Biological Principles
Lab: Photosynthesis
Name: ______________________________________
PURPOSE
 To visualize the various photosynthetic pigments present in spinach leaves.
 To test the effect of various light conditions on a leaf’s ability to synthesize and store starch.
INTRODUCTION
Photosynthesis is a complex chemical process that converts the kinetic energy from the sun to the
potential energy stored in sugar. The various chemical reactions that occur in photosynthesis can be
summarized by the following equation:
6 CO 2 + 6 H2O light

 C6H12O6 + 6 O 2
Carbon dioxide and water serve as reactants in photosynthesis, and along with light energy from the
sun, are used to produce glucose and oxygen. The light energy needed for photosynthesis is “captured”
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by colored molecules called
pigments, which absorb light at specific wavelengths. We will be looking at
four photosynthetic pigments in this lab: chlorophylls a and b, carotenes and xanthophylls.
Module 1 utlilzes a technique called paper chromatography, a process for separating molecules that
have been dissolved in solution. We will use paper chromatography to separate the four photosynthetic
pigments found in spinach leaves. The pigments will be applied to the bottom of a paper strip. The tip of
that strip is then placed in a chromatography solvent. The solvent will begin to move up the paper via
capillary action. As the solvent moves, the pigments will dissolve in the solvent and flow along with it up
the strip. Each type of pigment molecule will move differently, and this movement is determined by two
specific characteristics of the pigments: the pigment’s solubility in the solvent and its tendency to adsorb
to the paper. These characteristics are determined by a number of factors, including the size, polarity and
solubility of the different pigment molecules. Pigments that adsorb more tightly move less than those that
adsorb less tightly. There will be four bands of color that form: a thin, yellow band of carotenes, a wide,
light yellow band of xanthophylls, a blue-green band of chlorophyll a, and a green-yellow band of
chlorophyll b.
We can compare the mobility of two or more pigments by calculating each pigment’s Rf value
using the following formula:
Rf =
Distance moved by pigment
Distance from pigment origin to solvent front
Module 2 will demonstrate the importance of light and chlorophyll in photosynthesis. We will be
testing for the presence of starch in variegated leafs (part green and part white) from two geranium
 in the light and one which has been kept in the dark for several hours.
plants, one which is
Glucose is produced by photosynthesis, but we will be testing for starch because the test is much
easier to perform. Iodine and starch react to produce a blue-black color that is easily recognizable. If you
do not remember the structure and function of starch, review them at this time.
MODULE 1: Separation of pigments from spinach leaves
MATERIALS
 test tube
 test tube rack
 cork stopper with hook attached
 chromatography strip
 paper towel
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spinach leaf
a quarter
chromatography solvent
5-ml pipette
pipette pump
PROCEDURE
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1. Lay the strip of chromatography paper on the lab bench.
2. Place the leaf approximately 1 cm above the notch in the strip.
3. Run the coin over the leaf, leaving a line of pigment on the strip. Go over the same line five times
to ensure a dark, even line of pigment. Let the strip air dry for a few seconds between each
application.
4. Pipet 2 ml chromatography solvent into the test tube.
5. Hang the paper strip on the cork hook.
6. Place the strip into the test tube so that the paper below the notch is immersed in the solvent.
7. Let the solvent and pigments migrate up the strip until the solvent front is 1 cm from the top of the
strip.
8. Remove the strip and lay it on the paper towel to dry.
9. Answer Questions 1-7 in the lab report.
MODULE 2: Demonstration of the importance of light and chlorophyll in photosynthesis
MATERIALS
 variegated geranium in light
 variegated geranium in dark
 hotplate
 500-ml beaker
 250-ml beaker
 water
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alcohol
large forceps
paper towels
two Petri dishes
wax pencil
Lugol’s iodine
PROCEDURE
1. Answer Questions 8-10 on your lab report.
2. On the front bench, your instructor will boil 250 ml water in the 500-ml beaker.
3. Pour about 50 ml alcohol into the 250 ml beaker. Keep this away from the hot plate.
4. Label one Petri dish “light” and the other “dark.”
5. Remove one leaf, including the petiole (the stem-like structure of the leaf) from the plant that has
been stored in the light. Also get a leaf from the plant that has been stored in the dark. To help
keep the leaves straight, remove the petiole from the “dark” leaf. Look at the leaves and record
your observations with sketches in the first column of the table on your lab report.
6. To soften leaves and facilitate chlorophyll extraction, boil them in water for 1 min.
7. With the forceps, transfer the leaves to a paper towel and blot them to remove excess water.
8. Put the leaves in the beaker of alcohol and very carefully lower this beaker into the one with the
boiling water. Use care to avoid spilling the alcohol onto the hot plate.
9. When the alcohol starts to boil, turn the hotplate to low. Your instructor will assist ypu.
10. After 3 min, turn off the hotplate, remove the beakers and transfer each leaf to its corresponding
Petri dish. Record your observations with sketches in the second column of the table.
11. Add just enough tap water to cover the leaf and add about 10 drops of Lugol’s iodine.
12. After 2 min, rinse the leaves with water and record your observation in the third column of the
table.
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13. Answer the remaining questions on your lab report.
CLEAN-UP
 Discard the paper chromatography strip, paper towel, and the spinach leaf in the trash.
 Pour the chromatography solvent waste into the waste container. Do not put it down the sink!
 Discard the test tube in the glass disposal box.
 Discard the leaves and Petri dishes in the trash.
 Return all equipment to the instructor’s bench.
 Wipe down the lab bench with cleanser and paper towels.
 Push in your chair when you leave.
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