Chloroplast Pigment Analysis Lab
Name ___________________________________________ Block _________ Date _____________________
When you look at chloroplasts under a microscope or examine a plant leaf, the only color which appears to be
present is a green pigment called chlorophyll. However, there are other pigments contained within a leaf cell.
Yellow and orange pigments, not normally seen, are usually present within the plastids.
Objectives: In this investigation you will:
1. Separate the pigments from one another by using a technique call CHROMOTOGRAPHY.
2. Identify the pigment by their colors and positions on the chromatogram.
3. Determine the Rf value of each pigment
Materials:
Filter Paper
Chromatography Solvent
Spinach Leaf
Quarter ($0.25)
Metric Ruler
Paper Clip
Pencil
Colored Pencils
Rubber Stopper
Graduated Cylinder
Scissors
Goggles
Background:
The leaves of most plant contain many different pigments, or light absorbing compounds. Chlorophyll, the
main pigment found in green plants, is needed for photosynthesis. The primary purpose of chlorophyll is to
capture light energy, which is converted to chemical energy during photosynthesis. Chlorophyll is the most
abundant and important photosynthetic plant pigment and exists in several forms, including chlorophyll a and
chlorophyll b. Both types of chlorophyll absorb blue and red light and reflect green light (that is why plants
appear green).
Chlorophyll often hides the other pigments present in leaves. In autumn chlorophyll breaks down, allowing
xanthophylls, which reflect yellow light, and carotene, which reflects orange light to show their colors. Other
pigments may also be present in leaves as well.
The individual pigments in a mixture of pigments from a leaf maybe separated by the technique of paper
chromatography. Chromatography means color writing. The separation takes place by absorption and
capillary action. The paper holds substances by absorption. Capillary action pulls the substances up the paper
at different rates based on the solubility of each substance. The more soluble pigments will travel the fastest.
Pigments are separated on the paper and show up as colored streaks. The pattern of separated components on
the paper is called a chromatogram.
The relative rate of pigment migration is the Rf value. The Rf value for each pigment can be determined from
the chromatogram. The Rf value is the ratio of the distance a pigment moved on the chromatogram compared
to the distance the solvent moved.
In this lab, you will separate the pigments found in a spinach leaf. It may take more than one try to get this
right, so do not be frustrated, and keep trying. Follow the directions carefully, as they are complicated and
some of the substances you are using are dangerous!
Procedure:
With the scissors, cut out a strip of filter paper from the center of the filter paper circle that is 1cm wide and 12
cm long.
1 cm
Notch (cut a
small triangle
into the side of
the filter paper)
12 cm
Figure 1
2. Cut the bottom of the strip of filter paper in to a point as shown above in Figure 1
3. Cut a notch on both sides of the strip as shown in Figure 1.
4. PUT ON YOUR GOGGLES! Add the chlorophyll pigment between the two notches on the filter paper in
the following manner:
a) To add chlorophyll, obtain a piece of spinach leaf and lay it across the top of your filter paper. Be careful
not to apply any pressure to the leaf, as any pressure will result in a trace amount of green pigment
b) Using the edge of a quarter, roll the quarter across the leaf between the notches on the paper (press hard
but not too hard, you do not want to tear the filter paper or the leaf). Allow the pigment time to dry. Repeat
rolling across the paper between the notches at least 4 times side to side allowing the pigment to dry. The
more pigment you add to the paper, the easier your chromatogram will be to see.
5. Once filter paper has had time to dry hook the straight edge of the filter paper to the paper
clip which is attached to the rubber stopper.
6. Take your graduated cylinder to the fume hood and carefully pour 15 ml of
chromatography solvent in to the graduated cylinder.
7. Carefully place the strip in to the graduated cylinder, making sure that the filter paper
does not touch the sides of the graduated cylinder.
8. The pointed end of the filter paper is to be placed in to the solvent. (You may have to
adjust the length of the filter paper by folding, cutting the flat edge of the strip of filter paper)
Do not let the solvent touch the pigment in between the notches or you will have to start
over again!!!
Place strip in grad cylinder, so that just the very tip of arrow is touching the solvent!
9. Watch the solvent rise up the paper, carrying the separating pigments as it goes. After 5 -7
minutes you will notice an obvious change in the paper. When the solvent reaches near the
top of the filter paper remove the paper, place it on a paper towel on the desk and let it dry. Immediately
place a pencil mark on the chromatogram at the highest point the solvent traveled to (the solvent will dry
and you will not be able to see this later – so you must mark it).
10. Observe the bands/circles of pigment. Identify the pigments by their colors: Carotene (orange)
Xanthophylls ( Yellow) Chlorophyll a ( Yellow - light green) Chlorophyll b (Blue - dark green). Using the
pencil, draw a circle around your orange pigment mark, then place a dot at the approximate center of this
circle. Do the same for each of the four pigments – make sure to distinguish between the two green
pigments. Use the drawing in Figure 2 and draw (in color) the approximate locations of the solvent and the
4 pigments.
11. Using your chromatography strip (the real one – not the one drawn here) measure in
millimeters from the tip of the paper to the highest point the solvent traveled up the strip(the
first pencil mark you made). Record Migration Distance of the Solvent in Table A below
12. Next measuring from the starting point of the pigment (at the notches) measure in
millimeters the distance to the center point (pencil dot) for each of the pigment band.
Record Migration Distance of each pigment in Table A below
Band Number
Pigment
Table A
Color
Migration
Distance (mm)
Rf Value
1 (Top)
2
3
4 Bottom
Solvent
N/A
N/A
N/A
Use the following formula to calculate the Rf Values for each pigment to one number after the decimal. Record
you data in Table A above
Rf=
13.
14.
Distance substance’s pigment traveled
Distance solvent traveled.
Dispose of the waste materials according to you teachers direction. Make sure to put the
chromatography solution in the USED CHROMATOGRAPHY SOLUTION container!
Clean up your work area and wash your hands before returning to your seat
Analysis:
Answer the following questions on a separate sheet of paper. It is not necessary to copy the questions. Answers
MUST be in complete sentences!
1. Describe the appearance of the filter paper at the conclusion of the experiment.
2. Is chlorophyll composed of one or several pigments? How could you tell?
3. What did we learn about pigments from performing the chromatography lab? Predict why plants would
have several pigments versus just one type.
Examine your chromatogram strip. Each color band is a different pigment. Listed in order from top to
bottom on an ideal chromatogram strip are:
Carotene - Orange in color
Xanthophylls - Yellow in color
Chlorophyll a - Light Green
Chlorophyll b - Dark Green
Note: Chlorophyll a and Chlorophyll b are very close to each other in color.
4. Name the pigments that are present in the spinach leaf.
5. Looking at the pigments present on your chromatogram, which pigments a present in the smallest
amounts from your sample.
6. Which pigments a present in the greatest amounts from your sample?
7. Which of the pigments migrated the farthest distance? How can you tell?
8. Which chlorophyll forms are more soluble? How can you tell?
Use your textbook to answer the following questions:
9. In which organelle would you expect to find leaf pigments
10. How might the distribution of leaf pigments differ in a leaf that is dark green compared to a leaf that is
light green in color?
11. Many leaves change color in the fall. How is it possible for this color change to happen? Base your
answer on your new knowledge of pigments present in chloroplasts. ( Hint: Chlorophyll a- and
Chlorophyll b- are easily broken down by the cooler fall temperatures).