Extraction of Photosynthetic Pigments from Isolated Chloroplast and Hill Reaction

Authors: Daniel O. Amoah, Raphael F. Adolfo, Girlie T. Abasola

Biological Sciences Department

De La Salle University- Dasmarias
City of Dasmarias- Cavite
Hill reaction is used to determine the rate of photo reduction of 2, 6-dichlorophenolindophenol (DCPIP) when
isolated chloroplast split water molecules to produce electrons and oxygen. The experiment was aimed at isolating
chloroplast from the oregano leaves, separating the pigments presents in it and highlighting the principles of
photosynthesis through hills reaction. In the experiment, chloroplast in oregano leaves were isolated by
centrifuging green leaf suspension extracted by acetone which was further separated into various Photosynthetic
pigments by chromatography. The isolated chloroplasts were taken through hills reaction while being subjected to
heating, 3-(3,4-dicholorphenyl)-1,1-dimethylurea electron acceptor inhibitor (DCMU) and darkness. Chlorophyll
a, chlorophyll b, xanthophylls and carotene pigments were separated from the isolated chloroplast each having
different absorbance at different wavelengths. The final concentration of the dye in the positive reaction was
found to be 1.18 x 10-5 M which is 11.8% of the initial concentration of DCPIP used.
Key words: DCPIP, DCMU, chromatography, absorbance, wavelengths.
Introduction
Cells get nutrients from their environment. Virtually
all organic material on Earth has been produced by
cells that convert energy from the Sun into energycontaining macromolecules. This process, called
photosynthesis, is essential to the global carbon
cycle and organisms that conduct photosynthesis
represent the lowest level in most food chains.
Most living things depend on photosynthetic cells to
manufacture the complex organic molecules they
require as a source of energy. Photosynthetic cells
are quite diverse and include cells found in green
plants, phytoplankton, and cyanobacteria. During the
process of photosynthesis, cells use carbon dioxide,
water and energy from the Sun to make sugar
molecules and oxygen. These sugar molecules are
the basis for more complex molecules made by the
photosynthetic cell, such as glucose. Through
respiration, cells use oxygen and glucose to
synthesize energy-rich carrier molecules, such as
ATP. The overall equation for photosynthesis is as
follows:

Photosynthesis consists of both light-dependent

reactions and light-independent reactions. In plants,
the "light" reactions occur within the thylakoids of
chloroplast, where the chlorophyll pigments reside.
When light energy reaches the pigment molecules, it
energizes the electrons within them, and these
electrons are shunted to an electron transport chain
in the thylakoid membrane. Every step in the
electron transport chain then brings each electron to
a lower energy state and harnesses its energy by
producing ATP and NADPH. Meanwhile, each
chlorophyll molecule replaces its lost electron with
an electron from water; this process essentially splits
water molecules to produce oxygen. The ATP and
reduced carrier are used in the dark reactions to
convert carbon dioxide to carbohydrates.
Chloroplasts is the structure in a green plant cell in
which photosynthesis occurs. As such the

chloroplasts take active part in the process of

photosynthesis and it is of great biological
importance. A typical plant cell might contain about
50 chloroplasts per cell. The word chloroplast is
derived from the Greek word "chloros" meaning
"green" and "plastes" meaning "the one who forms".
These organelles conduct photosynthesis. They
absorb sunlight and convert it into sugar molecules
and also produce free energy stored in the form of
ATP and NADPH through photosynthesis.
Chloroplasts are unique organelles and are said to
have originated as endosymbiotic bacteria.
The Hill reaction is the transfer of electrons from
water to an electron acceptor in the presence of light
and chloroplasts. It can also be described as a lightdriven transfer of electrons from water to Hill
reagents (non-physiological oxidants) against a
chemical potential gradient (3). The hills reaction
was discovered by the biochemist Robin Hill from
the University of Cambridge in 1937. It
demonstrates that oxygen (O2) is produced in plants
in a process that is separate from the process that
converts carbon dioxide (CO2) to sugars. Hill's
observation made a basic underlying refinement to
our modern understandings of the process of
photosynthesis.

indication to know if there has indeed been the

release of electrons. The chemical reaction involved
in the reduction of DCPIP is shown below.
.

