EXPERIMENT 9

PHOTOSYNTHESIS: Pigments of the Chloroplasts

Photosynthesis uses energy absorbed by several photosynthetic pigments. The light

energy used for photosynthesis is not absorbed by just a single type of pigment. Instead,
several different photosynthetic pigments with different absorption spectra absorb the energy
that is eventually used for photosynthesis. In photosynthetic organisms including plants, protists,
and bacteria, these photosynthetic pigments include chlorophylls, carotenoids, and phycobilins.
Each of these absorbs light at different parts of the visible light spectrum and has a different
function in the plant.

Chlorophylls: In plants photosynthetic pigments two chlorophylls predominate: chlorophyll a

and chlorophyll b. These two molecules differ only slightly in their molecular structure. Both have
a complex ring structure similar to that of the heme group of hemoglobin. In the center of each
chlorophyll ring is a magnesium atom, and attached at a peripheral location on the ring is a long
hydrocarbon “tail,” which can adhere the chlorophyll molecule to proteins in the hydrophobic
portion of the thylakoid membrane. They are polar (water-soluble) pigments that act as electron-
transporters in photosynthesis and provide green colors to plants. Chlorophyll a produces a
bright green to blue-green band, and chlorophyll b produces a dull olive to yellow-green band.
Chlorophyll a is more soluble than chlorophyll b and will travel farther up the paper with the
solvent (carbon tetrachloride) used in the experiment.

Carotenes: Carotenes are non-polar (fat-soluble) hydrocarbons. This class of pigments ranges
in color from yellow to red. They are also essential as antennal pigments in photosynthesis.
Carotene itself produces a yellow-orange band.

It is possible to separate these pigments from one another by the use of paper
chromatography. Paper chromatography is a laboratory technique that separates components
within a mixture by using the differential affinities of the components for a solvent and for the
paper through which they pass.
Principle of Paper Chromatography:
• Capillary Action – the movement of liquid within the spaces of a porous material due to
the forces of adhesion, cohesion, and surface tension. The liquid is able to move up
the filter paper because the cohesive forces (attraction between the liquid molecules),

Naira Mitz Ampuan BS Biology student bitchy_princess24@yahoo.com

 together with adhesive forces (attraction between the liquid and the paper) are
stronger than the force of gravity.
• Solubility – the degree to which a material (solute) dissolves into a solvent. Solutes
dissolve into solvents that have similar properties (Like dissolves like).This allows
different solutes to be separated by different combinations of solvents. Separation of
components depends on both their solubility in the mobile phase and their differential
affinity to the mobile phase and the stationary phase.

Because pigments have unique attributes that define them, several color bands would be
expected if there is more than one present. Based on the bands formed on the filter paper, the
retention factor, or Rf, value can be calculated for each pigment. This is done by dividing the
distance the pigment traveled by the distance the solvent traveled.

Eqn. 1 Rf = distance pigment traveled

distance solvent traveled

MATERIALS:
 Papaya leaf tissue (Carica papaya)
 chromatography paper
 chromatography solvent (carbon
tetrachloride)
 chromatography chamber with lid
 ruler
 mortar and pestle
 capillary pipette
 calcium carbonate (added during the
leaf extraction process to prevent
the chlorophyll from degrading to
pheophytin)
 anhydrous sodium sulfate

Naira Mitz Ampuan BS Biology student bitchy_princess24@yahoo.com

METHODOLOGY:
1. (Preparation of pigment solution) In a mortar, the following were added: 1g of papaya
leaf tissue, and 15mL acetone. Using the pestle, the mixture is ground for 5 minutes or
until the solution is dark in color. This step is to disrupt the plant cell walls.
2. (Application of the pigments) Using the capillary pipette, a line of the pigment solution
was applied onto the chromatography paper. After dying the stain, the procedure is
repeated. It was first ensured that the pigment stain is TOTALLY dry before running the
chromatogram.
3. (Developing the chromatograms) The stained paper was placed into the developing
chamber. The lid was placed. The solvent was allowed to migrate upward until the
solvent front is 1cm from the top of the paper. Then immediately, the paper was removed
and the solvent front was marked.
4. (Obtaining Rf values) After allowing the chromatogram to dry, the distances traveled by
each pigment band were measured. After which, Rf values were computed using the
above stated equation.

