AP Biology Photosynthesis Labs
Thomases
Name:
Per:
Transpiration Labs (Poster required)
Procedure 1: Calculating transpiration rate
Procedure 2 Measuring loss of plant mass through transpiration
Procedure 3 Floating Leaf Disk Assay for Investigating Photosynthesis
Procedure 4: Chromatography to visualize plant pigments
Analysis
You have 3 days to complete these procedures and analysis
Transpiration, the loss of water by plants, occurs mainly through the stomata of the leaves. Water can
move up a plant to the leaves by being pushed from the bottom or pulled from the top. Root pressure is
not sufficient to push water all the way from the bottom to the top of a large plant such as a tree. It is
more likely that water is pulled through the plant by the cohension-tension mechanism.
As water leaves the intercellular space between the mesophyll cells in the leaf and evaporates through
the stomata, it is replaced by water from within the cells themselves. Since water moves out of these
cells freely but solutes do not, the solute concentration within the mesophyll cells increases and the water
potential of the cells decreases. Water will then move into the mesophyll cells from surrounding cells with
higher water potentials, for example, the cells of the xylem. Thus, as a result of transpiration, a gradient
of differences in water potential from the xylem to the air outside the leaf is formed ---- water tends to be
pulled upward.
The upward transpiration pull on the fluid within the xylem causes tension (negative pressure) to form in
the xylem, pulling the walls of the xylem inward. Tension, since it is negative pressure, causes water
potential in the xylem to decrease. The decrease in water potential, transmitted through the column of
fluid in the xylem, all the way to the roots, causes water to move from the soil across the cortex of the root
and into the xylem of the stele.
The opening of stomata, which allows transpiration to occur, is also required for the entry of carbon
dioxide used in photosynthesis. A balance must be maintained between the two processes, transpiration
and photosynthesis, by regulating the opening of stomata.
Purpose: To determine how environmental conditions affect the rate of transpiration and to
determine the transpiration rate of a plant.
Materials for all 4 procedures:
 plants
 plastic bags
 tape
 chromatography
paper
 chromatography
solvent
 test tube with stopper
 Coin








microscope
microscope slides
graph paper
fan
lamp
water misters
plastic cup
spinach