the concentration of the dye which is reduced in the

hills reaction can be calculated using the formula;
Using the formula:
Cr =

Co(Ao-Ac)
Ao

In Hills reaction, thylakoids are combined with the

Hill reaction assay buffer containing 2, 6dichlorophenolindophenol (DCPIP) and exposed to
light. At 30 second intervals the absorbance of the
DCPIP is measured. When the absorbance values are
plotted versus time, the slope of the line is a measure
of the rate of Photosynthetic electron transfer from
water to DCPIP. Hill assay buffer is slightly
hypotonic and causes the chloroplast outer
membranes to rupture, releasing the stromal
enzymes and allowing the thylakoids to be exposed
to the Hill reaction buffer. To conveniently measure
rate of the Hill reaction there is the need to use a dye
that changes color as it is reduced. 2,6dichlorophenolindophenol (DCPIP), which is blue in
its oxidized form turns colorless in its reduced form.
As such DCPIP do not only serve only as an
artificial electron acceptor but more so a clear

where:
Cr = the dye reduced in moles/liter
Co = the original dye concentration in moles/liter
Ao = the absorbance of the mixture at the start
Ac = the absorbance of the mixture at the end of the
experiment.

The rate of hills reaction can be affected by

temperature stress, presence of inhibitor and
darkness. When isolated chloroplasts are heated in
hills reaction, they are denatured and are made to
lose their overall shape and function and hence their
efficiency. They are rendered inactive (killed by
boiling)
and
cannot
reduce
DCPIP by
photosynthesis. Under normal circumstances, DCPIP
will change colors as chloroplasts split water

molecules in the presence of sunlight, but upon

boiling or intense heating, the amount of DCPIP will
virtually stay the same since there will be no or little
reduction of DCPIP.
3-(3,4-dicholorphenyl)-1,1-dimethylurea (DCMU) is
an electron acceptor inhibitor that prevents DCPIP
from being reduced. DCMU is a very specific and
sensitive inhibitor of photosynthesis. It blocks
the plastoquinone binding site of photosystem II,
disallowing the electron flow from where it is
generated, in photosystem II, to plastoquinone. This
interrupts the photosynthetic electron transport
chain in photosynthesis and thus reduces the ability
of the plant to turn light energy into chemical energy
(ATP and reductant potential). DCMU only blocks
electron flow from photosystem II, it has no effect
on photosystem I or other reactions in
photosynthesis, such as light absorption or carbon
fixation in the Calvin cycle. However, because it
absorbs
electrons oxidized from
water
in photosystem II, it creates an electron deficiency
in photosystem I and as such effectively shuts down
"linear" photosynthesis by blocking the reduction of
NADP+ to NADPH. In hills reaction in prevents the
reduction of DCPIP.
Darkness is another major factor that affect hills
reaction. Chloroplasts kept in the dark do not receive
any light energy whatsoever, therefore the water
molecules are not provided the energy to split to
produce oxygen and release electrons. This situation
is contrasted by chloroplasts incubated in the light
are able to keep flow of electrons and hence
continue the process of photosynthesis.

Methodology
The materials and reagents used in the experiment
were oregano leaf sample, pre-chilled mortar and
pestle, cheese cloth, chromatography papers, test
tubes, carbon paper, scissors, microscope centrifuge,
cuvette, water bath, ice baths, spectrophotometer,
pure acetone, petroleum ether, distilled water, 0.0035
NaCl, 5 x 10-7 M 3-(3,4-dicholorphenyl)-1,1dimethylurea (DCMU), 0.1 M K2HPO4 Buffer (pH
6.5), and 1 x 10-4 M dichlorophenolindophenol

(DCPIP).
The experiment was categorized into five (5)
procedures; Isolation of chloroplast, extraction of
photosynthetic pigments, elution of pigments,
determination of chlorophyll content, and hill
reaction.
To isolate the chloroplast, the leaves were washed
with distilled water, the veins of the leaves were
removed and they were cut in small pieces. The cut
leaves were placed in pre-chilled mortar and 20 mL
off pure acetone was added. The leaves was grind
thoroughly using pestle with subdued lighting. The
homogenate was filtered using cheesecloth and the
filtrate was collected from a 100 mL beaker. The
green suspension was transferred to a cold 50 mL
centrifuge tube and it was centrifuge for 1,500 rpm
for 1 minute at 4 degree Celsius to pellet the
unbroken cells and fragments. The supernatant
liquid was decanted into a clean centrifuge tube and
was centrifuged again for 4,000 rpm for 10 minutes.
The pellet formed during the centrifugation
contained chloroplasts. It was decant and the
supernatant was discarded. The chloroplasts pellet
were suspended in 5.0 mL of cold 0.035 M NaCl. To
disrupt the packed pellet a vortex was used. It was
then observed under oil immersion objective of the
microscope.
To extract the photosynthetic pigments, five strips of
chromatography paper with a puncture on top were
obtained. Using a pencil, the chromatography paper
was marked at 1.5 cm from the bottom end by
drawing a light line. Using the capillary tube, crude
chlorophyll suspension was applied along the line. It
was dried and the application was then repeated for
10 times. The 250 mL beaker was wrapped with
carbon paper. 20 mL 9:1 petroleum ether was
poured into the beaker including acetone. Then the
string was inserted into the punctured hole at the top
end of paper. It was lowered at the bottom edge
(with extract) towards the solution. Same steps were
done for the other strips. The beaker was covered
with petri dish for the chromatogram to develop.
Chromatogram development was allowed to occur
until the solvent had reached 0.5 -1 cm from the top
of the paper. The chromatogram was then removed
and was dried.

