Plant Pigment/ Paper Chromatography Lab

Laiba Sairah Ninan

AP Biology
Ms. Ranney
Period 1, 2
I. Title: Plant Pigment/ Photosynthesis Lab

II. Purpose: The purpose of the lab is to separate plant pigments using
chromatography.

III. Procedures:
1. Cut chromatography paper strip. Use scissors to cut off the two bottom of the
strip.
2. Measure 2 cm from the bottom of the paper strip.
3. Lightly draw a pencil line across the strip.
4. Place the leaf over the pencil line and roll a quarter over the line.
5. Repeat until there is a very dark green line over the pencil line at least 15 times.
6. Place 1 cm of solvent in a graduated cylinder.
7. Slowly lower the chromatography paper into flask. Do not allow pigment to
touch solvent.
8. Let solvent rise until it reaches the mark at top of paper.
9. Stop just before solvent front reaches top.
10. Remove chromatography paper and immediately draw a pencil line across the
top of the strip where the solvent front got to before it dries.
11. Draw a pencil line at the top of each pigment, showing how far that pigment
traveled.

IV. Observations/Data:
Band Number Name of Color of Migration Rf value
Pigment Pigment Distance (mm)
Solvent N/A 66 1.0
1 (top) Xanthophyll Yellow 66 1.0
2 Carotene Yellow - 9 .14
orange
3 Chlorophyll A Green 6 .09
4 (bottom) Chlorophyll B Gray – green 2 .03

V. Discussions/Analysis: I have edited this to show you how to turn the questions
into a good discussion/analysis paragraph.
1. What factors are involved in the separation of the pigments?
The factors that are involved in the separation of the pigments in this lab are
solubility, size of particles, and their attractiveness to the paper are involved in
the separation of the pigments. Beta Carotene was the lightest molecular weight
and does not contain any available oxygens to form hydrogen bonds with the
paper. Therefore it stayed with the solvent front and traveled up the paper the
furthest. Conversely chlorophyll molecules were heavier moleclular weight and
formed hydrogen bonds with the paper and so did not travel as far on the paper.
2. Would you expect the Rf value of the pigment to be the same if a different
solvent were used? Explain.
The Rf values for the pigments that were measured during this lab were unique
to the solvent used (a chromatography mixture of acetone and alcohol provided
by Carolina Biological). However, the magnitude of the values will be relative to
each other so the order of pigments (least soluble, slowest moving to most
soluble, fasting moving will be the same No, the different solubilities of the
pigments would change the Rf values. For example, chlorophyll b is only soluble
to fat solutions.

3. Name each of the pigments you were able to identify and describe their roles in
plant cells.
Chlorophyll a, being the main photosynthetic pigment, has a primary purpose to
convert light energy to chemical energy used by the plant itself. Chlorophyll b
absorbs light in a region of the spectrum apart from the dominant chlorophyll,
and transfers the energy it produces to chlorophyll a. Along with chlorophyll b in
transferring their energy produced to the dominant chlorophyll, two other
pigments that are found in plants are carotenes and xanthophylls, which are
orange and yellow respectively. Since chlorophyll is such a dominant pigment in
green plants, this domination hides the color of the carotenes and xanthophylls in
the leaves.
4. Which pigment traveled furthest? Why? Which pigment traveled least? Why?
The pigment that traveled the furthest was carotene xanthophyll because it was
the most soluble in the solvent. Chlorophyll b traveled least because it was the
least soluble in the solvent.
5. How does the amount of each pigment explain why leaves are certain colors?
The pigment inside the leaf absorbs all colors except the one shown. If a leaf is
green then it absorbed all colors except green. This lab showed that there was
much more green pigments (cholorophyll a and b) than yellow pigments because
the green bands were the darkest and thickest on the paper. That is why spinach
leaves are green.
6. How could you use paper chromatography to determine if the leaves of 2
different plants had the same or different pigments?
We could use the same Using paper chromatography experiment performed for
this lab to determine if a different plant had the same pigments. We could
compare the Rf values from the spinach leaf to the Rf values from those of the
different plant to determine if they had the same pigments. , the pigments that
give a leaf its color can be separated and observed to determine the R f value of
each pigment and their function during photosynthesis.
What happen to leaf pigments in the autumn? Why do leaves change color?
 In autumn, chlorophyll starts to break down which allows the other brilliant plant
pigment colors to show. These pigments include the red, orange, and yellow
colors.
7. Propose a reason for the results obtained by Abby and Melissa’s experiment in
the courtyard.
By spraying the leaf green, yellow, a light pigment that is not used by the plant
in photosynthesis, Abby and Melissa did not interfere with photosynthesis that
was occurring naturally in the plant because the plant was still able to receive
and absorb red and blue lights. Conversely it appears that the light paint coating
may have helped the plant by insulating the leaf from the decreased temperature
of the fall so that the enzymes that are involved in photosynthesis could continue
working after those in the leaves that were green stoped. actually helped the
plant to grow more.

VI. Conclusion:
Paper chromatography proved to be an accurate successful method of separating
and observing the various colors of plant pigments. The pigments dissolved in the
solvent and migrated upward. The colors were observed and their migration
distances were measured & recorded. The Rf value of each pigment was determined
by dividing its migration by the migration of the solvent. It was determined that 4
pigments were present in the original spot --- carotene, xanthophyll, chlorophyll a,
and chlorophyll b. Xanthophyll Carotenewas the most soluble, while chlorophyll b
was the least soluble.