1 Photosynthesis Introduction Much like cellular respiration by ill20582




Much like cellular respiration, photosynthesis is a complex series of chemical reactions.
For introductory students, it is important not to get confused in the many stages of the
process and forget the overall importance of what is occurring: the conversion of light
energy into chemical energy (food). All life on earth depends on photosynthesis to
accomplish this task. Additionally, the oxygen produced during the photosynthetic
process is vital for almost all organisms.

As we saw in the respiration lab, it is helpful to have a summary equation for the overall
photosynthetic process.

                        Summary Equation for Photosynthesis

        6CO2 + 12H2O + Light energy                      C6H12O6 + 6O2 + 6H2O

The process of photosynthesis can be divided into two sets of reactions, the light
reactions and the Calvin Cycle (sometimes called dark reactions or light independent

Light Reactions
It is during the light reactions that the initial energy of the sun is captured by pigment
molecules collectively called chlorophyll. Most photosynthetic organisms actually have
a mixture of pigments with chlorophyll a designated the primary pigment and other
pigments such as chlorophyll b, carotenes and xanthophylls called accessory
pigments. Each pigment has a slightly different molecular structure that allow them
each to absorb light better at various wavelengths which also gives them characteristic
colors. Chlorophyll a is a blue-green pigment, chlorophyll b is yellow-green, carotenes
are yellow-orange and xanthophylls are yellow. During the lab today you will be
separating an extract into the different pigments using a technique called Thin Layer
Chromotography (TLC).

After the light energy is absorbed by the chlorophyll pigments, this energy is used to
excite electrons in the chlorophyll and a series of reactions occur that will eventually
make ATP molecules that will later be used in the Calvin Cycle. These chlorophyll
pigment electrons need to be replaced and it is the reactant water that is split during the
light reactions to provide the missing electrons. A by product of the water splitting is the
oxygen you see in the summary equation therefore one can roughly estimate the
process of photosynthesis by measuring the production of oxygen.

Calvin Cycle
The light reactions is not only responsible for producing ATP but also shuttles electrons
to the Calvin Cycle using the electron shuttle NADPH. During the Calvin Cycle,
inorganic carbon dioxide is reduced in a series of endergonic steps using the electrons
from NADPH and ATP to eventually make organic sugar that the plant can then use to
power cellular respiration or put into storage for future energy needs. Just as we could
measure photosynthesis by the production of oxygen, we can also crudely measure its
progress by the uptake of carbon dioxide.

Photosynthesis Worksheet

I. Thin Layer Chromatography (TLC)

This procedure will allow us to visualize the different types of chlorophyll pigments
discussed in the introduction that are present in plants. Overall, chromatography is a
technique that separates molecules from each other based on their solubility in certain
solvents. The more soluble the pigment is in the solvent, the faster and farther it can
move up chromotography paper. The size of the molecule can also affect the rate it
moves up the paper with smaller molecules progressing further up the paper than larger

Obtain: thin layer chromatography plate (take care to only handle plate on sides); small
plastic ruler; glass capillary tube; plant pigment extract

a.) Draw a faint horizontal line in pencil about 2 cm from the bottom edge of the TLC
plate. Draw a small “X” in the center of the line.

b.) Use a capillary tube to apply a small amount of plant pigment to the “X”. Do this by
dipping the end of the capillary tube into the pigment and then barely touching it to the
paper and immediately removing it.

c.) Allow this drop to dry completely (several minutes) and then repeat part b.) 10-15
times. The resulting spot should be very dark and small. Take your time, allowing for
complete drying between each addition of pigment drop.

d.) Locate the TLC running chambers. They have already been filled to the correct
level with the solvent . The solvent is a very flammable and toxic substance. Do not
directly breathe the fumes.

f.) Place your TLC plate into the slots inside the running chamber with the pigment spot
at the bottom. Check before placing the plate into the chamber that the solvent level will
be BELOW the pigment spot. You want the solvent to move up the plate and dissolve
the pigment slowly, not immerse the pigment immediately in the solvent.

g.) You will see the solvent begin to move up the TLC plate, hit your pigment spot and
begin to dissolve the pigment. Watch the solvent front (top edge of the part wet by the
solvent ) and when it reaches the top of the plate but BEFORE it runs off of it, remove
your plate from the running chamber.

h.) Immediately mark the location of the solvent front with a pencil. You must do this
before the rapidly drying solvent dries.

i.) Allow the plate to dry completely at your workspace.

j.) Mark the top edge of each pigment spot with a pencil. Depending on the quality of
the pigment extract, you may see up to 4-5 different spots of pigment.

