Photosynthesis Lab
Notes From the teacher
Day 1:
Before class:
 Review the lab. Discuss Part A and watch teacher demonstration.
 Complete the pre-lab for Part A:
o Title and date of the lab (remember to add this lab to your table of contents)
o Purpose  1-2 sentences describing the goal of Part A of this lab
o Pre-Lab Questions you need to number the question, rewrite the question, and then answer it
for full credit
o Lab Procedure  Write a procedure for the experiment for Parts A. First list the materials
that you will be using, then use your own words to describe the steps in experiment.
o Data Tables Draw or cut out Data Tables 1.1 and 1.2.
In class:


Complete Part A of the lab.
Fill out the data tables, complete the calculations in your lab notebook, and answer the analysis
questions. If you do not have time to finish the analysis section, you can complete it for homework.
Make sure to copy the question and then answer it to get full credit.
Day 2:
Before class:


Review the lab. Discuss Part C and determine what variable your group would like to test.
Complete the pre-lab and watch this video: http://www.youtube.com/watch?v=vw8baZO89oc&feature=gv
o Title and date of the lab (remember to add this lab to your table of contents)
o Purpose  1-2 sentences describing the goal of Parts B and C of this lab; use complete
sentences
o Hypothesis  write TWO clear and concise if/then statements for the experiment (for Part B I
have given you the variable, for Part C you come up with your own variable – so decide that
before you can write your second hypothesis)
o Pre-Lab Questions you need to number the question, rewrite the question, and then answer it
for full credit
o Lab Procedure  Write a procedure for the experiment for both Parts B and C. First list the
materials that you will be using, then use your own words to describe the steps in experiment. I
have outlined the procedure for Part B, but you need to come up with one for Part C once you
decide on the variable you are testing. You may use a numbered list.
o Data Tables Draw or cut out Data Table 1.3. You can make Data Table 1.4 (for Part C) either
in your lab notebook or make it on the computer and then cut it out and tape it in.
In class:




Review the lab with the teacher.
Complete the procedure for Part A and Part B of the lab.
Begin the analysis portion of the lab:
o Create 2 graphs
o Remember for the analysis questions you need to number the question, rewrite the question,
and then answer it for full credit.
Finish the analysis section of the lab for homework.
PHOTOSYNTHESIS LAB
OVERVIEW
Photosynthesis fuels ecosystems and replenishes the Earth’s atmosphere with oxygen. Like all enzyme-driven
reactions, the rate of photosynthesis can be measured by either the disappearance of substrate or the
accumulation of product (or by-products).
The general equation for photosynthesis is:
6H2O + 6CO2 + light energy → C6H12O6 + 6O2
There are two ways to measure the rate of photosynthesis:
 Production of O2 (How many moles of O2 are produced for one mole of sugar synthesized?)
 Consumption of CO2 (How many moles of CO2 are consumed for every mole of sugar synthesized?)
In this investigation, you will use a system that measures the accumulation of oxygen.
We will also explore a process called chromatography. This is a procedure that can separate pigments so that
you can examine them. Pigments are extremely important in photosynthesis because they are responsible for
absorbing the photons of light and passing that energy onto the electrons. This is a critical step in the light
reactions.
OBJECTIVES
-
-
To design and conduct an experiment to explore the effect of certain factors, including different
environmental variables, on the rate of cellular photosynthesis
To connect and apply concepts, including the relationship between cell structure and function
(chloroplasts); strategies for capture, storage, and use of free energy; diffusion of gases across cell
membranes; and the physical laws pertaining to the properties and behaviors of gases
To understand how chromatography separates two or more compounds that are initially present in a
mixture.
To separate pigments and calculate their Rf values
Figure 1. Structure of a Leaf
PART A  Plant Pigment Chromatography
INTRODUCTION
Paper chromatography is a useful technique for separating and identifying pigments and other molecules
from cell extracts that contain a complex mixture of molecules. The solvent moves up the paper by capillary
action, which occurs as a result of the attraction of solvent molecules to the paper and the attraction of solvent
molecules to one another. As the solvent moves up the paper, it carries along any substances dissolved in it.
The pigments are carried along at different rates because they are not equally soluble in the solvent and because
they are attracted, to different degrees, to the fibers in the paper through the formation of intermolecular bonds,
such as hydrogen bonds.
