Activity I- Plant Pigment Chromatography
Part I.
In this part, students will utilize paper chromatography to separate plant
pigments. They will then calculate Rf values for each plant pigment.
Background Information:
The plant life around us is full of wonderfully rich and vibrant colors. From
red to violet, the entire color spectrum can be found in plant materials. These
colors come from a surprisingly small number of plant pigments. The three
primary groups of plant pigments are anthocyanins, carotenoids, and chlorophyll.
Each of these absorbs light at different parts of the visible light spectrum and has
a different function in the plant.
Anthocyanins attract insects for pollination by absorbing light in the UV
spectrum, serve as a sunscreen to protect plants from sun damage, and provide
protection against predators because of their foul taste.
Carotenoids and chlorophyll both serve as light-harvesting pigments in the
photosynthetic processes of plants.
It is possible to separate these pigments from one another by the use of
paper chromatography. In this process, plant tissue is applied to filter paper and
an organic solvent is allowed to move up the filter paper. Some pigments have a
higher affinity for the solvent than others and move at different rates up the filter
paper. Because of these
differences, several color bands would be expected if there is more than one
pigment present. Based on the bands formed on the filter paper, the retention
factor, or Rf, value can be calculated for each pigment. This is done by dividing
the distance the pigment traveled by the distance the solvent traveled.
Eqn. 1
Rf = distance pigment traveled
distance solvent traveled
Materials:
chromatography chamber
chromatography paper
chromatography solvent
ruler
pencil
penny
leaf (variety)
scissors
2 10-ml syringes
Punch
Beakers
.1% sodium bicarbonate solution
Phenol Red
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Procedure:
1. Obtain all materials and bring them to your lab station.
2. Cut chromatography so it fits in your chromatography chamber. The lid
must close.
3. Make a light pencil line about 2.0 cm from the end of the paper you just
cut.
4. Use the coin to extract plant pigments from the
leaf. Take the leaf and place it over the pencil line
you have drawn. Roll the penny over the leaf at
the same position as the pencil line. You should
see plant pigment across the pencil line you have
drawn. Move the leaf around and repeat this step
8-10 times using different parts of the leaf.
5. Pour about 1.0 cm of solvent into the bottom of the chamber. Place the
chromatography paper in the solvent being careful not to let the solvent
touch the plant pigment line you have made.
6. When the solvent is about 1-2 cm from the top of the filter paper remove
the filter paper and make a pencil mark where the solvent stopped before
the solvent evaporated. Pay attention. . . the solvent moves fast!
7. Allow the paper to dry. Once the paper dries, draw a pencil line at the
bottom of each distinguishable pigment band on the filter paper.
Depending on the leaf you used, you should see 4-5 bands.
Example of filter paper for a green leaf
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Student Data and Calculations:
After the filter paper has dried, you are ready to begin calculating R f values
using Equation. 1. Using your ruler, fill in the following tables and calculate Rf
values for one of your pieces of filter paper.
Band Distances
Band
Distance (mm)
Band Color
1
2
3
4
5
Distance solvent moved in mm
_________________________
Rf values for pigments
Pigment
Carotene (yellow orange)
Rf
Xanthophylls (yellow)
Chlorophyll A (bright
green)
Chlorophyll B (olive or
yellow green)
Anthocyanin (red)
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Questions and Applications:
1. Did you find all five pigments in your plant sample? If not, suggest a
reason why the pigment may have been missing.
2. Compare your Rf values to the group next to you. Are your values close?
Calculate the percent difference between the two Rf values for each
pigment.
3. The accepted Rf values for each pigment in this solvent are as follows:
carotene - 0.98
chlorophyll a - 0.59
chlorophyll b - 0.42
xanthophylls – 0.67
Calculate the percent error using these values as the accepted value and
your values as the experimental value.
