1. Activity of Various Photosynthetic Pigments in Common Plants
By Dave Porter
BIO156 – General Biology II
Wednesday Lab 2:00-4:50pm
Professor Mary Jo Witz
March 28th
, 2008

2. I.) ABSTRACT
Without the process of photosynthesis – a two-stage process that produces oxygen gas
and glucose from carbon dioxide, light energy, and water – the Earth would be much different
than it is today. This experiment investigated the effects of the absence of light on
photosynthesis, the regional production of starch in leaves, as well as the polarity and absorbance
of main pigments involved in photosynthesis. A leaf of a geranium (Pelargonium sp.) was
placed in a lit environment and another leaf of the same plant was placed in total darkness, both
for a week. The leaves were boiled in 80% ethanol and dyed with I2KI (iodine). The leaf
subjected to darkness could not photosynthesize effectively, so it remained colorless, while the
leaf in the lit environment stained purple, signifying the production of starch – a photosynthetic
product. A leaf of Coleus blumei was boiled and stained with the same chemicals, and the plant
stained purple only along the part of the leaf that was green to begin with. This result was due to
the presence of chlorophyll in the green region and its absence in other regions. Different
pigments produce different colorations in leaves. Paper chromatography was used on a solution
of ground spinach (Spinacea oleracea) leaves and the absorption spectra of notable pigments
were recorded. Each pigment tested had a peak absorbance between 420-440nm, as did the
action spectra of all four pigments together. This means that light in the violet-blue region of the
visible light spectrum is the most effective in driving photosynthesis – it is absorbed by
chloroplasts at the highest efficacy.
II.) INTRODUCTION
Photosynthesis is an intricate process that produces six moles oxygen, six moles of water,
and one mole of a sugar monomer from six moles of carbon dioxide, twelve moles of water, and

3. light energy (Campbell and Reece, 2005). It is a two-stage system, consisting of the light
reactions and the Calvin cycle. Both of these processes occur in the chloroplast.
Defining the light reactions, photons absorbed by chlorophyll power the transfer of
hydrogen and electrons from water molecules to an electron acceptor called nicotinamide
adenine dinucleotide phosphate (NADP+
) (Campbell and Reece, 2005). This splitting of water
releases diatomic oxygen. The NADP+
molecule is reduced to NADPH by the addition of a
hydrogen ion and a pair of electrons. The light reactions also produce adenosine triphosphate
(ATP) (Campbell and Reece, 2005).
The Calvin cycle begins with the absorption of CO2 from the atmosphere and the
subsequent process of carbon fixation – powered by the enzyme rubisco – the incorporation of
carbon into organic molecules (Nester et al. 2007). NADPH uses its reducing power to convert
the fixed carbon to carbohydrates by addition of electrons and the energy evolved from ATP
breakdown (Campbell and Reece, 2005). Nester et al. (2007) reveal that the Calvin cycle “turns”
six times and every six-turn cycle produces one molecule of the six-carbon sugar fructose at the
expense of 18 ATP and 12 NADPH. The sugar monomer is converted into sucrose and either
transported where needed or stored in the form of starch, a polysaccharide (Morgan and Carter,
2005).
The purpose of this experiment was to study where starch is produced in photosynthesis,
and to identify and analyze pigments involved in its production. Starch is important in the
development and growth of plants (Morgan and Carter, 2005).
Starch was predicted to be found in the group of Pelargonium leaves in the lit
environment and not the experimental group. The dark environment does not allow photons to
reach the chloroplasts, so the light reactions cannot begin. Since photosynthesis takes place in

4. chloroplasts, starch should be detected near them; chloroplasts are characteristically green, so
phenotypically speaking, starch should be present wherever there is green coloration. This was
the basis of the prediction that starch would only be found in the green region on the leaf of C.
blumei.
During paper chromatography, the most polar molecule, chlorophyll b, was predicted to
advance the least distance on the paper, and the least polar molecule, beta carotene, the furthest.
These predictions of relative polarity were based on the structures of the four pigments under
investigation provided in published material (Morgan and Carter, 2005). Based upon additional
published material, the peak ABS of chlorophyll a, chlorophyll b, and carotenoids is between
420-480nm (Campbell and Reece, 2005). The predicted wavelength range encompassing the
peak ABS of all four pigments coincided with this documented range.
III.) MATERIALS AND METHODS
The instructor, before lab, prepared leaves of Pelargonium; one was left in the sunlight
for seven days while the second one was placed in a dark environment for seven days where
sunlight could not reach it (Morgan and Carter, 2005). The leaves were then slowly boiled in an
80% ethanol solution until almost all color was lost (Morgan and Carter, 2005). These leaves
were cooled, rinsed with distilled water and then stained with I2KI.
A leaf of C. blumei was gently boiled in 80% ethanol before it was stained with I2KI.
Observations were made regarding the presence or absence of photosynthetic activity in differing
regions of the leaf after it was subjected to the stain.

