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Lab #8: Photosynthesis

OBJECTIVES:
After completion of this exercise, you should be able to:
1. Identify the structures of a monocot and dicot leaf involved in photosynthesis.
2. Demonstrate how leaf pigments are extracted from leaves.
3. Identify the pigments involved in photosynthesis by thin layer
chromatography and/or paper chromatography.
4. Determine the wavelengths most useful for photosynthesis by
determining the absorption spectrum of a pigment extract.
5. Describe the roles played by light and pigment in photosynthesis.
6. Demonstrate how light intensity affects the rate of photosynthesis using
spinach leaf disks.

INTRODUCTION:

hotosynthesis is a fundamental biochemical process that sustains all life on Earth.

Photosynthetic plants, protists and bacteria are the producers in a food chain
manufacturing foods for themselves and the consumers (animals and fungi). Even
carnivorous animals are ultimately dependent on photosynthesis since carnivores eat
herbivores who have fed upon plants. In addition to producing food materials (sugar and
starch), photosynthetic organisms are the source of 20 % of the air that is oxygen which
makes respiration possible.
What is common to all photosynthetic organisms is the green pigment
chlorophyll, a compound that can be activated by light. Photosynthesis is the conversion
of carbon dioxide and water into organic compounds. The energy that drives this process
comes from light-activated chlorophyll. What sets photosynthetic organisms apart from
the rest of the living world is their ability to synthesize from inorganic compounds and
from the energy of the sun all the organic compounds they require for building and
maintaining their protoplasm. A summary equation for photosynthesis is presented
below:
6 CO2 + 12 H2O + Light + Chlorophyll

C6H12O6 + 6 O2 + 6 H2O

Or balanced as:
6 CO2 + 6 H2O + Light + Chlorophyll

C6H12O6 + 6 O2

Organisms having this independent nutritional status are known as autotrophs

(self-feeders). All other living organisms must depend upon the organic compounds
synthesized by autotrophs; these compounds provide them with energy (through
respiration and fermentation) and protoplasmic building blocks. Organisms having this
dependent nutritional status are called heterotrophs. Why autotrophic organisms must be
the first link in any food chain and assume a central and crucial role in the community of
life should now be clear to you.

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In this lab exercise, you will first examine leaf structure where photosynthesis
begins. You will next extract the pigments from a leaf and learn how to separate these
pigments by a technique called chromatography. You and your team will also determine
the wavelengths of light important in photosynthesis and measure the rate of
photosynthesis using spinach leaves. This lab exercise will conclude with two activities
involving starch production in covered Geranium and Coleus leaves.

Exercise 8.1 Structure of a Leaf

The leaf is the principal organ of photosynthesis. The flattened shape is an
adaptation to expose large numbers of chloroplasts to sunlight. The leaf must also provide
a moist cell surface for exchange of gases with the environment yet prevent excessive
water loss. Lastly, the leaf must contain extensions of the vascular system of the stem to
move the products of photosynthesis from the leaf to the stem and roots and to move
water and minerals from the roots into the leaves. As you examine examples of monocot
and dicot leaves in cross-section, ask yourself which of its structural features are
particularly important in making it an efficient site for photosynthesis.
Materials Needed:
Compound microscope
Model of leaf structure
Samples of live monocot and dicot leaves

Prepared slide with leaf cross section of Ligustrum, Lilium, or generalized

dicot
Prepared slide with leaf cross section of Zea mays or a generalized monocot

Procedure:
1. Obtain a prepared slide of a leaf cross section of Ligustrum (privet hedge), Lilium
or generalized dicot. View under low and high power, using Figure 8.1 (A) as a
guide to identify the structures mentioned below.
2. Note that the leaf is composed of a complicated arrangement of cells. You will
first notice sections of vascular bundles that are recognized as veins in the whole
leaf. A vascular bundle is composed of two types of vascular tissue: xylem and
phloem. These are continuous with the same tissues in the stem and root. Observe
from the top of the leaf down.
a) The cuticle, a waxy layer on the upper and lower leaf surface. In
Ligustrum it is thicker on the upper surface. What is its function? Why is
it thicker on the upper surface?
b) The upper epidermis forms a protective layer and secretes a waxy
substance called cutin. The continuous layer of cutin forms the cuticle.
How many cells thick is the upper epidermis? Does this layer contain
chloroplasts?

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Figure 8.1: Leaf cross-sections. (A) Diagram of a dicot such as Ligustrum or Lillium.
(B) Diagram of a monocot such as corn (Zea mays).
c) The mesophyll is the region between the upper and lower epidermal
layers excluding the vascular tissue. The cells that makeup the mesophyll
or parenchyma are characterized by thin walls, a well-developed central
vacuole, and a majority of the leafs chloroplasts. Chloroplasts contain
several different green or blue-green chlorophyll pigments plus yellow or
orange pigments called carotenoids. The electron micrograph of a
chloroplast (Figure 8.2)

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Figure 8.2: Structure of the chloroplast.

reveals groups of membranes which look like a stack of coins called a
granum. Note that chlorophyll is found between the layers known as
thylakoids. What photosynthetic reactions occur in the chloroplasts?
Would you consider an isolated chloroplast a living unit? Because the
mesophyll contains so many chloroplasts, it is the primary photosynthetic
tissue of the plant. It has two distinct regions: palisade mesophyll,
cylindrical cells located nearest the upper epidermis; and spongy
mesophyll, a region of irregularly shaped cells beneath the palisade layer,
containing many intercellular air spaces.
d)

The lower epidermis. Do these cells contain chloroplasts? Locate very

small cells called guard cells that surround stomata (pores) leading to
intercellular spaces in the mesophyll. Are there chloroplasts in the guard
cells? How do you think guard cells function?