Beta carotene, the most abundant carotene in plants, is carried along near the solvent front because it is
very soluble in the solvent being used and because it forms no hydrogen bonds with cellulose. Another
pigment, xanthophyll, differs from carotene in that it contains oxygen. Xanthophyll is found further from the
solvent front because it is less soluble in the solvent and has been slowed down by hydrogen bonding to the
cellulose. Chlorophylls contain oxygen and nitrogen and are bound more tightly to the paper than are the other
pigments.
Chlorophyll a is the primary photosynthetic pigment in plants. A molecule of chlorophyll a is located at
the reaction center of photosystems. Other chlorophyll a molecules, chlorophyll b, and the carotenoids capture
light energy and transfer it to the chlorophyll a at the reaction center. Carotenoids also protect the
photosynthetic system from the damaging effects of ultraviolet light.
PRELAB QUESTIONS
1. What is paper chromatography?
2. What is the primary pigment in photosynthesis?
3. Why do beta carotene and xanthophyll travel up the paper at different rates?
MATERIALS:
Filter Paper
Spinach Leaves
Scissors
Coin
Glass Cylinder with top
Pencil
Chromatography Solvent
Ruler
PROCEDURE:
1. Obtain a glass cylinder that has a top and put about 1 cm of chromatography solvent in the bottom.
Immediately screw the top on so that the solution does not evaporate. (This solution is similar to nail
polish remover – it is highly volatile and will evaporate extremely quickly if not covered)
2. Get a piece of filter paper and cut one end into a point. Draw a pencil line (NOT PEN) 1.5 cm above the
point.
3. Use a coin to extract the pigment from the spinach leaf cells. Place a small section of the leaf on the top
of the pencil line. Make sure the leaf is shiny side up. Use the ribbed edge of the coin to crush the cells.
Be sure that the pigment line is on top of the pencil line. You should repeat this procedure 8-10 times,
being sure to use a new portion of the leaf each time.
4. Place the filter paper in the cylinder so that the pointed end is barely immersed in the solvent. Do not
allow the pigment to be in the solvent!
5. Put the lid back on the cylinder. The solvent will travel up the filter paper by capillary action and will
bring the pigments along with it. When the solvent is about 1 cm from the TOP of the filter paper,
remove the paper and IMMEDIATELY mark the location of where the solvent got to before it
evaporates. This is called the solvent front.
6. Mark the BOTTOM of each pigment band with your pencil. Measure the distance (in millimeters) each
pigment migrated from the bottom of the pigment origin to the bottom of the separated pigment band.
Record your data in Table 1.1. You may be able to observe 4 or 5 pigment bands.
DATA TABLE:
Table 1.1- Distance Moved by Pigment Band (millimeters)
Band
Number
1
2
3
4
5
Distance
(mm)
Band Color
Distance Solvent Front Moved =
___________ mm
** Remember to measure from the bottom of the band
ANALYSIS SECTION Part A:
Calculations  You will calculate the Rf values for each of the pigments:
The relationship of the distance moved by a pigment to the distance moved by the solvent is a constant
called Rf. It can be calculated for each of the four pigments using the following formula:
Rf =
Distance pigment migrated (mm)
Distance solvent front migrated (mm)
Record your Rf values in Table 1.2. Be sure to show your calculations in your lab notebook!
Table 1.2 – Rf values for Each Pigment
Rf Value
Pigment
Carotene (very faint yellow)
Xanthophyll (yellow)
Chlorophyll a (bright green to blue green)
Chlorophyll b (yellow green to olive green)
Questions  Copy the question and answer it in your lab notebook for full credit.
1. What factors are involved in the separation of the pigments?
2. Would you expect the Rf value of a pigment to be the same if a different solvent were used? Explain
why or why not.
