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 thylaoid
membranes of chloroplasts and provide the green color of leaves. The chlorophylls are
tetrapyrrole pigments and include a porphyrin head with a centrally chelated !g
"#
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 lacing the !g
"#
atom.
$t plays a critical role in photosynthetic electron transport, but its presence at high levels in a
pigment extract can be an artifact of the breadown 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. $n plant leaves, carotenoids
are accessory pigments and play some role in capturing light energy used in photochemical
reactions. %owever, their most important role may be in the prevention of photooxidation of
chlorophyll by absorbing excess light energy. Carotenoids are isoprenoids with &' 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 non-polar
carotenes.
Thin layer chromatography is a techni(ue 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. $n our experiment, we will use thin layer
chromatography to separate pigments contained in a spinach leaf extract. We will use strips of
plastic coated with silica gel as the hydrophilic stationary phase. The mobile phase will be a
hydrophobic mixture of organic solvents )petroleum ether, acetone, and chloroform*. When a
pigment extract is placed on the thin layer strip, and the mobile phase is allowed to pass through
it, pigments present in the extract will dissolve and migrate with the hydrophobic solvent
depending upon their affinity for it relative to the hydrophilic silica. +s the chromatogram
develops, distinct spots of pigment with different colors will appear on the silica. The distance
moved by a particular pigment relative to the distance moved by the solvent )the solvent front* is
called the ,f value. ,f values are characteristic for a specific set of conditions and can be used to
identify the compound.
The objective of this experiment is to separate, and identify pigments contained in plants. We
will first use thin-layer chromatography to separate and identify plant pigments. We will then
use spectrophotometry to examine the absorbance spectra of chlorophyll a and chlorophyll b.
PROCEDURES:
Thin Layer Chromotagrahy
-ollow directions on handout )if available* on Thin .ayer Chromatography. We will use
concentrated extractions of chlorophyll and carotenoids. ,ecord your data on this handout.
Determination o! A"#or"an$e Se$tr%m
/* $dentify the chlorophyll a and chlorophyll b pigments to further analy0e. Collect each
pigment separately from each of three silica gel strips by scraping the silica gel spot from the
strip and collecting the scrapings on a piece of white paper. -or each pigment, combine your
scrapings from each of the three strips into one /1 m. test tube for chlorophyll a and one tube for
chlorophyll b.
"* +dd & m. of acetone to each tube to elute the pigments from the silica gel scrapings. Cap the
tubes and shae for / minute.
2* +llow the silica gel to precipitate.
&* 3se a pasteur pipet and bulb to transfer at least 2 m. of solution to a /' m. glass test tube.
+void disturbing the silica gel.
1* Proceed to the spectrophotometer. 4et the instrument to read a"#or"an$e at &''nm and
blan it with a tube of pure acetone. Place your pigment extract in the holder, and tae an
absorbance reading. ,ecord the data for chlorophyll a in Table 2, and for chlorophyll b in Table
&. ,epeat this process at "1 nm wavelength intervals up to 5&'nm, reblaning the instrument
with pure acetone each time you change the wavelength. 6etween 5&' and 57' nm, reduce the
wavelength interval to 1nm, and reblan prior to each measurement. ,epeat the process for two
of the carotene pigments of your choice, measuring at "1nm intervals.
5* 3se 8xcel to plot your absorbance values )y-axis* versus wavelength )x-axis*. Place
absorbance spectra for all pigments on the same graph.
Ta"&e '( Searation an) )etermination o! igment#
Carotenoi) e*tra$t:
9istance that solvent moved: ;;;;;;;;;;;;; cm
Pigment
N%m"er
Di#tan$e
tra+e&e) ,in $m-
Ban) $o&or R! +a&%e Pro"a"&e i)entity o!
igment
Ch&orohy&& e*tra$t:
9istance that solvent moved: ;;;;;;;;;;;;; cm
Pigment
N%m"er
Di#tan$e
tra+e&e) ,in $m-
Ban) $o&or R! +a&%e Pro"a"&e i)entity o!
igment
Ta"&e .( 4ummary of ,f values and visible colors for chlorophyll plant pigments
Pigment name Vi#i"&e $o&or R!
Carotene <ellow '.=>
?anthophyll <ellow '.>5
?anthophyll ,ed '.>'
Phaeophytin a 9ar grey '.57
Phaeophytin b .ight grey '.5'
?anthophyll <ellow '.1'
Chlorophyll a/ .ight blue-green '.&>
Chlorophyll a 9ar blue-green '.&5
Chlorophyll b/ .ight yellow-green '.2'
Chlorophyll b 9ar yellow-green '."1
?anthophyll <ellow './1
Ta"&e /( 4ummary of ,f values for carotenoid plant pigments
Pigment name Vi#i"&e $o&or R!
-carotene <ellow-orange '.=7
-carotene <ellow orange '.=&
.ycopene ,ed-orange '.>/
.eutein <ellow-brown '.71
@iolaxathin <ellow-brown '.55
Aeoxathin <ellow-brown '.">
Ta"&e 0( ,ecord absorbance values of $h&orohy&& a at each of your selected wavelengths.
Wavelength
)nm*
+bsorbance
)+*
Wavelength
)nm*
+bsorbance
)+*
Ta"&e /( ,ecord absorbance values of $h&orohy&& " at each of your selected wavelengths.
Wavelength
)nm*
+bsorbance
)+*
Wavelength
)nm*
+bsorbance
)+*
Ta"&e 1( ,ecord absorbance values of $arotene igment ' 222222222222 at each of your
selected wavelengths.
Wavelength
)nm*
+bsorbance
)+*
Wavelength
)nm*
+bsorbance
)+*
Ta"&e 3( ,ecord absorbance values of $arotene igment . 2222222222222 at each of your
selected wavelengths.
Wavelength
)nm*
+bsorbance
)+*
Wavelength
)nm*
+bsorbance
)+*
DISCUSSION
/. What characteristics did you use to identify plant pigments separated on your
chromatogramsB %ow do these characteristics compare among pigmentsB 9id you find all the
pigments in Table /B 9id you find any pigments not shown in Table /B
". 9escribe 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 extractsB
2. Compare the absorption spectrum for chorophyll a and b that you obtained to the action
spectrum for photosynthesis shown in 4ig%re 5(1 in your textboo. %ow do they compareB
What can you conclude about the primary pigment)s* involved in photosynthesisB
+bsorption spectrum of Chlorophyll b +bsorption spectrum of Chlorophyll a
+bsorption spectrum of lutein +bsorption spectrum of violaxathin