3. Plants that grow in temperate climates have leaves that are structurally similar
to Ligustrum or the dicot that you examined above. These types of plants are
called C3 plants because of the nature of their biochemical pathway. In these

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plants, carbon fixation takes place in the mesophyll cells and produces a three
carbon compound, G3P.
4. Compare the structure of your leaf with that of the demonstration model.
Identify each of the model's components and know at least one function of
each.
5. Plants that survive in drier climates and carry on photosynthesis under intense
heat and low CO2 levels have special adaptations. These plants, such as corn,
sugar cane, and some grasses, are called C4 plants because their first product
after the addition of CO2 is a four carbon compound (oxaloacetate). Carbon
fixation occurs in the bundle-sheath cells that surround the vascular bundles
rather than in the mesophyll cells, as in C3 plants.
6 Obtain and examine a prepared slide of a corn (Zea mays) leaf [Figure 8.1
(B)]. Scan the slide under low and high power, and observe the vascular
bundles. Note that all the bundles are visible only in cross section because of
the parallel arrangement of veins in the leaf. How does the mesophyll layer
differ from that of the Ligustrum leaf? In corn, the mesophyll layer contains
enzymes needed to pump in CO2 under low carbon dioxide tension. Note that
the bundle-sheath cells in corn contain chloroplasts while those in Ligustrum
do not. Compare the size of the bundle-sheath cells in privet and corn. Why
would the size of these cells be important? What other structural adaptations
can you find in the corn cross section that would make it suitable for survival
in drier climates?
Answer the following questions at home:
1. (a) What is the function of the cuticle? _____________________________________
(b) Why is it thicker on the upper surface? __________________________________
2. How many cells thick is the upper epidermis? ________________________________
(a) Does this layer contain chloroplasts? __________________________________
3. Is an isolated chloroplast a living unit? ______
Explain:_________________________________________________________________
________________________________________________________________________
________________________________________________________________________
4. (a) Do cells of the lower epidermis contain chloroplasts? ________________________
(b) Are there chloroplasts in the guard cells? _________________________________
(c) How do you think guard cells function? ___________________________________

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______________________________________________________________________
5. How does the mesophyll layer of corn differ from that of the privet (Ligustrum)?
_______________________________________________________________________
6. Why would the size of the bundle sheath cell be important in corn?
_______________________________________________________________________
7. What other structural adaptations can you find in corn that would make it suitable for
survival in drier climates?
______________________________________________________________________
8. Using Figure 8.1 as a guide and label the diagram below:

Exercise 8.2 Extraction of Plant Pigments

A variety of pigments are found in plants. Anyone who has lived up North during
autumn's outburst of colors can recall. A pigment is a substance that absorbs light. If a
pigment absorbs all wavelengths of visible light it appears black. Other pigments absorb
some wavelengths and reflect others. Yellow pigments reflect light wavelengths in the
yellow portion of the visible light spectrum, green reflects in the green portion, etc. Some
colors are produced by only one pigment, but a great diversity of colors can be produced
by the cumulative effects of different pigments in cells. Green colors are produced by the
presence of chlorophylls a and b located in the chloroplasts. Yellow and orange colors
are produced by carotenoids and xanthophylls also in the chloroplasts. Blue, violet,
purple and dark red are usually produced by a group of water-soluble pigments, the
anthocyanins, that are located in the cell vacuoles and do not participate in
photosynthesis.
In plants, the chlorophylls are able to absorb the energy of sunlight and couple
this trapping process to the synthesis of carbohydrates. The other plant pigments
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mentioned above, absorb wavelengths of light different from those absorbed by the
chlorophylls; this energy is then transmitted to the chlorophylls. In order to separate the
variety of colored pigments in leaves, we must first extract the pigments by breaking
down the cell walls.
Materials:
Acetone, 5 mL
Petroleum ether, 5 mL
Distilled water, 5 mL
Test tube
Fine sand
Spinach or parsley leaves
Stopper for test tube
Glass rod
Test tube rack
Mortar and pestle
Procedure:
1. Go to the supply area and obtain 3-4 spinach (or parsley) leafs.
2. At your lab space, shred the spinach (or parsley) into small strips and place
them in the mortar.
..
3. Add a generous pinch of fine sand to the leaf shreds.
4. Grind the spinach until it is all pulp or a green paste.
5. Measure 4 mL of acetone into a 10 mL graduate cylinder and pour it down the
rod into the leaf pulp. Gently and carefully grind the pulp some more to
thoroughly mix the acetone.
6. Dispense 4 mL of distilled water into the 10 mL graduate cylinder and pour it
in the leaf pulp.
7. Carefully pour the mix with as little pulp as possible into the test tube.
8. Plug the tube with a stopper and shake it vigorously for 30 seconds.
9. Set the stoppered tube aside in the test tube rack for 3 minutes.
10. Add 4 mL of petroleum ether to the leaf suspension using the graduate
cylinder.
11. Stopper the tube and again shake vigorously for 30 seconds.
12. Set it in the test tube rack and watch as the solvents and pigments separate.
13. Leaf pigments are more soluble in non-polar (petroleum ether) solvents than
in polar (water-like) solvents such as acetone. The leaf pigments dissolved in
petroleum either floats on top of the water-acetone as a clear dark green layer.
14. The leaf pigments extracted here will next be separated from each other
because of their differential solubility by a technique known as
chromatography.

