203
Isolation and Spectrophotometric Characterization o f
Photosynthetic Pigments
RODNEY F BOYER
Department of Chemistry
Hope College
Holland, MI49423-3698, USA
Introduction
When students in the biochemistry laboratory are introduced to
the extraction and characterization of biomolecules, the tissue of
choice is most often of animal origin. Plant products are seldom
used; therefore, students rarely gain knowledge and appreci-
ation of the unique biomolecules present in the plant kingdom.
In order to fill in this gap, we have designed an experiment in
which students isolate a mixture of pigments by solvent
extraction of plant tissue, separate some of the components by
chromatography and identify them by visible region spectro-
photometry. Students who complete this experiment gain knowl-
edge and technical skills in biomolecule extraction, chromato-
graphic methods, spectrophotometric techniques and plant
pigment structure. Students will follow procedures which are
typical for the general extraction and characterization of lipids.
However, unlike most lipids (especially those in animals), the
plant pigments are highly colored and may be characterized and
quantified by visible spectrophotometry.
Theory
Many of the colors associated with higher plants (green leaves in
the spring and summer, yellow or red leaves in the fall, the
orange color of carrots, some colors in flower petals) are due to
the presence of pigment molecules. Review articles on plant
pigments have been recently written by Goodwin 1 and Lichten-
thaler. 2 General information on photosynthetic pigments can
also be obtained from the standard biochemistry texts by
Mathews and van Holde, 3 Stryer, 4 and Voet and Voet. 5
The major photosynthetic pigments of higher plants can be
divided into two groups, the chlorophylls and the carotenoids.
Both types of pigments are present in the subcellular organelles
called chloroplasts, where they are bound to proteins in the
thylakoids, the photochemically active photosynthetic biomem-
branes. Intact pigment-protein complexes, which are held
together by weak, noncovalent bonds, have been isolated from
chloroplasts and characterized b~( polyacrylamide gel electro-
phoresis and isoelectric focussing/The pigments are released in
a protein-free form by grinding plant tissue in solvents such as
acetone, methanol or hexane. Since the chlorophylls and the
carotenoids are readily soluble in organic solvents, they are
classified biochemically as lipids.
The most abundant plant pigments are chlorophyll a and
chlorophyll b which occur in a ratio (a:b) of approximately 3:1.
As is shown in Fig 1, the chlorophylls possess a porphyrin ring
with a coordinated magnesium atom at its center, a fused, 5-
membered ring and a C20 phytyl side chain. This nonpolar,
HzC=CH H C~ H2CffiCH H H-C-O
"3~c#. H~C~ceH
H H
a
o -oc.. -'Lo
CLN3H~I Offi~-OCH3
Chlorophyll a Chlorophyll b
Figure 1 Structures of chlorophyll a and chlorophyll b (From
Moore7)
BIOCHEMICAL EDUCATION 18(4) 1990
hydrocarbon side chain enhances the solubility of chlorophyll in
nonpolar solvents. The only difference between chlorophyll a
and b is the substituent on position 3 (ring 3): chlorophyll a has a
methyl group, whereas b has an aldehyde. The chlorophylls are
biosynthesized by condensation of ~-aminolevulinic acid in a
process similar to the synthesis of heme in mammals. In the
living plant cell, the chlorophylls serve as the primary photo-
synthetic pigments. They absorb light in the blue (450 nm) and
red regions (650-700 nm). Absorption of light, which excites an
electron in the chlorophyll molecule, provides energy for
initiation of the photosynthetic process producing NADPH and
ATP to be used for carbohydrate biosynthesis by the plant.
The second group of plant pigments, the carotenoids, can be
divided into two different types, (1) the carotenes which contain
only carbon and hydrogen, and (2) the xanthophylls which
contain carbon, hydrogen and oxygen atoms in the form of
hydroxyl or epoxide functional groups. The structures for several
major carotenoids are shown in Fig 2. Note that they all contain
40 carbon atoms and, because of the extensive conjugation,
would be expected to be highly colored. Most carotenoids are
yellow, red or orange, but some are green, pink and even black.
Many of the bright colors found in flower petals are due to the
presence of carotenoids (although some are due to antho-
cyanins). The yellow colors of fall foliage are due primarily to
preferential destruction of the green chlorophylls, revealing the
carotenoid color. 6
The percentage composition of carotenoids in plants varies
with growing conditions. The average weight % ranges are: 13-
carotene, 25-40%; lutein, 40-60%; violaxanthin, 10-20%; and
neoxanthin, 5-13%. 2 The carotenoids are biosynthesized by
condensation of acetylCoA through the mevalonic acid pathway.
The physiological function of the carotenoids is not completely
understood; however, two probable functions have been
described. First, the carotenoids, particularly the xanthophylls,
may participate in light adsorption for photosynthesis. It has
been shown that illuminated xanthophylls are able to transfer
excitation energy directly to chlorophyll a. Secondly, the
carotenes may inhibit the photo-oxidative destruction of chloro-
phyll, a process which consumes oxygen.
F-Carotene
Lutein
~ ~ ~ T ~ ~ ~ o OH
HO
Violaxanthin
OH
Neo~mnthln
Figure 2 Structures of major carotenoids: B-carotene, lutein,
violaxanthin and neoxanthin (From Lichtenthaler2)
204
I 1
Spectral Characteristics of Plant Pigments
The intense colors of the chlorophylls and carotenoids makes
them ideal candidates for absorption spectroscopy studies. In
fact, each plant pigment studied in this experiment has a unique
visible spectrum which can provide a positive identification. As
shown in Fig 3, chlorophylls a and b have absorption maxima in
the 600-675 nm range and in the 400-475 nm range. The o*
c
absorption maxima for each peak is very dependent upon solvent
polarity. For example, the two most intense absorption peaks for g I
chlorophyll a in diethyl ether are 660 nm and 428 nm, whereas, ,~ o.50o ! 0.500

