Lab: Photosynthesis
OVERVIEW
Green plants use sunlight to make glucose.
To do so, the plant must use carbon
dioxide and water in a process called photosynthesis. The glucose made
by plants is used by plants and animals as a source of energy. To release
the energy contained in the bonds
of glucose, the glucose must be converted to ATP. The process by which
ATP is made from glucose is called cellular respiration. Respiration
also produces waste products including
carbon dioxide and water, which are the same substances that serve as
raw materials for photosynthesis. For this reason scientists describe
the exchange of water, carbon dioxide,
glucose, and oxygen and a perfect cycle. In other words plants consume
what animals
produce, and animals consume what plants produce. Therefore it is hypothesized
that this
cycle could continue throughout the life of earth. This relationship
can be expressed by the following equation:
H2O + CO2 --------------->
C6H12O6
+ O2
In water, carbon dioxide dissolves to form
a weak acid. As a result, an acid-base
indicator such as bromothymol blue can be used to explore this relationship
between photosynthesis and respiration. Likewise the glucose produced
by plants, and the starches
made from this glucose, can be detected by iodine products. A third
product of
photosynthesis, oxygen, can be detected as bubbles in water.
In this series of experiments the raw
materials and finished products of
photosynthesis will be studied.
I. USING CHROMATOGRAPHY TO STUDY PLANT PIGMENTS
INTRODUCTION
Chlorophyll is the best known and most
common pigment in plant leaves. It comes
in two primary forms, “a” and “b”, both of which
are shades of green. Both chlorophylls
serve primarily to capture the energy in sunlight and convert it to
chemical energy in the
form of glucose. There are several other pigments in plant leaves, but
chlorophyll is so
abundant that it often hides them. In autumn, chlorophyll breaks down
first allowing other
pigments such as xanthophylls, carotene, and anthocyanin, to show their
colors.
The mix of pigments in a leaf may be
separated into bands of color for further study
using a technique called paper chromatography. Chromatography which
means "color
writing.", involves the separation of mixtures into individual
components. With this
technique the individual components of a mixture in a liquid form can
be separated. The
separation takes place by absorption and capillarity. The paper holds
the substances by absorption; capillarity pulls the substances up the
paper at different rates. Pigments are
separated on the paper and show up as colored streaks. In this lab a
common pattern that
results listed from top to bottom is carotenes (orange), xanthophylls
(yellow), chlorophyll b (yellow-green), chlorphyll a (blue-green), and
anthocyanin (red). This pattern of separated components on the paper
is called a chromatogram.
Once the “speed” at which
a pigment moves up the paper is known it can be
identified by calculating it Rf factor. To calculate the Rf of any pigment
use the
equation shown below:
Rf = Distance moved by pigment
Distance moved by solvent
The final step is to take the Rf value you calculated and compare it
to the Rf values of all
known pigments. Chemists have already studied the Rf value for thousands
of chemicals
and have their results stored in books that are available to anyone.
In this portion of the lab we will learn
the scientific technique of paper chromatography. Then we will use this
technique to separate and view the different pigments found in the plant
leaves.
| EQUIPMENT Fresh or thawed spinach leaves Chromatography paper (1 cm x 15 cm) Scissors Test tube (15-cm length or larger) Cork (with paper clip hook) Mortar and pestle 2 mL Ethyl alcohol 10 mL Graduated cylinder Glass stirring rod Chromatography solvent Test-tube rack or beaker Ruler |
Diagram
#1 |
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|
Diagram
#2 |
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| PROCEDURE CAUTION: Chromatography solvents are flammable and toxic. Have no open flames, and do not breath the fumes. CAUTION: Handle paper AS LITTLE AS POSSIBLE. Oil and sweat will interfere with movement of pigments through the paper. 1. Cut a strip of filter paper or chromatography paper so that it just fits inside a 15-cm (or larger) test tube. Cut a point at one end. Punch a hole in the other end with a hole punch. Draw a faint pencil line as shown in the picture. Locate a cork with a hook protruding from its bottom. Attach the paper strip to the hook on the cork so that it hangs inside the tube, as shown in Diagram #2. The sides of the strip should not touch the glass |
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2. Tear a spinach leaf into pieces about the size of a postage stamp.
Put them into a
mortar. Add about 2 mL ethyl alcohol to the
leaf pieces. Crush the leaves with the
pestle, using a circular motion, until the
mixture is completely ground. See Diagram
#1 above. The liquid in which the leaf pigments
are now dissolved is called the
pigment extract.