Exercise 8.3 Separation of Pigments by Thin-Layer

Chromatography
In this exercise, you will use commercially prepared strips, consisting of silica gel
on acetate sheet backing, to ensure uniformity of adsorbent thickness. There are several
advantages of thin-layer chromatography: first is the speed at which separation occurs;
secondly, it is especially useful when the compounds to be separated are very closely
related chemically and, therefore, difficult to separate.
In thin-layer chromatographic (TLC) separation, the compounds to be separated
are spotted along a line near one end of a plastic slide coated with an inert material such

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as silica. After the spot is allowed to dry, the same end of the slide is placed in a closed
chamber containing the chosen solvent. Chromatography takes advantage of subtle
differences in the relative solubility of various substances between a stationary phase and
a mobile phase. The greater the solubility of a given pigment in the mobile organic phase
relative to its solubility in the stationary phase (silica), the greater its tendency to remain
in the organic phase and the farther its ascent as the solvent rises by capillary action.
Thus, the different pigments in the original mixture will travel at different rates, and the
original spot will separate into a number of different bands. Each band will move a
certain fraction of the total distance traversed by the solvent front. This fraction is a
characteristic of the particular pigment making up the band and will always be the same
whenever this pigment is chromatographed in the same solvent system. If a different
pigment or solvent is used, the distance traveled by each compound would differ from
that in the original system. By careful choice of mobile solvent and supportive medium, it
is possible to separate the components of almost any mixture of compounds.
Materials Needed:
TLC strip, 1 x 3 inch
Chlorophyll extract
Capillary pipette
Coplin jar (chromatography chamber)
Procedure:
1. Obtain, from the supply area or your tray, a 1 x 3 thin layer chromatography
(TLC) strip. Handle it by the edges only and note its two sides: one shiny and the
other chalky white.
2. Make a small pencil dot (not ink) on the white chalky side 2 cm from the end and
halfway between the side edges.
3. Using the capillary pipette draw up a small amount of the clear green liquid from
the top layer in the grinding tube. CAUTION: you will only get one TLC strip so
don't ruin it by dripping or scratching!
4. Holding the pipette vertically, touch the tip to the pigment spot and lightly press
the pipette (not the bulb). Blow on the pigment spot as it forms to evaporate the
solvent.
5. Repeat this operation as often as necessary (at least 5 times) to produce a dark
green spot, allowing the pigment to dry between applications. Note the green
center of the spot and the orange-yellow ring around it. Blow the spot completely
dry.
6. With a paper towel, thoroughly dry the inside of the Coplin (TLC) jar. Blow it dry
also.
7. Measure the running solvent by dispensing 1 mL of acetone into the 10 mL
graduate cylinder and then adding 3 mL of petroleum ether to it up to the 4 mL
line. Without wetting the inside walls of the jar, empty the running solvent onto
the bottom of the TLC jar.
8. Carefully lower the TLC strip (pigment spot down) between the glass ribs inside
the TLC jar until it is standing in the solvent on the bottom. Replace the lid, note
the time, and do not move the jar during the run (see Figure 8.3).
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Figure 8.3:
Thin-layer
9. Observe through the rib-less
sidewal1
of thechromatography.
jar the rapid separation of the leaf
(A)
Application
of
pigment.
(B)
Chromatography
chamber.
pigments, occurring in a matter of minutes.
10. Note that the first color band moving up is orange-yellow. It consists of the
carotenes (pro-vitamin A), further down the blue-green of chlorophyll a, after
which follows the yellow-green of chlorophyll b, and finally the several bright
yellow bands of the xanthophylls. (The grey layer between carotene and chl. a is pheophytin, a
chlorophyll molecule lacking the central Mg2+ ion.)
11. When the uppermost orange-yellow band is within a centimeter of the top (about
7 to 10 minutes), lift the strip out, replace the lid, and blow the strip dry. Observe
how rapidly the fresh colors fade.
Answer the following questions:
A. How many different pigments were there in the extract?____________________
B. On the basis of color, try to identify them:

________________________________________________________
________________________________________________________
C. Remove the chromatogram, allow it to dry, and then attach to this page.

Exercise 8.4 Paper Chromatography of Photosynthetic

Pigments
Light must be absorbed before its energy can be utilized. A substance that absorbs
light is a pigment. The primary photosynthetic pigments that absorb light for
photosynthesis are chlorophylls a and b. However, chlorophyll is not the only
photosynthetic pigment; accessory pigments, such as carotenes and xanthophylls,
absorb light and transfer it to chlorophyll a. The light energy absorbed by accessory
pigments is transmitted to chlorophyll.
Paper chromatography is a technique used for separating dissolved compounds
such as plant pigments. When a solution of these pigments is applied to strips of paper,
the pigments adsorb onto the cellulose fiber matrix of the paper. When the tip of the paper
is immersed in a solvent, the solvent is absorbed and moves through the paper. As the
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solvent moves through the spot where the mixture of pigments was applied, the pigments
dissolve in the moving solvent - some pigments move almost as fast as the solvent, while
others move more slowly. This differential movement of pigments is the result of each
pigment having a characteristic tendency to stick to the cellulose fibers of the paper. A
pigment's molecular size, polarity, and solubility determine the strength of this
tendency. Pigments adsorbed strongest move slowly, while those weakly adsorbed move
fastest. Thus, each pigment has a characteristic rate of movement, and the pigments can
be separated from each other. Four bands of color will appear on the strip - a yellow band
of carotenes, a yellow band of xanthophylls, a blue-green band of chlorophyll a, and a
yellow-green band of chlorophyll b.
The relationship of the distance moved by the pigment to the distance moved by
the solvent front is specific for a given set of conditions. We call this relationship the Rf,
and define it as follows:
Rf =

Distance moved by pigment

Distance from pigment origin to solvent front

Thus paper chromatography can be used to identify each pigment by its

characteristic Rf. This Rf value is constant for a given substance in a particular solventmatrix mixture.
Materials Needed:
Spinach leaf
Filter paper strip and scissors
Test tube with stopper and
pin
Millimeter ruler

Solvent (9 parts petroleum

ether: 1 part acetone)
Test tube rack
Coin

Procedure:
1.
Obtain a strip of chromatography paper from the supply area. Handle the paper by
its edges so that oil on your fingers does not contaminate the paper. Make a faint
pencil-line across the paper approximately 2 cm from the tip of the paper (Figure
8.4).
2.

