PLANT PIGMENTS
OBJECTIVE:
The objective of this experiment is to separate and identify pigments contained in plants. We
will use thin-layer chromatography to separate and identify plant pigments, and
spectrophotometry to examine absorbance spectra of selected pigments.
INTRODUCTION:
Plant leaves contain a number of light-absorbing pigments, many of which are associated with
photosynthesis. Chlorophylls a and b are the primary photosynthetic pigments responsible for
capturing light energy for use in photochemical reactions. They are located in thylakoid
membranes of chloroplasts and provide the green color of leaves. The chlorophylls are
tetrapyrrole pigments and include a porphyrin head with a centrally chelated Mg2+ atom, and a
phytol tail derived from isoprene. Chlorophyll a differs from b in having a methyl group in place
of a formyl group on the porphyrin head. This difference confers a slightly higher affinity for
non-polar solvents to chlorophyll a, a property that is useful in the chromatographic separation of
these two pigments. Pheophytin is a grayish-green form of chlorophyll lacking the Mg2+ atom.
It plays a critical role in photosynthetic electron transport, but its presence at high levels in a
pigment extract can be an artifact of the breakdown of chlorophyll under acidic conditions.
Carotenoids are yellow to red pigments that are closely associated with chlorophyll in
chloroplast membranes. Common carotenoids include -carotene, that provides the color to
carrot roots, and lycopene, that provides the color to tomato fruits. In plant leaves, carotenoids
are accessory pigments and play some role in capturing light energy used in photochemical
reactions. However, their most important role may be in the prevention of photooxidation of
chlorophyll by absorbing excess light energy. Carotenoids are isoprenoids with 40 carbon
atoms, and are divided into a pure hydrocarbon class, the carotenes, and a class with two oxygen
atoms present in two hydroxyl groups, the xanthophylls. The additional oxygens confer greater
polarity to the xanthophylls, and enable their chromatographic separation from the more nonpolar carotenes.
Thin layer chromatography is a technique that allows the separation of a wide variety of
molecules based on their affinity for a mobile phase, that is usually hydrophobic, relative to a
stationary phase, which is usually hydrophilic. In our experiment, we will use thin layer
chromatography to separate pigments contained in a spinach leaf extract. We will use strips of
plastic coated with silica gel as the hydrophilic stationary phase. The mobile phase will be a
hydrophobic mixture of organic solvents (petroleum ether, acetone, and chloroform). When a
pigment extract is placed on the thin layer strip, and the mobile phase is allowed to pass through
it, pigments present in the extract will dissolve and migrate with the hydrophobic solvent
depending upon their affinity for it relative to the hydrophilic silica. As the chromatogram
develops, distinct spots of pigment with different colors will appear on the silica. The distance
moved by a particular pigment relative to the distance moved by the solvent (the solvent front) is
called the Rf value. Rf values are characteristic for a specific set of conditions and can be used to
identify the compound.
The objective of this experiment is to separate, and identify pigments contained in plants. We
will first use thin-layer chromatography to separate and identify plant pigments. We will then
use spectrophotometry to examine the absorbance spectra of chlorophyll a and chlorophyll b.
PROCEDURES:
Thin Layer Chromotagraphy
Follow directions on handout (if available) on Thin Layer Chromatography. We will use
concentrated extractions of chlorophyll and carotenoids. Record your data on this handout.
Determination of Absorbance Spectrum
1) Identify the chlorophyll a and chlorophyll b pigments to further analyze. Collect each
pigment separately from each of three silica gel strips by scraping the silica gel spot from the
strip and collecting the scrapings on a piece of white paper. For each pigment, combine your
scrapings from each of the three strips into one 15 mL test tube for chlorophyll a and one tube
for chlorophyll b.
2) Add 4 mL of acetone to each tube to elute the pigments from the silica gel scrapings. Cap the
tubes and shake for 1 minute.
3) Allow the silica gel to precipitate.
4) Use a pasteur pipet and bulb to transfer at least 3 mL of solution to a 10 mL glass test tube.
Avoid disturbing the silica gel.
5) Proceed to the spectrophotometer. Set the instrument to read absorbance at 400nm and
blank it with a tube of pure acetone. Place your pigment extract in the holder, and take an
absorbance reading. Record the data for chlorophyll a in Table 3, and for chlorophyll b in Table
4. Repeat this process at 25 nm wavelength intervals up to 640nm, reblanking the instrument
with pure acetone each time you change the wavelength. Between 640 and 670 nm, reduce the
wavelength interval to 5nm, and reblank prior to each measurement. Repeat the process for two
of the carotene pigments of your choice, measuring at 25nm intervals.
6) Use Excel to plot your absorbance values (y-axis) versus wavelength (x-axis). Place
absorbance spectra for all pigments on the same graph.
Table 1. Separation and determination of pigments
Carotenoid extract:
Distance that solvent moved: _____________ cm
Pigment
Number
Distance
traveled (in cm)
Band color
Rf value
Probable identity of
pigment
Rf value
Probable identity of
pigment
Chlorophyll extract:
Distance that solvent moved: _____________ cm
Pigment
Number
Distance
traveled (in cm)
Band color
Table 2. Summary of Rf values and visible colors for chlorophyll plant pigments
Pigment name
Visible color
Rf
Carotene
Yellow
0.98
Xanthophyll
Yellow
0.86
Xanthophyll
Red
0.80
Phaeophytin a
Dark grey
0.67
Phaeophytin b
Light grey
0.60
Xanthophyll
Yellow
0.50
Chlorophyll a1
Light blue-green
0.48
Chlorophyll a
Dark blue-green
0.46
Chlorophyll b1
Light yellow-green
0.30
Chlorophyll b
Dark yellow-green
0.25
Xanthophyll
Yellow
0.15
Table 3. Summary of Rf values for carotenoid plant pigments
Pigment name
Visible color
Rf
Yellow-orange
0.97
-carotene
Yellow orange
0.94
-carotene
Lycopene
Red-orange
0.81
Leutein
Yellow-brown
0.75
Violaxathin
Yellow-brown
0.66
Neoxathin
Yellow-brown
0.28
Table 4. Record absorbance values of chlorophyll a at each of your selected wavelengths.
Wavelength
(nm)
Absorbance
(A)
Wavelength
(nm)
Absorbance
(A)
Table 3. Record absorbance values of chlorophyll b at each of your selected wavelengths.
Wavelength
(nm)
Absorbance
(A)
Wavelength
(nm)
Absorbance
(A)
Table 5. Record absorbance values of carotene pigment 1 ____________ at each of your
selected wavelengths.
Wavelength
(nm)
Absorbance
(A)
Wavelength
(nm)
Absorbance
(A)
Table 6. Record absorbance values of carotene pigment 2 _____________ at each of your
selected wavelengths.
Wavelength
(nm)
Absorbance
(A)
Wavelength
(nm)
Absorbance
(A)
DISCUSSION
1. What characteristics did you use to identify plant pigments separated on your
chromatograms? How do these characteristics compare among pigments? Did you find all the
pigments in Table 1? Did you find any pigments not shown in Table 1?
2. Describe the similarities and differences between the absorption spectra for Chlorophyll a and
b. Which pigment appeared to be present in the greatest concentration in your extracts?
3. Compare the absorption spectrum for chorophyll a and b that you obtained to the action
spectrum for photosynthesis shown in Figure 7.5 in your textbook. How do they compare?
What can you conclude about the primary pigment(s) involved in photosynthesis?
Absorption spectrum of Chlorophyll b
Absorption spectrum of Chlorophyll a
Absorption spectrum of lutein
Absorption spectrum of violaxathin