in more polar methanol, the peaks are shifted to 665 nm and

432 nm.

l.(a:

[~.9"

B.B~

u :
~_~.~. -~_~Z:- \ ~" o
11
aoo.o ~o.o 4so.o sso.o
~Q. 4 Wavelength (nm)

Q.Z Figure 4 Visible absorption spectra for four carotenoids: ~-

carotene (--), lutein (--), violaxanthin (.-.), and neoxanthin ( . . . )
(From Lichtenthaler 2)
4i~Q ~BO Bgl~l 7BO
N z v m l mr, o c h ( h n )
measurement at wavelength, z. These equations are designed for
the use when 100% acetone is the solvent.

Practical Aspects of the Experiment

0.5
Plant pigments are isolated by careful extraction of plant tissue
(green or dried leaves) with 100% acetone. 7 The extraction may
w
be carried out with either a mortar and pestle or with a glass
a
u homogenizer. Plant cells have a tough wall and the addition of an
NgtLB
.¢1 abrasive agent such as quartz sand enhances the extraction
L
a
Nt~.2
process. Both types of plant pigments, chlorophylls and caro-
tenoids, are extremely light-sensitive and are readily destroyed
B.
by photo-bleaching; therefore, the extraction process should be
performed rapidly and in subdued lighting. If quantitative
determinations are to be made for each pigment, the absorption
,aa ~';=, sa'e 7e~
HIval mnl~thCnm:l data must be obtained immediately after extraction.
A wide range of chromatographic techniques, including
Figure 3 Visible absorption spectra for chlorophyll a (a) and column, paper, thin-layer and high-pressure liquid chromatog-
chlorophyll b (b) raphy, have been applied to the separation of photosynthetic
pigments. 2 There is great historical interest here, because
In contrast to the chlorophylls, which absorb light in two Tswett, in the first demonstration of chromatography in 1906,
regions of the visible spectrum, the carotenoids exhibit intense separated a plant pigment extract on CaCO3 and MgO columns. 8
absorption in just one, 350-500 nm. Fig 4 compares the In the present experiment, either paper or thin-layer chromatog-
absorption spectra of four common carotenoids. As with the raphy are used to separate the pigments. Both techniques
chlorophylls, the absorption maxima of the carotenoids vary provide an excellent resolution of the two chlorophylls and of the
with polarity of the solvent. 13-carotene in diethyl ether has a major carotenoids. After solvent elution of the pigments from
kma~ of 449.8 nm, but in the more polar acetone, the hmax is the paper or silica gel, the visible absorption spectrum of each is
454 nm. determined. Several types of plant tissue may be used. Some
In addition to its use in pigment identification, spectrophoto- recommendations include, fresh leaves (tree, plant, grass,
metric data may be used to measure the concentrations of spinach), green algae or mosses. For variety, students may be
pigments in a plant extract. Data from spectral analysis were asked to bring their own samples for analysis.
used to calculate absorption coefficients for each pigment in The approximate time requirements for each part of the
order to derive the following equations. 2 experiment are:

Ca = 11.24A661.6 - 2.04-4644.8 A Extraction of the Pigments - - 30 minutes,

Cb = 20.13A644.S - 4.19A6m.6 B Determination of Pigment Concentration-- 15 minutes,
Ca+b = 7.05A661.6 + 18.09A64a.s C Chromatography and Elution of Pigments - - 1 hour, and
Cx+c = (1000A470 - 1.90Ca - 63.14Cb)/214 D Measurement of Visible Absorption Spectra-- 1 hour.