Now place a spinach leaf on top of the chromatography paper. Roll a coin several
times on top of the leaf across the pencil line in your chromatography paper. You
need a concentrated green line.

3.

Place the chromatography strip in a test tube containing 5 mL of chromatography

solvent (9 parts petroleum ether: 1 part acetone). Position the chromatography
strip so that the tip of the strip (but not the stripe of plant extract) is submerged in
the solvent. You can do this by hooking the strip of paper with a pin inserted in
the tube's stopper (Figure 8.4). CAUTION: This solvent is highly flammable, so
extinguish all flames, hotplates, etc. before working with this solvent!

4.

Place the tube in a test-tube rack and watch as the solvent moves up the paper.

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5.

Remove your chromatography strip before the solvent reaches the top of the strip
(about 5 -10 minutes). Mark the position of the solvent front with a pencil, and set
the strip aside to dry. Observe the bands of color and draw your results.
****Repeat steps 1 5 several (4 to 5) times in preparation for the next
exercise. The more of these strips that are available the more intense the
colors will be for exercise 8.5. ******

6.

For one of these strips, measure the distance from the origin to the solvent front
and from the origin to each pigment band. Calculate the Rf value for each
pigment; enter results in the table provided below.
hook
cork

Cut paper
to
approximately
this length

Chromatogram
solvent

Fig. 8.4: Setup for Paper Chromatography

1. Make a diagram of your chromatogram in the diagram provided on the right. Identify
and label the color of each pigment band.
2. Complete Table 8.1 below:
Table 8.1: Rf Values for Pigments found in Chloroplasts
Pigment Band Distance from
Rf Value
Origin
Carotene
Xanthophyll
Chlorophyll a
Chlorophyll b
3. What does a small Rf value tell you about the characteristics of the
moving molecules?

_____________________________________________

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_____________________________________________
_____________________________________________
4. Which are more soluble in the chromatography solvent: xanthophylls
or chlorophyll a?

_____________________________________________
_____________________________________________
_____________________________________________

5. Would you expect the Rf value of a pigment to change if we altered the composition of
the solvent? Why or why not?

________________________________________________________
________________________________________________________
________________________________________________________
6. If yellow xanthophylls were present in the extract, why did the extract appear green?

________________________________________________________
7. Is it possible to have an Rf value greater than 1? Why or why not?

________________________________________________________
________________________________________________________
________________________________________________________
________________________________________________________

Exercise 8.5 Determining the Absorption Spectrum for Leaf

Pigments
In Exercises 8.3 and 8.4, you used chromatography to isolate and separate
pigments that were involved in photosynthesis.
Which of these pigments do you think support photosynthesis? ________
In this exercise, you will work in teams of 4 students to carry the previous
investigation a step further and plot the absorption spectrum of leaf pigments that you
separated by paper chromatography. The absorption spectrum is the absorption pattern for
a particular pigment, showing relative absorbance at different wavelengths of light. For
example, we know that chlorophyll a is a green pigment, and thus reflects or transmits
green wavelengths of light. However, we dont know the relative proportions of
wavelengths of light absorbed by chlorophyll a. This information is of interest because it
suggests that those wavelengths showing greatest absorbance are important in
photosynthesis.

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The absorption spectrum can be determined with an instrument called a
spectrophotometer. A spectrophotometer measures the proportions of light of different
wavelengths absorbed and transmitted by a pigment solution. It does this by passing a
beam of light of a particular wavelength through the pigment solution being tested. The
spectrophotometer then measures the proportion of light transmitted or absorbed by that
particular pigment and shows the reading on the calibrated scale.
Before measuring the absorption spectrum of the four pigments separated by
paper chromatography, consult the diagram of the electromagnetic spectrum in your text
and predict the wavelengths of light at which absorption will be greatest for each
pigment. Record your predictions below in Table 8.2.

Table 8.2: Predicted Wavelengths of Greatest Absorption for Photosynthetic Pigments

Pigment

Wavelength of Greatest
Absorption in nm (predicted)

Chlorophyll a
Chlorophyll b
Carotene
Xanthophylls
Materials Needed:
Spectrophotometer
2 small test tubes or cuvettes
Kimwipes
150-mL beaker to hold test tubes
Acetone
20-mL beaker to elute pigments
5 spec 20 tubes labeled C, X, A, B, T (for carotene, xanthophylls,
chlorophyll a , and chlorophyll b, total pigment) filled with acetone.
Procedure:
1. Cut out the pigments you separated by paper chromatography in Exercise 8.4
and placed them in the tubes at the instructors table as follows:
Tube labeled C -- carotene
Tube labeled X -- xanthophylls
Tube labeled A -- chlorophyll a
Tube labeled B -- chlorophyll b
Tube labeled T -- total pigment solution
2. The instructor will decide when the tubes have enough pigments dissolved in
each tube. Following this, measure the absorption spectrum and record your
measurements in the space provided.