60 mL 0.2% sodium
bicarbonate solution
with a drop of liquid
soap added.
hole puncher
syringe 10cc or
larger
Procedure 1 Calculating transpiration rate
1. Carefully remove one average sized leaf from your plant seedling.
2. Lay each leaf on a piece of graph paper and carefully trace it scale. Tape these
diagrams to your poster.
3. Calculate the total surface area of all of your leaves in cm 2 by using the squares
in the graph paper.
4. Coat the bottom of one average sized leaf with clear nail polish
5. When the nail polish is almost dry, put transparent tape over the nail polish and
use gentle pressure to get a good leaf print.
6. Carefully remove the tape and put the tape onto a glass slide. Make sure you
leave a tab on the tape so you can remove it from the slide when you are done.
7. Under a microscope, count the number of stomata you see. Based on the size or
your viewing field, calculate the number of stomata /mm 2 area. Based on this,
calculate the number of stomata on your leaf.
8. Take a photograph of your stomata and add this to your notebook.
9. Using the data from procedure 2, calculate the rate of transpiration of your plant
per stomata. Show your work.
Transpiration Procedure 2 Measuring loss of mass through transpiration
1. Take your plant out of the pot and cover the roots tightly with a plastic bag.
2. Measure and record the mass of your plant daily for the duration of your lab.
3. You and your partner(s) must propose an environmental condition under which
you conduct your experiment. Suggestions:
a. Room temperature and light
b. Simulate wind by placing the plant about 2m from a fan. Use a low or
medium setting because too much wind will cause the stomates to close.
c. Increase the vapor pressure of the air by misting the plant and covering it
with a plastic bag.
d. Increase the temperature of both the leaf and air, and increase the light
intensity by placing a flood light at a prescribed distance.
e. Reduce the leaf surface by removing half the leaves from the shoot.
Place amount of petroleum jelly over the exposed cuts.
f. your group may propose other environmental conditions.
4. At the end of day 3, use your data to calculate the rate of transpiration of your
plant per stomata/day. Show your work.
Transpiration Procedure 3 - Floating Leaf Disk Assay for Investigating
Photosynthesis
Explanation: The following experiment will allow you to quantitatively investigate
photosynthesis, understand this technique, and then design your own experiment. For
today’s experiment, you will see how the addition of light changes the rate of
photosynthesis. The bicarbonate serves as dissolved source of carbon dioxide for
photosynthesis. The soap wets the hydrophobic surface of the leaf allowing the solution
to be drawn into the leaf.
1.
Use a hole puncher cut 10-12 uniform leaf disks of spinach for each trial. Avoid
major veins.
2.
Remove gases from the leaf disks and infiltrate them with sodium bicarbonate
solution:
a. Remove the piston or plunger of the syringe.
b. Place the leaf disks into the syringe barrel.
c. Replace the plunger, being careful not to crush the leaf disks.
d. Push on the plunger until only a small volume of air and leaf disk remain in the
barrel (< 10%).
e. Pull a small volume (~ 5 mL) of sodium bicarbonate solution into the syringe. Tap
the syringe to suspend the leaf disks in the solution.
f. Holding a finger over the syringe opening, draw back on the plunger (~ 1 mL) to
create a vacuum.
g. Hold this vacuum for about 10 seconds. While holding the vacuum, swirl the leaf
disks to suspend them in the solution. Let off the vacuum. The bicarbonate
solution will infiltrate the air spaces in the leaf causing the disks to sink. You will
probably have to repeat this procedure several times (4-7X) in order to remove
enough gas to get the disks to sink.
3.
Pour the disks and solution into a 100 mL beaker or plastic cup.
4.
Add bicarbonate solution to a depth of about 20 mL. or until the cup is about ½ full.
Use the same depth for each trial. Shallower depths work just as well.
5.
Have your lab partner set up a second trial. This one will be placed under a light
source.
6.
Place your first sample away for the supplemental light source, and your second
sample under the light source.
7.
Start timing as soon as your sample is set up. At the end of each minute, record
the number of disks that have floated to the top. Then swirl the disks to dislodge
any that are stuck against the sides of the cups.
8.
Continue until all of the disks are floating.
9.
Why did the disks float?
10. Graph your results
Procedure 4 Using Chromatography to identify plant pigments
1. Cut a strip of chromatography paper so it just fits inside test tube without touching the
sides. Cut a point on one end.
2. Bend a paper clip and attach it to a cork stopper.
3. Attach the paper strip so it hangs inside the tube without touching the sides or bottom.
4. Place 5 mL of ethyl alcohol into your test tube.
5. Lightly mark the filter paper with a horizontal line just where the widest part of the point
meets the rest of the filter paper.
6. Mark the filter paper with pigment by placing a leaf over the line and scrubbing it with a
coin. Make your pigment mark heavy and dark.
5. Suspend the filter paper into the test tube so the VERY TIP is BARELY TOUCHING
the ethyl alcohol.
7. Watch as the alcohol wicks up the filter paper through capillary action. The INSTANT
the alcohol reaches the top of the filter paper, remove it and allow it to dry, (about 5-7
minutes)
Note: Some color streaks may be very light and cannot be seen well until the paper dries.
8. Take a photograph of your chromatography paper for your poster.
9. Observe and measure the bands of pigment. The order, from the top, should be
carotenes (orange), xanthophylls (yellow), Chlorophyll b (yellow green), Chlorophyll a
(blue-green) and anthocyanin (red).
10. Measure the distance in mm from the starting point (pigment rubbed area) to the
approximate center of each pigment band. Then measure the entire distance traveled by
the solvent. Do this for at least 3 pigments.
Calculate the Rf value as a decimal fraction for each pigment.
Each pigment has an Rf value, the speed at which it moves over the paper compared
with the speed of the solvent.:
Rf =
Distance moved by the pigment from the starting point
Distance moved by the solvent from the starting point
Data Table
Pigment
Color
Pigment
migration
distance in mm
Solvent
migration
distance in mm
Rf value
Analysis – please type your answers and place them on your poster.
1. Describe the pathway of water movement from xylem cells in the stem to the air
surrounding the leaf.
2. During transpiration, water moves down a water potential gradient. What is meant by
the term water potential gradient?
3. The slope of a line of best fit is equal to rate. What is the rate of water loss by
transpiration for the seedling you used in class? Show your work.
4. If the transpiration of several bean plants is measured during an experiment, how
could the data be standardized so that it could be meaningfully compared?
5. Every experiment must have a control set-up. In an experiment involving transpiration,
the control group should consist of plants at normal room conditions. Is this control set-up
considered a positive or negative control? As well, explain why a control set-up is
essential in this experiment.
6a. If a plastic bag and normal room conditions were used, (lights and heat off at
night), how would the following measurements be affected?




Rate of Transpiration
Total Water Loss Per Unit of Time
Water Potential of the Environment
Water Potential Gradient between the Mesophyll Cells in the Leaf and the
Environment
6b. If a 200 watt flood light was used to simulate a hot, sunny day, how would the
following measurements be affected? (Assume that stomates do not close.)




Rate of Transpiration
Total Water Loss Per Unit of Time
Water Potential of the Environment
Water Potential Gradient between the Mesophyll Cells in the Leaf and the
Environment
6c. If a fan was used to simulate a slightly windy day, how would this affect the following
measurements? (Assume that stomates do not close.)




Rate of Transpiration
Total Water Loss Per Unit of Time
Water Potential of the Environment
Water Potential Gradient between the Mesophyll Cells in the Leaf and the
Environment
6d. If a plastic bag and misting sprayer were used to create a humid environment as
is found in tropical rain forest biomes, how would the following measurements be
affected?




Rate of Transpiration
Total Water Loss Per Unit of Time
Water Potential of the Environment
Water Potential Gradient between the Mesophyll Cells in the Leaf and the
Environment
7. With intense sunlight, drought, and intense wind a plant’s guard cells may lose turgor
pressure and its stomates may close.


What is an advantage of a plant’s stomates closing?
What is a disadvantage of a plant’s stomates closing?
8. What happens in the light independent reactions of photosynthesis when carbon
dioxide is in low concentration and oxygen is in high concentration?
9. What biochemical pathway do desert plants have to keep carbon dioxide levels at
adequate levels when their stomates are closed?
10. What biochemical pathway do tropical plants have to minimize photorespiration when
stomates are closed?
11. Why do you think the pigments traveled at different rates through the
chromatography paper?
12. Where are plant pigments located?
13. How does the location of the pigments relate to their function?
14. Are all the pigments you saw responsible for the plant's color?
15. Which pigment was responsible for the leaf's original color?
16. What practical use can be found for plant pigment chromatography? (Cite sourcesMLA)