure: Starch Composition (LM page 37)
Table 3.7 Starch Composition
Tube
Color Change
Conclusions
1
None
No sugar is present.
Green
Maltose is present.
Water
Amylase
2 Starch
Amylase
Conclusions (LM page 38)
• From your test results, you may conclude that starch is composed of what kind of chemical? sugar
17
3.3 Lipids (LM page 38)
Test for Lipids (LM page 38)
Experimental Procedure: Test for Lipids (LM page 38)
1. Place a small drop of water on a square of brown paper. Describe the immediate effect. The water
sits on top—may evaporate away.
2. Place a small drop of vegetable oil on a square of brown paper. Describe the immediate effect. Oil
soaks into the paper and creates an oil spot.
Table 3.8 Test for Lipids
Sample
Results
Conclusions
Water spot
Does not penetrate paper
Substance is not an oil.
Oil spot
Penetrates paper
Substance is an oil.
Emulsification of Lipids (LM page 39)
Experimental Procedure: Emulsification of Lipids (LM page 39)
Tube 1: 2. Is vegetable oil soluble in water? no
Tube 2: 2. Describe how the distribution of oil in tube 2 compares with the distribution in tube 1.
There is no longer a separation into two separate layers. The oil is distributed throughout the tube.
Microscopic Examination (LM page 40)
Table 3.9 Emulsification Observations (Microscopic)
Tube
Contents
Observations
Conclusions
1
Oil
Water
If any oil droplets are visible, they are large.
Oil does not mix with water.
2
Oil
Water
Emulsifier
Oil droplets are numerous and small,
due to the effect of the emulsifier.
Emulsifier causes oil to mix with water.
Conclusions (LM page 40)
• Explain the correlation between your macroscopic observations (how the tubes look to your
unaided eye) and your microscopic observations. The cloudy layer in tube 2 is due to suspended, small
oil droplets.
3.4 Testing the Chemical Composition of Everyday Materials
and an Unknown (LM page 41)
Experimental Procedure: Chemical Composition (LM page 41)
3. Record your result as positive or negative in Table 3.10. Results will vary depending on the material
provided by the instructor.
Conclusions (LM page 41)
• Was there any material that tested positive for only one of the organic compounds? e.g., sucrose
Explain. Sucrose is a pure carbohydrate.
• What types of foods would you expect to test positive for more than one of the organic
compounds studied in this laboratory? e.g., milk has carbohydrate, fat, and protein in it
18
LABORATORY REVIEW 3 (LM page 42)
What type of bond joins amino acids to make peptides? peptide
What type of protein speeds chemical reactions? enzyme
What is —NH2? amino group
If iodine (IKI) solution turns blue-black, what substance is present? starch
If Benedict’s agent turns red, what substance is present? glucose
What is the function of starch in plant cells? stored energy
What is starch composed of? sugar
What is the function of fat in animal cells? stored energy
What molecules are released when fat is hydrolyzed? glycerol, fatty acids
What can be added to make fat disperse in water? emulsifier
Are fats polar or nonpolar? nonpolar
If biuret reagent turns purple, what substance is present? protein movement
A student adds iodine to egg white and waits for a color change. How long will the student have
to wait? forever
14. To test whether a sample contains glucose, what reagent should be used? Benedict’s
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
Thought Questions
15. Why is it necessary to shake a abottle of salad dressing before adding it to a salad? The oil in salad
dressing is not soluble in water, and shaking mixes the otherwise separate layers.
16. An unknown sample was tested with both biuret reagent and Benedict’s reagent. Both tests result
in a blue color. What has been learned? No protein and no sugar were present.