3. What type of chlorophyll does the reaction center contain? What are the roles of the other pigments?
PART B  Presence of CO2
INTRODUCTION
Because the spongy mesophyll layer of leaves (shown in Figure 1) is normally infused with gases (O2
and CO2), leaves — or disks cut from leaves — normally float in water. What would you predict about the
density of the leaf disk if the gases are drawn from the spongy mesophyll layer by using a vacuum and replaced
with water? How will that affect whether or not the leaf floats? If the leaf disk is placed in a solution with an
alternate source of carbon dioxide in the form of bicarbonate ions, then photosynthesis can occur in a sunken
leaf disk. As photosynthesis proceeds, oxygen accumulates in the air spaces of the spongy mesophyll, and
the leaf disk will once again become buoyant and rise in a column of water. Therefore, the rate of
photosynthesis can be indirectly measured by the rate of rise of the leaf disks. However, there’s more going on
in the leaf than that! You must also remember that cellular respiration is taking place at the same time as
photosynthesis in plant leaves. (Remember that plant cells have mitochondria, too!) What else could be going
on that might affect this process? Aerobic respiration will consume oxygen that has accumulated in spongy
mesophyll. Consequently, the two processes counter each other with respect to the accumulation of oxygen in
the air spaces of the spongy mesophyll. So now you have a more robust measurement tool — the buoyancy of
the leaf disks is actually an indirect measurement of the net rate of photosynthesis occurring in the leaf tissue.
In parts B and C of this lab, we will first work through the procedure to test the effect of the presence of CO2
has on the rate of photosynthesis. Next, you will test a variable of your choosing to see how the rate of
photosynthesis is affected.
For Part B, you will learn how the floating leaf disk technique can measure the rate of photosynthesis by
testing a variable that you know affects photosynthesis. The variable we will test in Part B of the lab is the
presence of CO2. We know that Photosynthesis uses CO2, so we will make one setup that has a CO2 source,
and one that does not.
Later, in Part C of this lab, you will apply this technique to test a variable that you choose. It is
important for you to develop a few skills during this part of the investigation in order to carry out your own
investigation. For the floating disk technique, the most challenging skill is getting the disks to sink.
The general idea behind this lab experiment is that when immersed in water, oxygen bubbles are usually
trapped in the air spaces of the spongy mesophyll in the plant leaf. By creating a vacuum in this experimental
procedure, the air bubbles can be drawn out of the spongy mesophyll, and the space is refilled by the
surrounding solution. This allows the leaf disks to sink in the experimental solution. If the solution has
bicarbonate ions (carbon source) and enough light, the leaf disk will begin to produce sugars and oxygen
through the process of photosynthesis. Oxygen collects in the leaf as photosynthesis progresses, causing the leaf
disks to float again. The length of time it takes for leaf disks to float again is a measure of the net rate of
photosynthesis. This process is shown in Figure 2.
Figure 2. Creating a Vacuum in a Leaf Cell
PRELAB QUESTIONS
1.
2.
3.
4.
5.
6.
What is the equation for photosynthesis?
Draw the “Big Picture” for photosynthesis. (This was drawn on the board during class)
What is the variable we are testing in Part B of this lab?
What is the purpose of using the soap in the solutions?
Why do we need to flush out the air between the mesophyll cells for this lab to work?
Which leaf disks do you anticipate to rise faster, the ones in the “With CO2” cup or the ones in the
“Without CO2” cup, and WHY?
7. What gas is released during photosynthesis?
8. What are some factors that can affect the rate of photosynthesis?
MATERIALS:
Sodium Bicarbonate (0.2%)
Spinach Leaves
Timer
Liquid Soap Solution
Hole Punch
Light Source
2 plastic 10mL syringes
2 plastic cups
PROCEDURE:
1. Obtain two plastic cups. For one cup, label it “With CO2” and pour the 0.2% bicarbonate solution to a
depth of about 3 cm. For the other cup, label it “Without CO2” and fill it about 3 cm high with water.
This second cup will be your control.
2. Using a pipette, add ONE DROP of a dilute liquid soap solution to the solution in each cup. It is
critical to avoid suds. If either solution generates suds, then dilute it with more bicarbonate or water
solution. The soap acts as a “wetting agent” — it wets the hydrophobic surface of the leaf, allowing the
solution to be drawn into the leaf and enabling the leaf disks to sink in the fluid.
3. Using a hole punch, cut out 10 leaf disks for each cup. Use the Baby Spinach Leaves for this part of the
lab. Avoid major leaf veins. See Figure 3.
Figure 3. Hole punching a leaf to create leaf disks.
4.