3. Use a glass rod to touch a drop of the pigment extract to the center
of the pencil line on
the paper strip. Let it dry. Repeat 3-4 times,
to build up the pigment spot. NOTE: You
must let the dot dry after each drop is added.
The drying keeps the pigment dot from
spreading out too much.
4. Pour approximately 5 mL chromatography solvent into the test tube.
Next place the paper
and cork assembly inside also. Adjust it so
that the paper point just touches the solvent
(but not the sides of the tube). The pigment
dot must be above the level of the solvent.
Watch the solvent rise up the paper, carrying
and separating the pigments as it goes. At
the instant the solvent reaches the
top (bottom edge of the whole you punched earlier),
remove the paper and let it dry. Quickly mark
the approximate centers and label the
colors of the bands of pigment since the color
often fades as they dry.
5. Record the colors and pigment names in the data chart.
6. Measure the distance in cm from the starting point to the center
of each pigment band.
Then measure the entire distance traveled by
the solvent. Use the dried chromatograms
you made. Record these distances in the data
chart.
7. Calculate the Rf value as a decimal fraction for each pigment that
is visible on your chromatogram. Record
the Rf values in the data chart.
8. Compare your results for both leaves with those of other students.
9. Answer any questions in the data sheet pertaining to this portion
of the lab.
II. CONSUMPTION OF CARBON DIOXIDE DURING
PHOTOSYNTHESIS
INTRODUCTION
As shown in the equation in the lab overview
green plants use sunlight to make
glucose. To do so, the plants must take in carbon dioxide and water
to be used as raw
materials in the process of photosynthesis. Once produced this glucose
can be used as a
source of energy by both plants and animals. To do so the glucose must
be “burned” in a
process of respiration. Respiration reverses the action of photosynthesis,
thereby causing
the release of both carbon dioxide and water back into the environment.
This portion of the lab will try to study
the production of carbon dioxide by animals
and the consumption of carbon dioxide by plants. In water, carbon dioxide
dissolves to
form a weak acid called carbonic acid. This reaction is described by
the following equation.
H2O + CO2
-------> H2CO3
(Carbonic acid)
This weak acid will alter the pH of the
water and therefore also alter the appearance
of indicator chemicals such as bromothymol blue. When a solution a neutral
(pH = 7),
bromothymol blue appears blue to greenish blue. In acidic solutions
(pH < 7), the color
becomes yellowish. In this lab we can study the buildup and depletion
of carbon dioxide in
a water source by studying these color changes that occur in the bromothymol
blue.
| EQUIPMENT | DIAGRAM #2 |
| large test tube (with stopper) |
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| PROCEDURE | |
| 1. Fill the large test tube ¾ full with bromothymol
blue solution.. 2. Obtain a straw, insert one end into the solution in the test tube, and gently blow bubbles into the liquid. (Hold your thumb over the mouth of the test tube if it overflows with bubbles.) Keep blowing until there is a change in the appearance of the bromothymol solution. |
3. Record the changes in the appearance of the bromothymol blue in
the data sheet.
4. Place a sprig of Elodea (or another aquatic plant) into the test
tube you had been blowing
air into. See Diagram #2 above.
5. Place the test tube with the Elodea in it under a light source for
24 hours.
6. Record the changes in the appearance of the bromothymol blue in the
data sheet.
7. Answer any questions in the data sheet pertaining to this portion
of the lab.
III. PRODUCTION OF OXYGEN DURING PHOTOSYNTHESIS
INTRODUCTION
As shown in the equation in the lab overview
green plants use sunlight to make
glucose. To do so, the plants must take in carbon dioxide and water
to be used as raw
materials in the process of photosynthesis. A byproduct of this process
is the production
of oxygen. Plants, like animals, need to utilize some of this oxygen
during respiration
(“burning” of glucose) to produce the energy needed to power
their cells. Plants produce
more oxygen than they need, however, resulting in the release of this
excess oxygen.
In this lab we will study the process
of oxygen production by plants during
photosynthesis. A good method for studying oxygen production is to use
water plants.
In this way the production of oxygen can be observed. Specifically we
will observe the
movement of plants from deep in the water to the surface as they fill
with oxygen and
become buoyant. This lab utilizes spinach leaves that have been cut
into small circular
discs. These discs can be sunk to the bottom of a dish, and over time
will slowly rise if photosynthesis is allowed to occur.