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3. The instructions that follow are for the spectrophotometer present in lab. The
machine should have been turned on prior to class by your instructor.
Rearrange the tubes to A, B, X, C, & T to agree with the column order on
Table 8.3 (next page).
a. Select the beginning wavelength using the up and down arrows. Begin
your measurements at 400 nm. Insert the tube labeled blank from the
supply counter into sample holder and close the lid.
b. Zero the instrument by pressing 0 Abs/100% T. You are now ready
to make your first reading.
c. To begin your readings, remove the blank tube and insert tube of
solution A. Close the cover and record the reading of absorbance at
this wavelength. Remove tube of solution A, and read tubes B, X, C,
& T accordingly.
d. Insert the blank tube and set the wavelength to 420 nm (use the up and
down arrows). Again, zero the instrument by pressing 0 Abs/100%
T.
e. Remove the blank and insert solution A and take your second reading
at 420 nm. Repeat for B, X, C, & T as in step c above.
f. Continue to make readings by increasing the wavelength by 20-nm
increments, inserting the blank and zeroing every time the wavelength
is changed, until you reach 720 nm.
4.
Record the class data in the chart provided below.
Table 8.3: Absorbance of Photosynthetic Pigments

Wavelength
(nm)
400

Chlorophyll a

Chlorophyll b

Xanthophylls

Carotene

Total Pigment

420
440
460
480
500
520
540
560
580
600
620
640

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660
680
700
720
5. Using the readings recorded in Table 8.3, plot the absorption spectrum for
each pigment in the graph below. Choose appropriate scales for the axes,
determine dependent and independent variables and plot data points. Draw
smooth curves to fit the values plotted. Prepare a legend and use colored
pencils to identify each of the four pigments. (Sample data from previous labs
can be seen in Appendix 8.A-8.D)

6. Which pigment is most important in the process of photosynthesis? ______________

Support your answer with evidence from your results:

_____________________________________________________________
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_____________________________________________________________
_____________________________________________________________
_____________________________________________________________
7. Chlorophyll b and the carotenoids are called accessory pigments. Using data from
your results, speculate about the role of these pigments in photosynthesis:

_____________________________________________________________
_____________________________________________________________
_____________________________________________________________
_____________________________________________________________
_____________________________________________________________

Exercise 8.6: The Necessity of Carbon Dioxide in Photosynthesis

Carbon dioxide (CO2) is essential for photosynthesis. It is required in the dark
reactions or Calvin-Benson cycle that occurs in the stroma of the chloroplasts. In this
experiment you will test the necessity of CO2 in photosynthesis using Elodea and an
indicator for the presence of CO2.
Materials:

3 test tubes
Soda straw
Test tube rack
150-watt flood lamp with
ring stand
Bromothymol blue

Previously heated water

Elodea sprig
Procedure:
1. Pour the previously heated (to remove any air present) water into a
small beaker. Place 10 drops of bromothymol blue (pH range of 6.2
7.6) into the beaker. Decant about a third of the colored water into a test
tube labeled #3. Blow air with the straw through the remaining water in
the beaker until it turns yellow. Pour an equal volume of this solution
into two test tubes. Label these test tubes as 1, and 2. (Bromothymol blue
is an indicator here for the presence of CO2. Bromothymol blue turns yellow in an
acid such as carbonic acid (H2CO3) formed when CO2 combines with water. When no
CO2 is present, the indicator is blue.)

2. Place a sprig of Elodea in tube #1.

3. Attach a 150-watt flood lamp to the support stand so that the lamp is
approximately 25 cm from the base of the stand. Place all three tubes
under bright light for 30 minutes.
4. After 30 minutes, record your data and answer the questions below.
Complete Table 8.4
Table 8.4: Color changes before and after exposure to light

Tube
Tube #1
Tube #2
Tube #3

Color before exposure to light

Color after exposure to light

1. Have any color changes taken place? Which? _____________________________

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2. Has the control tube #3 changed color? __________________________________
3. Has tube #2 changed color? _________________________________________
a. What does this indicate? _______________________________________
4. Has tube #1 with the plant changed color? _______________________________
5. Is there any CO2 in the liquid of tube #1 after exposure to light? ______________
6. What can you conclude happened to the CO2?

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Exercise 8.7: Measuring Photosynthetic Rate in Spinach Leaf.

In the light-dependent reactions of photosynthesis, light energy is used to split
water molecules. Some of this energy is captured by adding phosphate (Pi) to ADP to
make ATP and by adding hydrogen to NADP+. O2 is released as a by-product. The
rate of these reactions can be estimated by measuring O2 production in disks cut from
spinach leaves.
Leaf tissue is riddled with gas-filled intercellular spaces (Fig.8.5), so they float
when they are placed in solution. But when leaf disks in a solution are subject to a
vacuum, the gases in the leaves are pulled out and the spaces are filed by the liquid.
Since fluids are heavier than gas, replacing the gas causes the leaf disks to sink to the
bottom of the flask. Then as O2 is produced by the light reactions of photosynthesis, it
diffuses into the intercellular spaces and replaces the liquid with gas. When enough
O2 has accumulated, each leaf disk will regain its buoyancy and float to the surface .
Your team will use this technique as a method of measuring photosynthesis. Sodium
bicarbonate (NaHCO3), which supplies CO2 for photosynthesis, will be the solution
used to infiltrate the leaf disks.

Fig. 8.5 Leaf disk method for measuring the rate of photosynthesis.
Materials:

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Spinach leaves
Cork borer (#4)
250 mL Erlenmeyer flask
3 Petri plates per team with one pair of forceps
Bottle of 0.2% (or higher) NaHCO3 solution
Vacuum-flask setup