5. Paper chromatography was performed on a solution of S. oleracea pigment extract
(Morgan and Carter, 2005). A 9:1 petroleum ether and acetone solvent was used. Rf values were
calculated.
Samples of each pigment were cut from the chromatography papers and pooled in
separate beakers of 10mL of acetone (Morgan and Carter, 2005). The absorption spectrum of
each pigment solution was recorded starting at a wavelength of 400nm and increasing at intervals
of 20nm terminating at 720nm (Morgan and Carter, 2005). Each individual pigment had an
absorption spectrum measured. Data from all groups were then compiled.
IV.) RESULTS
The Pelargonium leaf that was subjected to a week in a lightless environment, boiled in
80% ethanol, and then stained with I2KI was nearly colorless. The Pelargonium leaf that was left
in a lighted region for a week, boiled in 80% ethanol, and then stained with I2KI was purple in
color.
After subjected to I2KI, the C. blumei leaf showed purple colors where the green regions
of the original, unstained leaf were. All other regions were colorless after the staining.
The absorption spectrum of each individual pigment in S. oleracea showed a peak
between 420-440nm and a lesser peak between 640-680nm (Fig. 1). The action spectrum of the
combined pigment solution showed a similar result (Fig. 2). The average Rf value of chlorophyll
b was the lowest value of the four pigments, while the average Rf of beta carotene was the
greatest (Table 1).

6. 0.000
0.050
0.100
0.150
0.200
0.250
0.300
0.350
400 440 480 520 560 600 640 680 720
Wavelength (nm)
ABS
Chlorophyll a
Chlorophyll b
Xanthophyll
Beta carotene
Fig. 1: The absorption spectrum of four photosynthetic pigments found in leaves of S. oleracea.
0.000
0.100
0.200
0.300
0.400
0.500
0.600
0.700
0.800
400 440 480 520 560 600 640 680 720
Wavelength (nm)
ABS
Fig. 2: The action spectrum of four photosynthetic pigments in leaves of S. oleracea.

7. Table 1: The average Rf values of four photosynthetic pigments extracted from leaves of S.
oleracea.
Pigment Rf value
Chlorophyll b 0.29
Chlorophyll a 0.48
Xanthophyll 0.61
Beta carotene 0.98
V.) DISCUSSION
The leaf of Pelargonium that was left in total darkness for a week appeared white when
stained with iodine. This was the case due to the lack of starch production. As mentioned
before, photosynthesis produces sugar monomers that can be stored as starch or transported for
use (Morgan and Carter, 2005). Light reduction due to either biotic or abiotic forces leads to a
lower photosynthetic rate (Ralph et al. 2007). Therefore, no starch is produced; explaining why
iodine, a reagent that tests for starch presence, did not stain the leaf purple. The leaf of
Pelargonium left in an environment with ample light did stain purple – signifying that starch was
present at the time of staining.
The idea that reduced light limits photosynthesis can be directed to the study of
photosynthetic rates in leaves of the same plant exposed to direct sunlight and leaves in shade.
Leaves exposed to the sunlight experienced a greater rate of photosynthesis than leaves growing
in the shade (Lichtenthaler et al. 2007). Evidence from studies shows that leaves growing with
ample sunlight are between 40-65% thicker than leaves growing in shade (Lichtenthaler et al.
2007). Since photosynthesis is occurring at a more expedient rate, it would be valid to conclude
that leaves must be larger in area and thickness in order to maximally absorb light rays as well as
store starch. Research by Ralph et al. (2007) shows that seagrasses such as Thalassia testudinum