To elute of pigments, the 5 strips of bands were cut

according to color. The number of colors was
counted and the same number of tubes was prepared
containing 4 mL of acetone. The colored bands were
put in one tube. The tubes were shaken for 5 to 10
minutes to produce partially purified pigments in
each tube.
The chlorophyll content was determined using
spectrophotometer. In this, the solution of the eluted
pigments was transferred to separate cuvette using a
pippetor. A blank solution consisting pure acetone
was also placed in a cuvette. The absorbance of each
sample from 350 nm to 700 nm at 25 nm interval
were recorded and interpreted.

measured every 10 minutes interval for 30 minutes.

The test tubes were dried before inserting them in
the spectrophotometer. The absorbance of the test
tube serving as the dark control wrapped in dark
paper and aluminum foil was measured at the end of
the experiment . .

Results and Discussion

Below are pictures of isolated chloroplast as seen
under the oil immersion objective.

To perform the Hill reaction, 5.0 mL portion of

diluted chloroplast suspension was heated in a
boiling water bath for 5 minutes to render them
inactive. Inactivated chloroplasts were removed
from the boiling water and were placed in an ice
bath to cool. The active chloroplast suspension was
remained in 4 degree Celsius.
5 test tubes were prepared each containing 3.0 mL of
0.1 M K2HPO4 Buffer. Active Chloroplast were
added to three (3) of these tubes and were covered
with foil to avoid premature photosynthetic reaction.
One of the three was immediately wrapped in dark
paper and aluminum foil to serve as the dark control.
DCMU was added to one of the active chloroplast to
serve as the inhibitor control while the other was
maintained at normal conditions and served as the
light control. Heated chloroplast were used in one of
the test tubes while the other contained no test
Chloroplast at all serving as the blank. 1.0 mL of
water was added to each of the test tubes.
The spectrophotometer was adjusted to 600 nm. 1
mL DCPIP dye was added to the test tubes
containing the light control, inhibitor, and heated
chloroplast. They were then mixed by swirling. A
sample of each was collected and put into a cuvette
and the absorbance was measured at 600 nm. After
the initial measurement, these test tubes were placed
in a beaker of ice water and it were illuminate with a
strong light. The absorbance of each tube was

Figure 1- isolated chloroplast observed under

microscope.

Below are pictures of the chromatogram obtained

from the experiment after the photosynthetic
pigments were extracted.

Figure 2. Chromatogram of pigments present in

oregano leaf.

Table showing Total Dye Reduced

Initial concentration of DCPIP = 1 x 10-4 M

Table 1.1
Positive
Control
Dark Control
Heat
Inhibitor

Table showing absorbance of the positive control

(light control), dark control, heated chloroplast and
inhibitor at different times
Time
(min)

Positive
control

Dark
control

heat

inhibitor

0.813

0.789

0.247

0.078

10

0.718

0.619

0.28

0.174

20

0.712

0.676

0.30

0.169

30

0.717

0.695

0.282

0.177

600nm absorbance of under various factors

0.9
0.8
Dark control

heat

0.5

ABSORBANCE 0.4
0.3
inhibitor

0.2
0.1
0
0

10 15 20 25 30 35

TIME (MIN)

Figure 3. Graph of absorbance of

chloroplast exposed to different factors.