Since the pigment extract rapidly deteriorates as a result of

where: Ca = concentration of chlorophyll a in micrograms per
milliliter of plant extract solution (l~g/ml), Cb = concentration of
chlorophyll b in ixg/ml, Ca+b = concentration of total chloro-
phyll in p,g/ml, Cx+c = concentration of total carotenoids
(xanthophylls and carotenes) in p,g/ml, and Az = absorbance
photo-oxidation, it is critical that students work rapidly, but
safely and efficiently. Part B must be completed immediately
after the extraction. Parts C and D may be completed at a later
time, but, in the meantime, the plant extracts must be stored
refrigerated in a concentrated form and under nitrogen gas.

BIOCHEMICAL EDUCATION 18(4) 1990


Materials and Supplies
Plant tissues (leaves, moss, algae, etc); acetone, 100%; mortar
and pestle or glass homogenizer; quartz sand; small funnel and
qualitative grade filter paper; test tubes, 100 mm x 12 ram;
microcapillaries or tapered capillaries; anhydrous sodium sulf-
ate, Na2SO4; UV-VIS spectrophotometer; glass cuvets, 3 or
1 ml; metal spatula or clean razor blade; materials for paper
chromatography (Whatman 3M paper, 13.5 cm x 7 cm; solvent
system, petroleum ether:acetone (9:1, v/v); chromatography
chamber, 250 ml beaker with watch glass; stapler; scissors);
materials for thin-layer chromatography (silica gel plates, 10 cm
x 10 cm; solvent system, petroleum ether:dioxane:2-propanol
(70:30:10, v/v/v); chromatography chamber).
Experimental Procedure
A Extraction of the Pigments This procedure should be com-
pleted in subdued lighting! Obtain 5 tree leaves or equivalent
amount of grass or other plant tissue, tear them into small pieces
and place in a mortar or homogenizer. Measure 15 ml of acetone
and pour into the container. Add approximately 0.5 g of pure
sand and grind the mixture thoroughly until the solution
C A U T I O N : USE OF A C E T O N E
Exposure to acetone by inhalation, ingestion, or skin
absorption is harmful. Contact with eyes will cause severe
irritation. First aid: eyes - - flush with copious amounts of
water for at least 15 minutes; skin - - flush with water for a
few minutes; inhalation - - remove to fresh air. Acetone is
volatile and extremely flammable. No open flames should be
in the vicinity of your work. In case of fire, use a CO2
extinguisher. Dispose of acetone extraction, and solutions in
the organic waste container.
becomes a deep green (about 10 minutes). The plant cell walls of
cellulose are tough, so you need to be persistent. After
extraction is complete, add 1-2 g of anhydrous sodium sulfate to
remove water from the extract. (What is the source of the
water?) Rapidly filter the extract by gravity through fluted filter
paper. Measure the volume of the filtered extract and rapidly
proceed to Part B.
B Determination of Pigment Concentration In order to
determine the chlorophyll and carotenoid content of your
extract, you must measure the absorbance at several wave-
lengths, 661.6 nm, 644.8 nm and 470 nm. If you are using a
single ,beam spectrophotometer, use a cuvet containing acetone
to zero the instrument. A double beam instrument should have a
cuvet containing acetone in the reference beam and the acetone
extract solution in the sample beam. If desired, it is instructive to
obtain a complete spectrum of the extract in the range,
400-700 nm. This can be compared to the spectrum obtained for
each of the individual pigments in Part D.
C Chromatography and Elution of Pigments The general
procedures for paper and thin-layer chromatography are similar.
Obtain a piece of chromatography paper (13.5 x 7 cm) or a thin-
layer plate (10 x 10 cm). With a pencil, draw a very light line
1.5 cm from and parallel to the long edge of the paper or TLC
plate. Using a Pasteur pipet or tapered capillary, evenly apply
the acetone extract along the line. Begin on the pencilled line
approximately 1 cm from the edge and stop approximately 1 cm
from the opposite edge. The narrower the line, the better your
separation will be. Allow the first coating of extract to dry and
repeat above procedure with another application of extract.
Repeat a third time if the line is not dark green. Allow the paper
to dry completely, roll it into a circle with the pencil line and
extract outside and on the circular edge. Place the paper (extract
line down) into a chamber containing no more than 1 cm of
BIOCHEMICAL EDUCATION 18(4) 1990
205
appropriate solvent, petroleum ether:acetone (9:1). A chamber
may be prepared by pouring 7 ml of solvent into a 250 ml