8. and eelgrasses (Zostera marina) are particularly sensitive to reductions in light availability –
small decreases can cause large declines in growth.
C. blumei is a multicolored plant. Pigments in the plant reflect and absorb different
wavelengths – this is what causes the leaf to have multiple colors. For example, chlorophylls a
and b in plants absorb red and violet-blue light, while reflecting green light; they both appear
green (Campbell and Reece, 2005). Carotenoids produce yellow, orange, and bright red
coloration in plants while anthocyanins in plant cell vacuoles produce blues, violets, pinks,
purples, and dark reds (Morgan and Carter, 2005). One can conclude that chloroplasts are not
present in regions of the leaf that are not green. The experimental results support this hypothesis,
because the once-green region was the only area to test positive for starch after iodine staining.
Photosynthesis did not occur anywhere else in the leaf, as these pigments are not able to
photosynthesize independently.
Pigment concentration and distribution in leaves can vary as shown in the work of
Lichtenthaler et al. (2007) who observed that chlorophylls a and b were present in significantly
higher concentrations in leaves of Acer pseudoplatanus and Tilia cordata exposed to direct
sunlight than in shaded leaves of each respective plant. Inferring a potential reason for this, the
plant is maximizing the effectiveness of where chlorophyll molecules are distributed; it would be
detrimental to the plant’s energy cost to have a higher concentration of chlorophyll where little
sun reaches. To be economical, plants have more chlorophyll in areas of higher photon intensity.
The absorption of light by leaves is regulated by factors such as pigment content and leaf
morphology (Ralph et al. 2007). In aquatic plants, decreasing the available light can cause a
decrease in chloroplast density and the chlorophyll a to b ratio, an increase in chlorophyll
content, and a decrease in UV blocking pigments (Ralph et al. 2007). These observations may

9. provide another insight as to why photosynthesis is impeded when light is restricted besides the
fact that the light reactions cannot begin without photon absorption.
Pigments were separated using paper chromatography in the third phase of the study.
The theory behind this is that the 9:1 petroleum ether and acetone is relatively nonpolar, and will
draw nonpolar substances further up the paper (Morgan and Carter, 2005). Since beta carotene
migrated the furthest distance, it is the most nonpolar molecule tested. This is validated in
looking at the structure of beta carotene. Since it is a long hydrocarbon molecule with no polar
functional groups, it is a nonpolar molecule by definition. Conversely, chlorophyll b is the most
polar, and rightly traveled the least distance up the paper, due to the saturation of the molecule
with many polar functional groups.
The absorption spectra of each individual pigment (Fig. 1) were checked against a similar
figure of data to verify correctness. A figure in published material shows the relationship
between ABS and wavelength and includes experimental data for chlorophylls a and b and
carotenoids (Campbell and Reece, 2005). The data collected from this study and the published
data coincide fairly well. Errors can be explained due to the improper cleaning of cuvettes and
inadequate rinsing with distilled water. Another likely error was the failure to recalibrate the
spectrophotometer appropriately. These incorrect procedures skew data by changing ABS values.
The action spectrum of the four pigments in this study was measured as well (Fig. 2), and
coincides to a respective graph in published material (Campbell and Reece, 2005). Similar peaks
were observed in Fig. 1. These results can be used to make an inference that light in the violet-
blue portion and to a lesser extent the red portion of the visible light spectrum is the most
effective in driving photosynthesis.
VI.) WORKS CITED

10. Campbell NA, Reece JB. Biology. 7th
ed. Pearson Education Inc.; 2005. 1231 p.
Lichtenthaler HK, Ač A, Marek MV, Kalina J, Urban O. Differences in pigment composition,
photosynthetic rates and chlorophyll fluorescence images of sun and shade leaves of four
tree species. Plant Physiology and Biochemistry [database]. 2007;45(8):577-588.
Available from: ScienceDirect database by subscription. http://www.sciencedirect.com/
Morgan JG, Carter MEB. Investigating biology laboratory manual. 5th
ed. Pearson Education
Inc.; 2005. 792 p.
Nester EW, Anderson DG, Roberts Jr. CE, Nester MT. 5th
ed. McGraw-Hill; 2007. 811 p.
Ralph PJ, Durako MJ, Enriquez S, Collier CJ, Doblin MA. Impact of light limitation on
seagrasses. Journal of Experimental Marine Biology and Ecology [database].
2007;350(1-2):176-193. Available from: ScienceDirect database by subscription.
http://www.sciencedirect.com/