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150-Watt flood lamp with ring-stand.
Procedure:
1. Work in teams of 4 students. Attach a 150-watt flood lamp to the support stand so
that the lamp is approximately 25 cm from the base of the stand.
2. Fill a large beaker (1 -2 L) with cold water to act as a heat filter for the dish you will
place under the lamp. The lamp can produce a lot of heat, which may affect the rate of
photosynthesis. Set the beaker aside for the moment.
3. Pour 0.2% NaHCO3 solution into 3 Petri plates so that they are about 2/3 full. Pour
approximately 100 mL of 0.2% NaHCO3 solution into a 250 mL flask.
4. Get several spinach leaves (3 4 stacked together) and cut 40 50 disks with a cork
borer or other circular cutting instrument. Do not include the large veins. Use a paper
towel or Styrofoam board to cut on. As the disks are cut out, put them in the flask
with the bicarbonate solution.
5. Use a water aspirator or the fume hood aspirator, as directed by your instructor, to
sink the discs.
a) Attach the vacuum tubing to the sidearm of the flask. Put a rubber stopper
firmly in the mouth of the flask and press a piece of tape securely over the
hole in the stopper if it has one. Hold the flask steady while you apply the
vacuum.
b) Turn on the water faucet (or fume hood vacuum control) all the way. After
several seconds, you should see bubbles coming out of the edges of the
disks. Leave the disks under the vacuum for approximately 15 20
seconds, and then release the vacuum by peeling back the tape or by
turning off the vacuum control.
c) Swirl the flask and wait to see whether the disks sink. Remember, the
leaves will continue to float as long as they are under a vacuum.
d) You may have to apply the vacuum two or three times. It is not necessary
to sink all the disks. You will damage the spinach tissue if you
overaspirate. Keep the disks away from bright light by immediately
placing the flask in the desk drawer for a few minutes!!!! After a few
minutes check to see if the majority of the disks have sunk.
6.
Pour the contents of the flask into a large dish. Discard any disks that are
floating. Use forceps to carefully transfer 10 -15 disks to each Petri Plate. Put
lids on the Petri plates. Put one plate in a dark place such as a drawer; put
another one under the flood lamp. Set the larger beaker of water on top of the
Petri plate; then turn on the lamp. Place the third Petri plate on the lab bench
where it will receive only room light.
7.
Wait 20 minutes for photosynthesis to occur. While the experiment is running,
go on to the next exercise.
8.
After 20 minutes, count the number of disks that are either floating or turned on
edge in each Petri plate. Record your data in table 8.5 and answer the questions.
Table 8.5: Results of Subjecting Spinach Leaf Disks to Different Light Conditions

Light

# disks floating

% disks floating

Dark
Room Light

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Under Lamp
1.
What hypothesis is being tested with this experiment? _______________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
2.

What is the
a) independent variable in this experiment? ______________________________
b) dependent variable in this experiment? ________________________________

3.

What is the control treatment? __________________________________________

Exercise 8.8 Pigments in Photosynthesis.

A variety of pigments besides the chlorophylls are found in plants. A pigment is a
substance that absorbs light. If a pigment absorbs all wavelengths of visible light, it
appears black. Yellow pigments, for example, reflect light wavelengths in the yellow
portion of the visible light spectrum, green reflects in the green portion, etc.
Some colors are produced by only one pigment, but an even greater diversity of colors
can be produced by the cumulative effects of different pigments in cells. Yellow, orange,
and bright red colors are produced by carotenoids, also found in chloroplasts. Blue,
violets, purples and dark reds are usually produced by a group of water-soluble pigments,
the anthocyanins, that are located in cell vacuoles and do not contribute to
photosynthesis. Additional colors may be produced by mixtures of these pigments in
cells.
Working in teams of 4, you will use the I2KI test for starch to determine which pigment(s)
in Coleus leaf support photosynthesis. Before beginning the experiment, examine your
Coleus leaf and hypothesize about the location of photosynthesis based on leaf colors.
Hypothesis:

Materials:
o
o
o
o
o
o
o

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Coleus plant with multicolored leaves.

1 1000-mL beaker with 300 mL of water
1 400-mL beaker with 200 mL of Ethanol
Dropper bottle of I2KI solution
Petri plates
Scissors
Plastic wash bottled water

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Procedure:
1. Remove a multicolored leaf from a Coleus that has been in strong light for several
hours.
2. In Table 8.6, list the colors of your leaf, predict the pigments present to create that
color and predict the results of the I2KI starch test in each of the leaf.
3. Sketch the leaf outline in the next page, mapping the color distribution before the I2KI
test.
4. Set up the boiling alcohol bath. Place a 1000-mL beaker with 300 mL of water on the
hot plate. Carefully place the 400 mL beaker containing 200 mL of 80% alcohol
(ethanol)into the larger beaker of water. Turn on the hot plate and bring both beakers
to a boil. Adjust the temperature to maintain slow boiling. Caution!! Ethanol is
highly flammable! Do not place the beaker of alcohol directly on the hot plate!
5. Carefully drop the leaf, using forceps, into the boiling alcohol solution to extract the
pigments.
6. When the leaves are almost white, use forceps to remove them from the alcohol, and
place them in separate Petri plates. Rinse each leaf with distilled water and add
enough distilled water to just cover the leaf. Turn off the hot plates if all teams are
finished with boiling their leaves.
7. Add a few drops of I2KI solution to the water until a pale amber color is obtained.
I2KI reacts with starch to produce a blue-black color.
8. Wait about 5 minutes and sketch each leaf in the next page, showing which areas of
the leaf tested positive for starch.
A. Record the results of the I2KI test in Table 8.6.
Table 8.6:Pigments in photosynthesis

Color

Pigments

Predicted Presence
of Starch
(+ or -)

Actual results of
Starch Presence
(+ or -)

Green
Purple
Pink
White

B.

Which pigments supported photosynthesis?

Record your results in the Actuals column of Table 8.6

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C. Sketch the Coleus leaf as instructed in steps 3 and 8 of the procedure.
Before the I2KI Test

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After the I2KI Test

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N.B. The following section is done in preparation for lab 10 only if the
Virtual Fly Lab computer simulation will not be used. Even so, you
are advised to read the section to familiarize yourself with what is
involved when dealing with the life specimen.

