Lab Exercise 8- Photosynthesis
Light Dependent and Light Independent Reactions
Introduction:
Every living organism requires energy in order to carry out its normal functions. In every case
organisms generate this energy through metabolism of a food source resulting from photosynthesis.
Photosynthesis provides oxygen and food to other living organisms. Photosynthesis is the food
preparation process of green plants using carbon dioxide and water in the presence of chlorophyll
pigment and sun light producing sugar and water and liberating oxygen. Light energy is converted
into chemical energy during this process. The overall equation for photosynthesis is:
Light
6CO2 + 12 H2O ------------------ C6H12O6 + 6O2 + 6H2O
Chlorophyll
Photosynthesis takes place in the mesophyll region of the leaf which is containing the organelle called
chloroplast. There are 2 stages in photosynthesis. The first is the light reaction (light dependent)
which will occur in the grana and the second is dark reaction (light independent) which will occur in
the stroma of the chloroplast. A stack of thylakoids constitute a grana and the thylakoid portion of the
chloroplasts contain the primary pigments, i.e. Chlorophyll a (dark green) and chlorophyll b (light
green). Also present are accessory pigments such as carotene (orange) and xanthophyll (yellow).
It is the visible portion of the electromagnetic spectrum that supplies energy for photosynthesis). The
green portion is reflected by chlorophyll giving green plants their perceived color. Other colors are
absorbed as energy which is subsequently passed on to Photosystems II and I.
During the light reaction photolysis of water will occur, releasing oxygen and hydrogen (Figure 8.1).
Oxygen will come out through the stomata, openings present in the epidermis of the leaf and the
hydrogen will be accepted by the electron carrier NADP and used during dark reaction.
Figure 8.1 - Photosystems II and I
During the dark reaction (Figure 8.2), carbon fixation will occur resulting in the formation of sugar.
Excess sugars are stored by the plant in the form of starch.
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Figure 8.2 - Calvin Cycle
Objectives:
1.Isolate plant pigments by the technique know as paper chromatography.
2.
Explain an experiment that indicates light promotes photosynthesis.
3.
Describe an experiment that indicates CO2 is utilized during photosynthesis.
4.
Explain why chlorophyll is important for photosynthesis.
5.
Define all bolded and underlined words in the reading.
Lab Activity 8 - Photosynthesis
Light Dependent and Light Independent Reactions
Required Materials:
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4 healthy potted plant
2 Bell Jar
Soda lime in a small cup
Pair of scissors
Three 250ml beaker
Hot plate
Tongs
Water bottle
Boiling chips
Ethyl alcohol
50 ml beaker
Petri dish
Forceps
Iodine
Light screen
Coleus plant (plant with variegated leaves)
Spinach
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Mortar and pestle
Mortar and pestle
Sand
Acetone
6 Droppers
Chromatography strip (paper)
No. 2 pencil
Ruler
Micro capillary tube
Four Developing tub with lid
Marking pencil
Chromatography /Developing solvent
Assignment 1
Paper Chromatography
The purpose of this experiment is to demonstrate the presence various plant pigments in leaves.
Separation of various kinds of plant pigments:
a) Make an extract of pigment by grinding spinach leaves with a pinch of sand and 5 – 10 drops of
acetone solvent (obtain from your instructor) using the mortar and pestle.
b) Obtain a strip of chromatography paper longer than the tube. With a pencil, mark a dot on the strip
of paper 2cm above the bottom of the paper. (Figure 8.3)
c) Dip one end of a toothpick into the spinach mixture.
Carefully place the toothpick with pigment on the dot marked on the chromatography paper (Figure
8.6). Allow the dot to dry thoroughly and then apply more extract over the same dot. Repeat at
least 10 times making sure to allow the dot of pigment to air dry between applications. More
applications will give you better results.
Figure 8.3 - Pigment Extract Being Applied To Marked Chromatography Paper
d) Obtain a developing tube (large diameter test tube) with a rubber stopper. Obtain small amount of
“developing” solvent from your instructor and pour it into the tube. Make sure you immediately
place the stopper in the tube to keep the solvent from evaporating.
e) When completely dry, insert the paper, pigment end down, into the developing tube so that the paper
bottom will just barely touch the solvent. The pigment dot should be
above the solvent, not
in it.
f) Suspend the chromatography paper in the developing tube with the help of the stopper, closing
tightly. Set the tube in a test tube rack and leave undisturbed for at least 30minutes (Figure 8.4).
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Figure 8.4 - Cork Closed Tightly
g) After 30 minutes, remove the paper strip and place it on a paper towel. This is our chromatogram.
Replace the rubber stopper into the test-tube to prevent the solvent from evaporating. It can be
reused.
h) Examine your chromatogram. Locate the orange, yellow, dark green and light green bands and
identify each pigment. Staple the chromatogram onto assignment – 1 of your lab report at the
indicated area and identify the pigment bands and answer the questions
Assignment 2
Light and Photosynthesis:
The purpose of this experiment is to demonstrate that light is necessary for the production of
sugar (stored as starch) during photosynthesis.
