Pigment Chromatography
Prelab
Visible Light:
Visible light, also called white light, is composed of many different colors, or wavelengths. IN
turn, visible light makes up only a fraction of the overall Electromagnetic Spectrum, pictured
below.
Plant Pigments
Plants use only a small portion of visible light for photosynthesis. Their leaves contain
pigments, molecules which absorb discrete wavelengths (colors) of light. Photosynthetic
pigments absorb the wavelengths of blue and red best, and then transfer the captured light energy
to the reactions of photosynthesis. Pigments also reflect certain colors, which is why pigments
can appear green or yellow.
Chromatography
Molecules of different solubility can be separated out of solution using special solvents. The
most soluble molecules will be carried by the solvent the furthest. This process of separation is
called chromatography.
Rf values- The Rf value represents the ratio of the distance the pigment traveled compared to the
solvent front. Each plant pigment has a distinct value that can be used to identify it.
The Rf is calculated by dividing the distance each band moves by the distance the solvent front
moves. The Rf value will then always be between 0 and 1.
Distance substance (pigment) traveled
Rf =
Distance solvent traveled
Example: Using the sample chromatography above, what is the Rf of the…
a. …first band?
1.6 cm / 10.6 cm = 0.15
b. …second band? 4.1 cm / 10.6 cm = 0.39
Pre-Lab Questions
1. What is another name for white light?
2. What is a pigment?
3. Do pigments reflect the same color they absorb? Explain.
4. Which wavelengths (colors) do plants absorb best?
5. Calculate the Rf’s for the three remaining bands on the sample chromatography.
6. Explain how chromatography works.
Laboratory-Separation of Photosynthetic Pigments using Chromatography
Materials
Large test tube, fitted cork stopper, paper clip, chromatography solvent (9 PetEther: 1 acetone),
ruler, chromatography paper, spinach leaf, paper towel, pencil, scissors, goggles
Procedure
1. Obtain a strip of chromatography paper at least as
long as your test tube.
2. Use scissors to cut the bottom end of the paper strip
3. Draw a faint pencil line 2.5 cm above the
end of the paper strip.
See Figure 1.
4. Take a leaf of spinach and fold it in half, and then
in half again.
5. Using a quarter, roll the quarter (with the ridged edge)
over the leaf and onto the paper, exactly along the
pencil drawn line of your chromatogram until it turns green.
See Figure 2
6. Pour the solvent into your test tube until it reaches a height
of no more than 1.5 cm.
7. Attach the prepared chromatogram to a cork using a
paper clip and position the strip so that when inserted
into a test tube, the end just touches the bottom of the tube.
Adjust the height by moving the strip either up or down on the cork.
8. Place the paper strip into the tube containing the solvent.
9. DO NOT LET THE LINE OF CHLOROPHYLL
TOUCH THE SOLVENT!!!
See Figure 3.
Figure 1
Figure 2
Figure 3
10. Do not move or shake the tube for at least 15 minutes. Remove the paper chromatogram
from the test tube when the level of solvent almost reaches the top of the paper strip. MARK
THIS LINE WITH A PENCIL BECAUSE IT WILL DRY CLEAR.
11. Examine the chromatogram for the presence of different bands of color.
Data Collection
1. When your strip is dry, tape or staple your chromatogram to this sheet (or draw it), here.
2. Calculate the Rf value of each pigment and record in Data Table 1.
Band #
Pigment Color
Distance (cm)
Rf Value
Solvent Front
1
2
3
4
Data Table 1
Rf Value
.99 - .95
.4
.2
.1
Common Pigments
Carotene
Xanthophyll
Chlorophyll a
Chlorophyll b
Data Table 2
Analysis Questions
1. What is a chromatogram?
2. What does each band (color) really represent?
3. Why can pigments be separated?
4. Are spinach leaves composed of one or more pigments? What proof do you have?
5. How many different pigments can be seen on your chromatogram? Using Data Table 2,
name the pigment that is represented by each band color.
Continue onto the next page…
Conclusion
Using the graph below to support your answer, write a conclusion that addresses the following:

What is the main pigment(s) used by spinach, and other “green” plants, to absorb light
energy during photosynthesis?

Which color(s) does chlorophyll absorb the most?

Which color(s) does chlorophyll absorb the least, and therefore, is reflected?

At what time of the year do the other pigments (called accessory pigments) become visible,
such as red, orange, and yellow? Why?

Why is it more advantageous for a plant to contain many pigments, and not just chlorophyll?
Revised from: Spectrum Publications