RESULTS AND DISCUSSIONS:

Band Distances and Rf values
Band Distance Band Color Pigment Rf
(cm)
1 6.8 Yellow orange carotene 0.97

2 5.4 Blue green Chlorophyll a 0.77

3 3.6 Yellow green Chlorophyll b 0.51

Solvent front (in cm) = 7.0

It is noted that there is a trend in the movement of the color bands, representing the
characteristic polarity of the pigment. Using a nonpolar solvent (carbon tetrachloride), the
nonpolar color bands (pigments) move farther. That is to say, carotenes (nonpolar) travels
further the two polar chlorophylls. Such that,

Continuum on Distances Traveled:

Naira Mitz Ampuan BS Biology student bitchy_princess24@yahoo.com

 Carotene > Chl a > Chl b
Polarity Continuum:
Carotene < Chl a < Chl b

Moreover, as the Rf value increases, the degree of polarity decreases, such that those
located at the top of the chromatogram is the most nonpolar. It is to be noted that the above
explained phenomenon is only applicable for experiments utilizing a nonpolar solvent. For the
case of a polar solvent, an opposite outcome is expected. That is, the pigment band that travel
the farthest, the one with the highest Rf value, is the most polar.

GUIDE QUESTIONS
Q: Define Fluorescence.
A: Fluorescence is a process in which an atom or molecule emits radiation in the course of a
transition from a higher to a lower electronic state. A more restricted definition, applicable
particularly to atomic processes, excludes the special case, known as resonance radiation, in
which the wavelength of the emitted radiation is the same as that of the exciting radiation. The
term fluorescence is further restricted to phenomena in which the time interval between the
acts of excitation and emission is small, of excitation and emission is small, of the order of
10-8 –10-3 second. This distinguishes fluorescence from phosphorescence, where the
time interval between absorption and emission may extend from 10-3 second to several
hours. The phenomenon of fluorescence was known by the middle of the century. It was the
British scientist Stokes who first made the observation that the fluorescing light has longer
wavelengths than the excitation light, a phenomenon that has become to be known as Stokes
shift.
One of the most prominent processes is the chlorophyll fluorescence, which is readily
observed when a solution of chlorophyll (green leaves) is illuminated with a strong source of
continuum radiation. The red colour characteristics of fluorescence readily appear in a sample
cuvette. Light energy is absorbed by chlorophyll within plant tissues and used to drive
photochemistry of photosynthesis and thus become chemical energy available to the plant for
growth. Light in the waveband 400-700 nm is absorbed by chlorophyll and used for
photochemistry. This light is termed photosynthetic ally Active Radiation (PAR). Although
fluorescence emission from whole leaf system is too weak to be viewed with the naked eye, it
can be observed from the illuminated extracts of a chlorophyll solution. Peak fluorescence
occurs in the red region of the spectrum (685 nm) and extends into the infrared region to around

Naira Mitz Ampuan BS Biology student bitchy_princess24@yahoo.com

800nm. The fluorescence from chlorophyll has been used extensively in the past to characterize
and investigate agricultural plants.

Q: Define Phosphorescence.
A: Phosphorescence is a specific type of photoilluminescence related to fluorescence. It
corresponds to light emissions accompanying radiative electronic transitions from triplet to
ground states of pigment molecules. Unlike fluorescence, a phosphorescent material does not
immediately reemit the radiation it absorbs. The slower time scales of the reemission are related
with “forbidden” energy state transitions in quantum mechanics. As these transmissions occur
less often in certain materials, absorbed radiation may be reemitted at a lower intensity for up to
several hours.
In simpler terms, phosphorescence is a process in which energy absorbed is released
relatively slowly in the form of light. This is in some cases the mechanism used for glow-in-the-
dark materials which are charged by exposure to light. Unlike the relatively swift reactions in a
common fluorescent tube, phosphorescent materials used for these materials absorb the energy
and store it for longer time as the subatomic reactions required to reemit the light occur less
often.

CONCLUSIONS:
Results of the paper chromatography demonstrate that the chloroplasts of a Carica
papaya leaf contains of three types of plant pigments- chlorophyll a, chlorophyll b, and carotene.
Researchers also conclude that among the three pigments, carotenes are the most non polar.
Between the two chlorophylls, although they have similar polarity (attributed to their similar
structures) their solubility varies. Chlorophyll b is less soluble than chlorophyll a.

REFERENCES:
G.H.Krause and E Weis. Ann. Rev. plant. physiol. plant. Mol. Biol. Vol 42, 313-349 (1991).
WL Buller and Kitajima. Biochim. Biophys. Acta. Vol 396, 72-85 (1975).
E Weis and J A Berry. Biochim. Biophys. Acta.Vol 894,198-208 (1940).
E W Chappelle, J E McMurtrey III and M S Kim. Remote. Sens. Environ.Vol 36, 213-218
(1991).
A Rosema and H Zahn. Remote Sen. Environ.Vol.62, 101-108 (1997).

Naira Mitz Ampuan BS Biology student bitchy_princess24@yahoo.com