Chromatography of Plant Pigments
Student’s Name
Period S
Mrs. Rezendes
Date
Chromatography of Plant Pigments
Overview
Paper chromatography is a useful technique for separating and identifying pigments and
other molecules from cell extracts that contain a complex mixture of molecules. The solvent
moves up the paper by capillary action, which occurs as a result of the attraction of solvent
molecules to the paper and the attraction of solvent molecules to one another. As the solvent
moves up the paper, it carries along any substances dissolved in it, in this case pigments. The
pigments are carried along at different rates because they are not equally soluble in the solvent
and because they are attracted, to different degrees, to the cellulose in the paper through the
formation of hydrogen bonds.
Problem:
How can the process of chromatography be used to separate and identify the plant pigments in
a leaf and how are they related to photosynthesis?
Hypothesis & Prediction:
Generate your hypothesis and prediction before continuing with the lab:
If pigment from a (spinach leaf/kale leaf) is applied to (chromatography paper/graph paper) and
placed in a (test tube/vial) containing the solvent (acetone/alcohol), then the pigment will
separate into different bands of color based on (solubility/melting point).
Materials:
Chromatography paper
Solvent
Spinach leaf
Glass vial with cover
Pencil
Coin with smooth edges
Ruler
Procedure:
1. Obtain a piece of chromatography paper and, using a pencil, lightly draw a baseline 1 cm
from the bottom of the page. (Touch the paper as little as possible, because skin oils
interfere with chromatogram development.)
2. Place a spinach leaf over the chromatography paper and roll the edge of a coin
over the leaf (using a ruler as a guide) so that the pigments of the leaf are driven
into the chromatography paper 1 cm from the bottom.
3. Apply the pigment streak at least five times, allowing the paper to dry between
each application, keeping the streak as narrow as possible (on the line).
4. Place chromatography solvent in a beaker so that the level of the liquid is below
the line that you made on the paper (does not touch the pigment). (Caution: Avoid
inhaling the solvent.)
5. Place the chromatography paper in the beaker so that the pigment streak end is
barely immersed in the solvent.
6. Cover the beaker with plastic wrap and do not disturb for several minutes.
7. When the solvent is about 0.5 cm from the top margin of the paper, remove the
paper and immediately mark the location of the solvent front before it evaporates.
Data & Calculations
Plant Pigment
Distance Pigment
Migrated (cm)
xanthophyll (yellow)
chlorophyll b (yellow green to olive green)
chlorophyll a (bright green to blue green)
carotene (yellow to yellow orange)
Distance Solvent Front Migrated (cm): __________
Calculate the Rf (Reference front) values for each of the four pigments using the formula:
distance pigment migrated
Rf =
distance solvent front migrated

Show calculations here:
Analysis & Conclusion:
A. Provide a summary of the lab
B. Provide an analysis of the data
C. Provide a discussion of error (experimental, equipment, and human)
During this lab, the process of (chromatography/photography) was used to show how pigments from
(spinach/kale) could be separated, using (alcohol/acetone) as a solvent. After the process, bands of
color were observed and (measured/erased). The pigments in the spinach separated because each
one dissolved at a (different/same) rate in the alcohol, so that some of them travelled quickly and
some travelled slowly along the paper.
The distance the pigment travelled was measured and compared to the distance travelled by the
solvent. This ratio is called the (Rf/RX) value.
Questions: (Choose the correct answer)
1. Describe what happened to the original spot of simulated plant pigment.
A. After the solvent soaked into the paper, the spot separated into different bands of color.
B. After the solvent soaked into the paper, the entire paper turned green.
2. a. Would a plant containing the pigments shown in the data table above be a
source for yellow dye?
A. No, because no yellow pigment was present.
B. Yes, the yellow pigment was hidden by the chlorophyll.
b. What other colors of dye might be available from the same plant? Explain
your answer.
A. No other colors would be available.
B. In addition to green, yellow and orange might be available.
3. What factors are involved in the separation of the pigments?
A. Separation of pigments into bands of color happens because of solubility.
B. Separation happens due to solubility and attraction of pigments to the chromatography paper.
4. a. Which of the four pigments, beta-carotene, chlorophyll a, chlorophyll b,
or xanthophyll, is the most soluble in the solvent system used? Which is the least soluble?
Explain your answers.
A. The one that moved the furthest is the most soluble because it dissolved quickly.
The least soluble stayed near the bottom.
B. All the pigments were equally soluble.
b. Which of the chlorophyll forms is more soluble? Why?
A. Chlorophyll A is the most soluble because it moved further.
B. Chlorophyll B is the most soluble because it moved the furthest.
5. If the experiment were run on a longer paper for a longer time, the separation
between chlorophyll a and chlorophyll b would become larger. Eventually, two
completely resolved spots would be seen. Why? Would you expect the Rf values
for the pigments to significantly change between a shorter and a longer run?
A. The Rf value is a ratio so it would not change with a longer paper.
B. The longer the time, the larger the Rf values.
6. What type of chlorophyll does the reaction center contain? What are the roles of the other
pigments?
A. The reaction center contains chlorophyll A.
B. The reaction center contains chlorophyll B.