Exercise 8.9 Preparation for Lab #10

Drosophila occurs naturally on rotting fruits; both the adults and larvae feed on
plant sugars and on the wild yeasts that grow in rotting fruit. The female lays eggs on this
fruit. Drosophila melanogaster is an insect that has a complete metamorphosis. This
means that it goes through 4 readily identifiable stages in its life cycle (see Figure 8.6).
The adult female fly lays its eggs in the soft medium. The eggs are white, very small, and
have two prong-like projections extending from one end. In about 2 days the eggs hatch
and the larvae appear as wormlike creatures that work their way through the medium
eating constantly. During this stage they molt twice. There are, therefore, 3 larval stages
separated by 2 molts; these periods of larval growth are called instars. The total larval
period lasts about 8 days. The larvae finally move onto the side of the vial and form
pupae (the pupa is immobile, non-feeding and protected by a case). During the pupal
stage of development the larval tissues reorganize to form adult flies. The pupal cases
darken as they mature, and in about 4 days, the adults hatch. Most of the flies hatch
during the early morning (approximately 6 A.M.) and are capable of mating within 8-10
hours. Thus any female left in a vial of hatching flies longer that this period of time will
probably have mated. In order to obtain virgin female flies, you must remove the females
and isolate them in a new vial of medium within 8-10 hours.

Figure 8.6: Life cycle of Drosophila. Letters a, b, and c indicate features found in the
adult male only.

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Materials:
o Instant Drosophila
medium and water
o Culture vials and plugs
o Dissecting microscope
o Flynap

Drosophila melanogaster
cultures: dihybrid flies, both
male and female
o
3 x 5 white card
o
Small paint brush
o
Fly morgue
o

Procedure:
1. Preparing the fly medium: Obtain a vial and add one scoop of fresh fruit fly
medium and add 15 mL of distilled water. Let dry before you add the flies.
2. Performing your cross: Obtain 3 male and 3 female flies from the F1 generation
that have been anesthetized with Flynap. Male flies can be differentiated from
females by their smaller size, wider abdominal bands, and blunt abdomen (see
Figure 8.7).
3. Place your flies in a vial (on its side) containing fresh medium and close with the
foam and lid provided in the supply counter. This is the F1 cross and will produce
the F2 generation. The F1 generation is heterozygous for two traits: wing shape
and eye color (SsVv x SsVv). KEEP VIAL ON ITS SIDE UNTIL ALL FLIES
ARE AWAKE OR THEY WILL GET STUCK IN THE MEDIA AND DIE.
4. Label the vial with the following information: cross, date, your name, lab section.
Your cultures should be grown at home at 20 22 C. Do not leave in your car.
5. Release the parents after 4 5 days, when larvae are seen in the medium.
6. Bring back to lab two weeks from today (i.e. the day you crossed them in lab).

Figure 8.7: Male and Female fruit flies. Compare the size, shape of the abdomen, and the strip pattern of
the male and female flies.
Acknowledgments:
Special thanks are given to the late Professor Bernard Fritze for allowing us to include the
extraction of chlorophyll techniques, Exercise 8.2, and the Thin Layer Chromatography technique used in
Exercise 8.3.
References:

Dickey, Jean. 1995. Laboratory Investigations for Biology. Benjamin/Cummings

Publishing. (Exercise 8.7)
Helms, D. R. 1994. Biology in the Laboratory. 2nd ed. Worth Publishers. (Exercise 8.5)
Morgan, J. D., and Carter, M. E. 1993. Investigating Biology. Benjamin/Cummings
Publishing. (Exercise 8.6)
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Appendix 8.A
Data from a previous lab.
Absorbance of Photosynthetic Pigments
Wavelength
(nm)

Chlorophyll a

Chlorophyll b

Xanthophylls

Carotene

Total
Pigment

400

.148

.150

.013

.039

.049

420

.211

.218

.106

.064

.7

440

.184

.225

.107

.082

.6

460

.092

.185

.071

.084

.35

480

.061

.081

.070

.077

.15

500

.013

.018

.079

.033

.03

520

.009

.013

.009

.008

.03

540

.013

.014

.006

.005

.03

560

.013

.016

.005

.000

.18

580

.018

.024

.005

.002

.16

600

.022

.028

.007

.000

.09

620

.031

.037

.008

.002

.11

640

.037

.057

.009

.001

.15

660

.136

.137

.032

.003

.35

680

.029

.026

.007

.000

.17

700

.003

.002

.002

.001

.10

720

.002

.001

.002

.001

.15

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Appendix 8.B
Data from a previous lab:
Absorbance of Photosynthetic Pigments
Wavelength
(nm)

Chlorophyll a

Chlorophyll b

Xanthophylls

Carotene

400

.082

.103

.042

.013

420

.096

.103

.028

.012

.068

440

.082

.117

.024

.010

.076

460

.042

.097

.026

.014

.056

480

.022

.044

.031

.019

.039

500

.012

.017

.016

.010

.020

520

.014

.006

.002

.003

.005

540

.009

.006

.10

.000

.002

560

.010

.009

.009

.007

.011

580

.009

.009

.003

.000

.007

600

.007

.007

.003

.007

.004

620

.017

.016

.004

.006

.012

640

.018

.022

.002

.000

.012

660

.064

.057

.000

.008

.034

680

.018

.017

.004

.001

.012

700

.000

.000

.002

.002

.000

720

.005

.001

.002

.000

.003

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Total
Pigment

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Appendix 8.C
Data from a previous lab:
Absorbance of Photosynthetic Pigments
Wavelength
(nm)

Chlorophyll a

Chlorophyll b

Xanthophylls

Carotene

Total
Pigment

400

.257

.244

.122

.063

.231

420

.360

.344

.178

.104

.339

440

.310

.351

.184

.132

.355

460

.115

.293

.126

.140

.027

480

.085

.120

.115

.124

.153

500

.019

.027

.028

.057

.039

520

.018

.022

.010

.011

.016

540

.019

.025

.008

.002

.016

560

.023

.029

.008

.003

.020

580

.036

.043

.011

.004

.033

600

.040

.048

.011

.003

.036

620

.058

.060

.014

.002

.049

640

.064

.094

.016

.003

.066

660

.239

.220

.052

.004

.199

680

.054

.046

.014

.002

.040

700

.005

.004

.002

.001

.002

720

.002

.002

.002

.001

.001

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Appendix 8.D
Data from a previous lab:
Absorbance of Photosynthetic Pigments
Wavelength
(nm)