A. Starch Test: (Figure 8.5).
Test the leaf for the presence of starch, using the following procedure, and record your observations
under assignment – 2 of the Lab Report.
Preparation of a leaf for the starch test: BE CAREFUL...ALCOHOL IS FLAMMABLE!
a) Add boiling chips to a 250ml beaker containing 100ml water. Remove a leaf from a green plant
that was exposed to light. Add the leaf to be tested to the beaker with water and boil for 3-5
minutes to rupture the cells.
b. Using the forceps, transfer the boiled leaf into a test tube. Cover the leaf with enough alcohol.
Carefully place test tube with boiled leaf & alcohol, inside a 250 ml beaker with 100 ml water.
Keep this set up on a hot plate and allow the water in the 250 ml beaker to boil to extract the
chlorophyll pigment from the leaf. (This way you are boiling the leaf in alcohol with the help of
water bath).
c) Then take the leaf out and place them in a Petri dish and completely cover the leaf with iodine
solution.
d) After 3 minutes rinse the leaf with water and examine the color of the leaf. If the leaf turned
blue/black or dark brown/black, it indicates the presence of starch. If the leaf do not turn
blue/black or dark brown/black, it shows that starch has not been stored in the leaf. Answer the
questions under Assignment 2 of the lab report.
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Leaf in water
Leaf in alcohol
Figure 8.5 – Starch Test
Leaf with iodine
B. Light Screen Experiment: A demonstration has been set up and will be discussed by your
instructor.
a) A potted plant was taken and few leaves of this plant were covered with light screen.
(Figure 8.6) and this plant was exposed to sunlight.
b) After few days, the light screens were removed from those leaves and were tested for starch.
c) After observing this demonstration set up, record your answers under assignment 2 of the lab
report.
Figure 8.6 Light Screen Experiment
Assignment 3
Chlorophyll & Photosynthesis (Figure 8.7)
The purpose of this experiment is to show that chlorophyll is necessary for the production of sugar
(stored as starch). A demonstration has been set up and will be discussed by your instructor.
a) A leaf was removed from a plant with green and white or a multicolored leaves. (Variegated leaf)
b) Tested for the presence of the starch.
c) After observing this demonstration set up, record your answers under assignment 3 of the lab
report.
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Figure 8.7 – Coleus Plant with variegated leaf
Assignment 4
Carbon dioxide and Photosynthesis (Bell Jar Experiment):
The purpose of this experiment is to prove that carbon dioxide is necessary for photosynthesis.
A demonstration has been set up and will be discussed by your instructor.
a) Two healthy plants were taken and labeled “A” and “B” (Figure 8.8).
b) Plant “A” was placed under a bell jar.
c) Plant “A” was exposed to air from which CO2 has been removed by soda lime cup placed inside
the bell jar
d) Plant “B” was exposed to air containing CO2.
e) Both the plants were then exposed to light.
f) Cut a leaf from each plant and carefully test these leaves for the presence of starch.
g) Record your observations in assignment 4 of the Lab Report. L
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Plant A
P Plant B
Figure 8.8 - Plants Without and With CO2
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tA
Lab Report 8 - Photosynthesis
Light Dependent and Light Independent Reactions
Name: ________________________________
Date:
____________________________
Class Index: ___________________________
Instructor: ____________________________
Before you begin filling out this lab report you must read Exercise 8 - Photosynthesis in your lab manual.
Complete Assignments 1-3 below. You can use your Lab Manual results and Textbook to complete the
information below.
Assignment 1
Paper Chromatography
The purpose of this experiment is to demonstrate the presence various plant pigments in leaves.
a) How many kinds of pigments are present in spinach leaves?_________________________________
b) Indicate the colors and identify each of the pigment.
Pigment
c) Attach your chromatogram strip here.
Color
↓ Identify each pigment seen.
d) What colors of visible light would you expect the chlorophyll pigment to absorb?
__________________________________________________________________________________
e) What colors of visible light does the chlorophyll pigment actually reflect?
_________________________________________________________________________________
f) Which organelle inside the mesophyll cells of the leaf contains the pigments? ____________________
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Assignment 2
Light and Photosynthesis:
A. The purpose of this experiment is to demonstrate that light is necessary for the production of
sugar (stored as starch) during photosynthesis.
Color of Leaf
before boiling
Color of leaf
after boiling
Color of leaf after
exposure to
iodine.
Is starch
present
in leaf.
Explain your observation.
Starch test
result
B. Leaf with Light Screen (Demonstration set up)
Will starch be present in the light screen covered portion of the leaf? Yes/No _____________________
Justify your answer: ___________________________________________________________________
____________________________________________________________________________________
Assignment 3
Chlorophyll and Photosynthesis (Variegated Leaf Experiment)
The purpose of this experiment is to show that chlorophyll is necessary for the production of sugar
(stored as starch).
Portion of the leaf
Color after Starch test
Presence/ absence of starch
With Chlorophyll
Without Chlorophyll
Assignment 4
Carbon dioxide and Photosynthesis (Bell Jar Experiment):
a) How does carbon dioxide get into a leaf? ______________________________________________
b) What does bell jar experiment prove? ________________________________________________
_______________________________________________________________________________
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