Draw the gases out of the spongy mesophyll tissue and infiltrate the leaves with the sodium bicarbonate
solution by performing the following steps:
a. Take out both syringes from your kit and make sure one is labeled “With CO2” and one is
labeled “Without CO2”.
b. Remove the plunger from both syringes. Place the 10 leaf disks into each syringe barrel.
c. Replace the plunger, but be careful not to crush the leaf disks. Push in the plunger until only
a small volume of air and leaf disk remain in the barrel (<10%). REMEMBER NOT TO
CRUSH THE LEAF DISKS!
d. Pull a small volume (5 cc) of sodium bicarbonate solution (that you added a drop of soap to)
from your prepared cup into the syringe labeled “With CO2”, and a small volume of water
(with your drop of soap) into the syringe labeled “Without CO2”. Tap each syringe to suspend
the leaf disks in the solution. Move the plunger to make sure no air remains and it is just
solution and leaf disks in the syringe at this point.
e. You now want to create a vacuum in the plunger to draw the air out of the leaf tissue. Create
the vacuum by holding a finger over the narrow syringe opening while drawing back the
plunger (see Figure 4). Hold this vacuum for about 10 seconds. While holding the vacuum,
swirl the leaf disks to suspend them in the solution. Now release the vacuum by letting the
plunger spring back. The solution will infiltrate the air spaces in the leaf disk, causing the leaf
disks to sink in the syringe. If the plunger does not spring back, you did not have a good
vacuum, and you may need a different syringe. You may have to repeat this procedure two to
three times in order to get the disks to sink.
Figure 4. Creating a vacuum in the plunger and leaf disks after they sank.
5. Pour the disks and the solution from the syringe into the appropriate clear plastic cup at the same time.
Disks infiltrated with the bicarbonate solution go in the “With CO2” cup, and disks infiltrated with the
water go in the “Without CO2” cup.
6. Place both cups under the light source and start the timer. At the end of each minute, record the number
of floating disks in each cup. Then swirl the disks to dislodge any that stuck against the side of the cups.
Continue until all of the disks are floating in the cup labeled “With CO2”. This should not take more
than 20 minutes. Record this data in Data Table 1.3.
DATA TABLES:
Table 1.3 - Results of Part B: Number of Floating Leaf Disks With CO2 vs. Without CO2
# OF LEAF # OF LEAF
TIME
DISCS
DISCS
(in
FLOATING FLOATING
minutes)
(WITH
(WITHOUT
CO2)
CO2)
1:00
# OF LEAF # OF LEAF
TIME
DISCS
DISCS
(in
FLOATING FLOATING
minutes)
(WITH
(WITHOUT
CO2)
CO2)
11:00
2:00
12:00
3:00
13:00
4:00
14:00
5:00
15:00
6:00
16:00
7:00
17:00
8:00
18:00
9:00
19:00
10:00
20:00
PART C  Design Your Own Experiment
The rate of photosynthesis can be affected by several factors. In this part of the lab, you are going to
create an experiment that tests one of these factors. The options that you have are:
- Change in Temperature
- Change in Age of Leaf (Baby Spinach vs. Mature Spinach Leaves)
- Color of Light (we have red, blue, green, and yellow filters, but keep in mind you will only
be able to test two colors because we are limited on lamps)
You first need to decide what variable you would like to test, and then, with your lab group, come up with a
procedure that would successfully test it. Write your procedure in your lab notebook, and design a data table.
ANALYSIS SECTION Part B and Part C:
Graph  You will create TWO graphs for this lab:
Make a line graph of your results for PART B. Include and appropriate title, key and correct scale for
your graph. There will be two lines on the graph, one for the With CO2 disks and one for the Without CO2
disks (make sure to identify which line is which!!). Use a ruler and make this neat. The x-axis (bottom) will be
Time, and the y-axis (side) will be # of Discs Floating. Be sure to label the axes.
Make another graph for PART C. The axes will be the same as they were for the first graph, but the
lines will be labeled differently based on your variable. Include an appropriate key, title, labeled axes, and
correct scale.
Questions  Copy the question and answer it in your lab notebook for full credit.
1. In Part B of your lab, which leaf disks rose faster, the ones With CO2 or the ones Without CO2? Why did
this happen?
2. What variable did you test in Part C of your lab?
3. What would happen if we covered the beaker and did not let light in? Would the disks rise? Why or
why not.
4. Why did we use a bicarbonate solution in this lab?