beaker. Cover with a watch glass. If a thin-layer plate is used,
place directly in a chamber containing petroleum ether:di-
oxane:2-propanol (70:30:10) and cover. When the solvent has
risen to within 0.5 cm from the top of the paper or TLC plate,
remove from the chamber and mark the position of the solvent
front. Allow the chromatogram to dry. Measure the position of
each pigment band and the solvent front and draw a full-scale
picture of the completed chromatogram in your notebook. Since
each chromatogram is complete in a few minutes, it is pbssible to
experiment with the amount of extract applied to the paper or
plate. If good separation is not attained and pigment bands are
very dark, try a chromatogram with less extract. On the other
hand, if the bands are light and difficult to distinguish, prepare
and develop a chromatogram with more extract. Each pigment is
eluted from the paper or TLC plate using acetone. With the
paper chromatogram, use a scissors to cut each colored band
from the paper. Cut each paper strip into smaller pieces and
place pieces from each band into separate, labeled test tubes
containing 4 ml of acetone. Cover the tubes with a cork and
allow to sit for 5-10 minutes with gentle mixing at 1-2 minute
intervals. Proceed directly to Part D. To extract the pigments
from the TLC plate, use a metal spatula or razor blade to scrap
the silica gel at each colored band into labeled test tubes
containing 4 ml of acetone. Cover with a stopper and allow to sit
for 5-10 minutes with mixing at intervals. Centrifuge or filter
each solution using fluted filter paper. Proceed directly to
Part D.
D Measurement of Visible Region Absorption Spectra Using
glass cuvets, measure the absorption spectrum of each fraction
from 400 to 700 nm. A cuvet containing acetone should be used
as reference. A Pasteur pipet may be used to transfer solutions
to and from the cuvet but be careful not to scratch the inside of
the glass cuvet with the pipet. After all spectra have been
obtained, dispose of the acetone solutions in the waste organic
contz~iner in the laboratory.
Analysis of Results
A Extraction of the Pigments Describe the color of the extract.
Explain the action of acetone in the extraction process. What is
the purpose of the sand?
B Determination of Pigment Concentration Using the ab-
sorbance data obtained on the crude extraction mixture and the
equations provided earlier, calculate the concentrations of
chlorophyll a, chlorophyll b, and total carotenoids.
C Chromatography and Elution of Pigments Calculate the
mobility of each pigment relative to the solvent front (Re):
distance from the origin migrated by a compound
Rf =
distance from origin migrated by solvent
Explain the relative order of the Rf values. Can you make any
general statements about the polarity of each pigment?
D Measurement of Visible Absorption Spectrum Prepare a
table of spectral data (wavelengths of major peaks and absorb-
ante) for each pigment. Using these data and the standard
spectra in Figs 3 and 4, try to assign the identity of each pigment
isolated. Now that each compound is identified, is there a
correlation between the experimental Rf value and polarity as
determined from the structure? Which pigments are more polar,
those near the top or those near the bottom of the chromato-
gram?
Study Questions
(1) What other solvents in addition to acetone could be used for
extracting plant photosynthetic pigments?
(2) Predict the relative Rf values for thin-layer chromatography
206
of a mixture of chlorophylls a and b, f3-carotene, lutein,
neoxanthin, and violaxanthin. Assume that the solvent is the
same as used in this experiment.
(3) Repeat Question 2 (above) except substitute paper chroma-
tography with petroleum ether:acetone (9:1) as developing
solvent.
(4) How does acetone disrupt the plant pigments from their
natural state as pigment-protein complexes in the chloroplasts?
(5) What extraction solvent system would you use if you wanted
to isolate the intact protein-pigment complexes? Describe
experimental techniques which could be used to determine the
molecular weight of each protein-pigment complex.
References
tGoodwin, T W (1983) 'Introduction to Plant Biochemistry', Second
Edition, Pergamon Press, Oxford, pp 96-103
2Lichtenthaler, H K (1987) in 'Methods in Enzymology', Academic
Press, New York, vol 148, pp 350-382
3Mathews, C and van Holde, K (1990) 'Biochemistry', Benjamin-
Cummings, Redwood City, CA, pp 643-669
4Stryer, L (1988) 'Biochemisty', Third Edition, W H Freeman, New
York, pp 517-533
5Voet, D and Voet, J (1990) 'Biochemistry', John Wiley and Sons, New