Chlorophyll a

Chlorophyll b

Xanthophylls

Carotene

Total
Pigment

400

.27

.12

.68

.06

.49

420

.36

.17

1.4

.08

.70

440

.34

.20

.6

.09

.60

460

.25

.17

.38

.08

.35

480

.13

.05

.12

.05

.15

500

.05

.01

.05

.02

.03

520

.03

.01

.04

.01

.03

540

.02

.01

.02

.01

.03

560

.03

.02

.04

.01

.18

580

.04

.02

.06

.01

.16

600

.04

.01

.06

.01

.09

620

.05

.01

.09

.01

.11

640

.06

.05

.16

.01

.15

660

.13

.06

.35

.01

.35

680

.06

.01

.17

.02

.17

700

.30

.02

.09

.02

.10

720

.10

.04

.15

.04

.15

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REPORT Lab #8
Date: ___________________

Name: __________________________

Photosynthesis
Exercise 8.1 Structure of a Leaf
1. (a) What is the function of the cuticle? _____________________________________
(b) Why is it thicker on the upper surface? __________________________________
2. How many cells thick is the upper epidermis? ________________________________
(a) Does this layer contain chloroplasts? __________________________________
3. (a) What photosynthetic reaction occurs in the chloroplasts? _____________________
(b) Is an isolated chloroplast a living unit? ______ Explain: _____________________
______________________________________________________________________
4. (a) Do cells of the lower epidermis contain chloroplasts? ________________________
(b) Are there chloroplasts in the guard cells? _________________________________
(c) How do you think guard cells function? ___________________________________
______________________________________________________________________
5. How does the mesophyll layer of corn differ from that of the privet (Ligustrum)?
_______________________________________________________________________
6. Why would the size of the bundle sheath cell be important in corn?
_______________________________________________________________________
7. What other structural adaptations can you find in corn that would make it suitable for survival in drier
climates? ______________________________________________________________________
8. Using Figure 8.1 as a guide and label the diagram below:

Exercise 8.3 : Separation of Pigments by Thin-Layer

Chromatography
1. How many different pigments were there in the extract?_________________________
2. On the basis of color, try to identify them:
________________________________________________________
________________________________________________________

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3. Remove the chromatogram, allow it to dry, and then attach to Lab Report sheet on the right.

Exercise 8.4 : Separation of Pigments by Paper Chromatography

1. Make a diagram of your chromatogram in the diagram provided on the bottom right.
the color of each pigment band.
2. Complete Table 8.2 below:
Table 8.2: Rf Values for Pigments found in Chloroplasts
Pigment Band
Distance from Origin
Carotene
Xanthophyll
Chlorophyll a
Chlorophyll b

Identify and label

Rf Value

3. What does a small Rf value tell you about the characteristics of the moving molecules?

4. Which are more soluble in the chromatography solvent: xanthophylls or chlorophyll a?

5. Would you expect the Rf value of a pigment to change if we altered the composition of the solvent? Why
or why not?

6. If yellow xanthophylls were present in the extract, why did the extract appear green?

7. Is it possible to have an Rf value greater than 1? Why or why not?

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Exercise 8.5 Determining the Absorption Spectrum for Leaf

Pigments
1. Complete Table 8.3:
Table 8.3: Absorbance of Photosynthetic Pigments
Wavelength
(nm)

Chlorophyll a

Chlorophyll b

Xanthophylls

Carotene

Total Pigment

400
420
440
460
480
500
520
540
560
580
600
620
640
660
680
700
720

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2.

Using the readings recorded in Table 8.3, plot in the graph below the absorption spectrum for each
pigment.

3. Which pigment is most important in the process of photosynthesis? ______________

Support your answer with evidence from your results:

________________________________________________________
________________________________________________________
4. Chlorophyll b and the carotenoids are called accessory pigments. Using data from your results,
speculate about the role of these pigments in photosynthesis:

________________________________________________________
________________________________________________________
________________________________________________________

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Exercise 8.6 The Necessity of Carbon Dioxide in Photosynthesis
1.

Complete Table 8.5

Table 8.5: Color changes before and after exposure to light

Tube
Tube #1
Tube #2
Tube #3

Color before exposure to light

Color after exposure to light

2.

Have any color changes taken place? Which? _____________________________

3.

Has the control tube #3 changed color? __________________________________

4.

Has tube #2 changed color? _________________________________________

a.

What does this indicate? _______________________________________

5.

Has tube #1 with the plant changed color? _______________________________

6.

Is there any CO2 in the liquid of tube #1 after exposure to light? ______________

7.

What can you conclude happened to the CO2?

________________________________________________________
________________________________________________________

Exercise 8.7 Measuring Photosynthetic Rate in Spinach Leaf

Disks.
Table 8.6: Results of Subjecting Spinach Leaf Disks to Different Light Conditions
Light

# disks floating

% disks floating

Dark
Room Light
Under Lamp
1.

What hypothesis is being tested with this experiment? _______________________

________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
________________________________________________________________________
2.

What is the
a) independent variable in this experiment? ______________________________
b) dependent variable in this experiment? ________________________________

3.

What is the control treatment? __________________________________________

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Exercise 8.8 Pigments in Photosynthesis
1. Record the results of the I2KI test in Table 8.7.

Color

Pigments

Predicted Presence
of Starch
(+ or -)

Actual results of
Starch Presence
(+ or -)

Green
Purple
Pink
White

2. Sketch the Coleus leaf as instructed in steps 3 and 5 of the procedure.

Before the I2KI Test

3.

After the I2KI Test

Which pigments supported photosynthesis?

Record your results in the Actuals column of Table 8.7

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