Analysis of the pigments:

k.) Measure the distance, in mm, from your original line with the X marked pigment spot
and each of the other pencil marks you made (all the pigment spots and the solvent
front). Put the measurements in the appropriate space in Table 1. Using these
measurements, calculate the Rf value of each pigment which is the distance each
pigment ran versus the distance the solvent ran.

                            Rf = Distance pigment moved mm
                                 Distance solvent front moved mm

Table 1: TLC Data

Spot              mm moved          Rf               Color of spot
Solvent front                      xxxxxxxxxxxxxxx xxxxxxxxxxxxxxx
Spot #1

Spot #2

Spot #3

Spot #4

Using the color descriptions of the different chlorophyll pigments found in the
introduction, attempt to identify which spot represent which pigment.

       Spot #1       _________________________

       Spot #2       _________________________

       Spot #3       _________________________

       Spot #4       _________________________

II. Measuring rate of photosynthesis in varying light intensities by oxygen production

In this experiment, you will use Elodea as a representative photosynthesizing plant.
You will be varying the intensity of light the Elodea is exposed to and counting oxygen
bubbles produced as an estimate of photosynthetic rate.

Obtain: sprig of Elodea, razor blade, test tube, lamp, meter stick


a.) With the razor blade, carefully cut a length of Elodea at least 10 cm long from the
tip. Save the 10 cm piece.

b.) Put the piece in the test tube upside down so that the cut end is at least 3 cm from
the top of the test tube.

c.) Fill the test tube with tap water so that the cut end is completely submerged.

d.) Using the meter stick, place the test tube with the Elodea 25 cm from the lamp.

e.) Wait a few minutes for the plant to adjust to the light level. You should begin to see
bubbles of oxygen coming from the cut end of the tip shortly.

f.) Once you see two or more bubbles/minute, begin the official count and record the
number of bubbles produced in 10 minutes in Table 2.

g.) Calculate the average bubble count/minute for the light intensity at 25 cm and
record it in Table 2.

h.) Move the tube to a distance of 50 cm and allow the plant to adjust. Repeat the
counting procedure from f.) above and record your results in Table 2.

i.) Move the tube to a distance of 75 cm. Repeat the same counting procedure and
record your results in Table 2.

Table 2: Oxygen production during photosynthesis

Distance                      Bubbles/10 min.              Average bubbles/min
25 cm
50 cm
75 cm

III. Uptake of Carbon Dioxide during Photosynthesis

This experiment will demonstrate the use of carbon during photosynthesis.

Obtain: 2 test tubes, phenol red solution (pH indicator that turns yellowish orange in an
acid and red in a neutral or basic solution), sprig of Elodea, lamp, meter stick, straw


a.) Fill both test tubes with phenol red solution.

b.) Using the straw, breathe bubbles into both test tubes just until the phenol red turns
yellow. The carbon dioxide from your respiration combines with the water in the phenol
red solution to form carbonic acid, thus lowering the pH and turning the pH indicator
yellowish orange. DO NOT continue to blow once the solution is yellowish orange.

c.) Place a sprig of Elodea in one test tube and place both tubes, side by side, 25 cm
from the lamp.

d.) Watch the color of the solutions until you detect a change in color from yellowish
orange to red. The time required for the color change can vary but is usually between
1/2 to 1 hour.

e.) Based on your results, fill out Table 3.

Table 3

Test tube                     Color before light exposure Color after light exposure
                              but after blowing in it
Elodea present
Elodea absent


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