4. Why do you think some pigments moved farther than others?
5. Why is it important for plants to have more than one pigment?
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Activity 2- A Measure of Photosynthetic Rate In Spinach
Leaf Disks
In this technique, the intercellular spaces of spinach leaf disks are infiltrated with
a sodium bicarbonate solution, which causes them to sink in the solution. As
photosynthesis occurs, oxygen collects in the intercellular spaces and refloats
the leaf disks.
Procedure
1. Using a punch made from a small diameter
soda straw (or hand punch), cut 10 leaf
disks from young actively growing spinach
leaves by supporting the leaf with your
index finger while pressing and using a
twisting motion of the straw.
2. Remove the plunger from a clean 10-ml
syringe. Place the10 disks into the body of
the syringe. Be sure the leaf disks are near
the tip of the syringe as you re-insert the
plunger so as not to damage the disks.
3. Insert the tip of the syringe into a beaker of
0.1% sodium bicarbonate solution and
draw about 8 ml into the syringe. The leaf
disks should be floating at this time.
4. Hold the syringe tip upward and expel the
air by depressing the plunger carefully.
5. Seal the tip of the syringe using the index
finger of your left hand. Pull back on the
plunger, creating a partial vacuum within
the syringe. If you have a good seal, it
should be hard to pull on the plunger and
you should see bubbles coming from the
edge of the leaf disks.
6. Simultaneously, release your index finger
and the plunger. Some of the leaf disks
should start to sink. Tap the side of the
tube to dislodge bubbles on the edges of
the disks.
7. Repeat steps 5 and 6 until all disks sink.
Do not overdo these steps!! You have
been successful if the disks sink to the
bottom. Don't repeat "just to be sure" as it
is possible to damage the cells of the
leaves.
8. Carefully remove your disks and place them into a beak full of DI water.
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9. Set the beakers up in front of the flood light. Your teacher should have
divided you into two groups. One light with a high wattage and one with a
low wattage.
10. Record the number of disks that are floating for 10 minutes at one
minute intervals.
Time (minutes)
# of disks
1
2
3
4
5
6
7
8
9
10
11. What did the disks produce that caused them to float?
Activity 3-Do Plants Consume or Release CO2? Both?
1. Background
In this lab, you will use phenol red as an indicator to show whether CO2 is being
consumed or produced in a reaction.
It is well known that in the presence of light plants perform photosynthesis. It is
less well understood that at the same time plants are also performing cell
respiration. To demonstrate this we will determine whether CO2 is consumed or
produced as Elodea is placed in either a light or dark environment. The change in
CO2 will be detected by the pH indicator phenol red.
Think about the chemical equations for respiration and photosynthesis. Which
one releases CO2 causing an increase and which one uses up CO2 which will
cause a decrease?
Phenol red is yellow under acidic conditions (high H ion concentration), pink
under basic or alkaline conditions (low H ion concentration) and orange under
neutral conditions. A change in CO2 will cause a directly proportional change in
H ion.
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If the CO2 concentration decreases, the H ion concentration will also decrease
and the solution will change to pink, becoming basic.
If the CO2 concentration increases, the H ion concentration will also increase
and the solution will change to yellow, becoming acidic.
Neutral solutions of phenol red will be orange.
2. Preparation
Create a solution of phenol red by adding concentrated phenol red to about 100
ml of water. The phenol red may change color as a result of adding water
(depending on how acidic your tap water is). Your goal is to make your solution a
neutral orange color. You can do this by gently blowing into the solution with a
straw. Once you have the solution at an orange color, transfer it to 4 test tubes
(they should be filled about 2/3 full with your orange solution).
Place a cut piece of elodea (cut end up) into two of the four tubes. The other two
test tubes will not have elodea and will serve as controls. One set will be placed
in the light, and one set in the dark (see data table).
Aluminum foil can be used to seal the test tubes to be set in the dark. All test
tubes should be stoppered or covered with parafilm to minimize reactions with
the air.
3. Data - Record the colors of the solutions in the test tubes after 1-2 days.
Elodea + PR/ dark
Elodea + PR / light PR / dark (control)
4. Analysis: Describe what happened and why.
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PR / light (control)