(An
alternative method is to place Elodea in an inverted test tube filled
with
water. As photosynthesis occurs Elodea releases the excess oxygen as
bubbles
which can be observed and counted.)
| EQUIPMENT | Diagram #3 |
|
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| PROCEDURE 1. Pour 100 mL of NaHCO3 solution into 250 mL Erlenmeyer flask. |
2. Obtain 3 petri dishes and label them as follows:
#1: Strong light (under light source)
#2: Indirect light (near windowsill)
#3: Little/No light (in cabinet or back
room)
Also place your names on each dish.
3. Obtain spinach leaves and cut 40-50 small round discs using a cork
borer. Stay away
from the larger veins when cutting the spinach
discs.
4. Pour the spinach discs into the NaHCO3 solution in the flask.
5. Take the flask to the vacuum pump. Place the cork assembly onto the
top of your flask,
attach the vacuum pump, and begin the vacuum.
See Diagram #3 above. As you
vacuum the spinach discs should sink to the
bottom of the flask. Once most of the discs
have sunk, you can stop the vacuum and take
off the cork assembly.
6. Pour approximately 1/3 of the spinach discs into each of the 3 petri
dishes. Use forceps
to discard any discs still floating on the
surface of the NaHCO3 solution.
7. Count the number of spinach discs in each Petri dish and enter this
data into the data sheet.
8. Place each Petri dish in the located indicated by its label (step
2 above) for 24 hours.
9. Count the number of spinach discs that are floating in each of the
Petri dishes, and enter
this data into the data sheet. Calculate the
percentage of discs that were floating by
the end of the 24 hours for each petri dish
and enter this data into the data sheet.
10. Answer any questions in the data sheet pertaining to this portion
of the lab.
III. PRODUCTION OF GLUCOSE DURING PHOTOSYNTHESIS
INTRODUCTION
As shown in the equation in the lab overview
green plants use sunlight to make
glucose. In many plants the food make by photosynthesis is stored in
the cells of the leaf
where it is produced. This food is stored in leaves as starch. This
makes it possible to show
that food-making occurs in the leaf by testing for that starch. Lugol’s
iodine is a chemical
that reacts with starch by turning it deep blue to black in color. To
be able to visually see
this color change in a leaf though, we must first remove the chlorophyll
from the leaf.
In this portion of the lab you will stimulate
starch production in some parts of a leaf
while preventing it in another part of the leaf. Then you will remove
the chlorophyll, and
use Lugol’s iodine to look for the presence of starch in both
parts of the leaf.
| EQUIPMENT |  |
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PROCEDURE
1. Obtain a plant that has been in a dark area for 24 hours.
2. Obtain some aluminum foil and cover approximate 1/3 of the left side
of one of the
leaves. Make sure to cover.both the top and
bottom sides of the leaf you have
selected. Secure the aluminum foil in place
with paper clips so that it stays tightly
against the leaf. When done the leaf should
look like the diagram above.
3. Place the entire plant in bright light for 24 hours.
4. After 24 hours remove the leaf from the plant, and take off the aluminum
foil.
5. Place the leaf in the 400 mL beaker of boiling water for 5 minutes.
This
softens the leaf so that the chlorophyll can be removed.
| 6. Use tongs to remove the leaf from the
boiling water. Immediately place the leaf into a 250 mL beaker of boiling ethanol (located inside a 600 mL beaker of boiling water). This setup is illustrated in Diagram #4. Continue to boil the leaf in the ethanol for 8 minutes or until it turns completely white. 7. Use tongs to remove the leaf from the boiling water. Immediately rinse the leaf in a beaker of tap water. 8. Now that the chlorophyll has been removed we can look for starch. Lay the leaf flat across the bottom of a Petri dish. Locate a beaker of Lugol’s iodine and pour some into the dish until it completely covers the leaf. |
DIAGRAM
#4 |
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9. Let the leaf soak in the iodine for 15 minutes so that every cell
in the leaf has a chance
to absorb the iodine.
10. Return the iodine to its beaker, and rinse the leaf by gently running
water into the Petri
dish.
11. Pour off all the water and spread the leaf across the bottom of
the dish again.
12. Now check for any signs of starch in the leaf. You probably will
not get any intense
blue-black colors, but you might see
some different shades of light and dark where
you had covered the leaf with aluminum
foil.
13. Draw any color pattern you observed in the leaf into the space provided
in the data
sheet. Then answer any questions in the
data sheet pertaining to this portion of the lab.