York, pp 586-594
6Goodwin, T W (1958) Biochem J 68, 503-511
7Moore, T C (1981) 'Research Experiences in Plant Physiology - - A
Laboratory Manual', Second Edition, Springer-Verlag, New York, pp
35-45
STswett, M (1906) Ber Dtsch Bot Ges 24, 384
A Simulation of Radioactive Tracer Flow
DESMOND S T NICHOLL l and PETER C L JOHN 2
1Biology Department
Paisley College o f Technology
Paisley P A l 2BE, Scotland
and
2plant Cell Biology Group
Research School o f Biological Sciences
The Australian National University
P O Box 475
Canberra A C T 2601, Australia
Introduction
The use of radiolabelled compounds in the study of metabolic
pathways has been of crucial importance in biochemistry.
Perhaps the best known example of the use of this technique is
the elucidation of the path of carbon during photosynthetic
fixation of CO2, which provided information about the pathway
which we now know as the Calvin cycle. Although this type of
work is time-consuming and complicated, there have been
reports of experiments suitable for class practicals in biochem-
istry. These include investigation of the Calvin cycle I and the
incorporation of radiolabelled inorganic and organic precursors
by E coli. 2
In undergraduate classes we have used a procedure similar to
that of Hawcroft 1 to illustrate the incorporation of 14CO2 by
Chlorella, and to monitor the distribution of label between
soluble and insoluble pools in the cell. We also provide a simple
simulation to illustrate and complement the experimental pro-
cess. This has proved valuable, particularly in the case of weaker
students who have difficulty grasping the concepts involved.
B I O C H E M I C A L E D U C A T I O N 18(4) 1990
In this paper we describe the simulation of radioactive tracer
incorporation and distribution. The simulation can be used in
isolation, and makes a useful introduction to the topic of
metabolic analysis by using labelled precursors. However, most
benefit will be derived if an actual experiment is carried out in
conjunction with the simulation. We therefore present a brief
description of a suitable experimental system which we have
found reliable.
Experimental System
The experiment involves photosynthetic incorporation of radio-
active CO2 by algal cells. Strains of Chlorella or Chlamydomonas
are probably most useful, although any photosynthetically
competent alga could be used. Details of suitable strains, media
and growth conditions can be found in references 3-5.
The experiment requires access to a scintillation counter and
facilities to work with 14COa. The labelling apparatus is simple to
construct, and modifications may be incorporated to suit
materials available. A key safety point is that release of 14CO2 to
the atmosphere should be prevented. A suitable apparatus is
shown in Fig 1.
[
U c
E
B . !G
/i I\
Figure I Labelling apparatus. Algal suspension (A) is placed in a
cell culture flask (B) and a magnetic flea added. Cells are
illuminated from above with fluorescent strip lights (C) and
agitated on a magnetic stirrer (D). The neck of the flask has a
rubber seal which is pierced with three syringe needles, one of
which is attached to a length of small-bore silicon tubing. A filter
(E) is made by plugging a syringe barrel with cotton wool. Solid
KOH pellets may be incorporated to trap any radioactive C02
released. The label is introduced by syringe (F). Samples (1 ml)
are withdrawn at G
The isotope used is sodium [14C] bicarbonate, which can be
used at a variety of dilutions. We have found that a final
radioactive concentration of around 37kBq m1-1 (1 i~Ci m1-1) is
adequate. The label is added to the cells and the experiment
timed from this point. Samples are removed at intervals and
processed using apparatus similar to that shown in Fig 2. If
possible this should be carried out in a fume-hood designated for
radioactive materials, although the system is designed to prevent
release of labelled CO2.
When all samples have been processed, 1 ml of each is
centrifuged in a microfuge to separate the soluble and insoluble
fractions. The remainder is retained for measuring total incor-
poration. After centrifugation the supernatant is removed to a
clean tube and the pellet resuspended in 1 ml acetic methanol.
This gives three samples for counting to determine total
incorporation, counts in soluble and counts in insoluble frac-
tions. A typical result is shown in Fig 3. This gives a good
indication of the incorporation of label and its distribution, but
does not identify the products of the labelling experiment.