Table 1.2

% dye
reduced
11.8%

1.19 x 10-5 M
-1.42 x 10-5 M
-1.27 x 10-4 M

11.9%
-14.2 %
-12.7 %

Discussion

The chart below shows summarizes the data

presented on table 1.1 above

0.7
Positive control
0.6

Total Dye
Reduced
1.18 x 10-5 M

isolated

In the first part of the experiment which involved

isolation of chloroplast, the whole experiment was
performed under subdued light. This was done to
avoid chlorophyll degradation or to prevent photo
leaching since chlorophyll degrades when exposed
to heat and light. The leaves were grinded with a
mortar and pestle to increase the surface area for the
extraction of the green pigment. The first part of the
centrifuging at 1500 rpm for 1 minute was done to
separate the heavy organelles (cellular debris) like
nucleus from the cell. The second centrifuging
separated the chloroplast from the other organelles.
The chloroplast pellets were suspended in 0.035 M
NaCl to prevent osmosis and rupturing of the
membrane or organelles as 0.035 M NaCl
is isotonic to chloroplast.
In the extraction of the photosynthetic pigments, the
chromatography papers were held with forceps and
not with bear fingers, as fingerprints can affect the
formation of the chromatogram. Again, the fingers
may be stained with some form of pigments which
may affect the formation of the chromatogram. 9:1
Petroleum ether: acetone was utilized as the solvent
placed in the beaker during the chromatography.
This was because different pigments have different
polarities, molecular weight, solubility and varying
affinities to different solvents. The pigment that is
more non polar is more likely to adhere to the
petroleum ether as it is highly non-polar. On the
other hand, the pigments which are slightly polar
may adhere more to the acetone. The pigments
moved up the chromatography paper via capillary

action and by this principle they were separated. As

the solvent move up the chromatography paper,
different pigments move along with them. And so
the pigment which has the highest affinity to the
polar solvent is expected to be at the topmost
followed by others. Again the beaker was wrapped
with carbon paper to prevent light from reacting
with the pigments. This was because photosynthetic
pigments becomes denatures when it reacts with
light and as such this can affect the chromatogram.
Four different pigments were eluted from the
chromatography paper. These were of the colors,
deep green, yellowish green, yellow and yellowish
orange. The deep green color pigment was found to
be chlorophyll a, the yellowish green was
chlorophyll b, the yellow pigment was xanthophyll
and the yellowish orange was carotene. This showed
that chlorophyll is not the only pigments found in
green leaves. Chlorophyll a and b have a larger
molecular size and molecular weight than carotene
and xanthophyll and as such they ascended the
chromatography paper slower than carotene and
xanthophyll. Carotene is nonpolar pigment whereas
xanthophyll are polar pigments due to presence of
additional hydroxyl groups on xanthophyll. Since
carotene was more soluble in the nonpolar solvents,
they moved at greater rate than that of xanthophyll
and appeared topmost. Chlorophylls a and b provide
the green color and absorb the light energy needed
for photosynthesis. However, other accessory
pigments, such as yellow xanthophyll and orange
carotenes are also present in the chloroplasts and
collect additional light energy for photosynthesis.
In the Hill reaction, the Positive control composed
of Buffer, Active chloroplast, and water had no
modified variable. This was why it showed the
highest absorbance as seen on the graph, it also
showed a high concentration of reduced DCPIP after
the experiment.
The chloroplasts placed under dark conditions and in
the absence of CO2 the artificial electron acceptor
was oxidized but not reduced terminating the
process. Again the absence of light prevents the
water molecule from being split to release electrons
which is required in the reduction of the DCPIP. In

this experiment the ideal results for a dark control

was not achieve. This was due the fact that the
absorbance was measured at 10 minutes interval as
the light controls. This allowed light to get into the
tube and for reaction to continue. This is what
accounted for the high value of reduced DCPIP in
the dark control.
The heated Chloroplast had a low absorbance. This
was due to the fact that heat denatures chloroplast
and render them inactive making them less
functional. If Chloroplast are less functional, its
activity to reduce DCPIP is as well reduced. The
negative value of reduced DCPIP concentration was
due to the fact that when the DCPIP was added to
the content, it took a while before the absorbance
was taken. Hence the slightly active heated
chloroplast had some time to reduce some part of
DCPIP before it the initial reading was actually
taken. Theoretically at zero minutes there
is supposed to be no reaction taking place between
anything in the tube, therefore the absorbance
reading obtained from different tubes should be the
same. However, absorbance readings obtained for
different tubes were significantly different hence it
can be concluded that the difference was due
to varying amounts of reduced DCPIP
Since reduced DCPIP can only be obtained due to
the occurrence of the Hill Reaction it can be said
that the time taken as the chloroplasts suspension
was added to when the zero minute reading was
taken allowed for the hill reaction to proceed. As
such it is important to immediately measure the
initial absorbance upon the addition of DCPIP.
For the inhibited reacted, DCMU (3-(3,4dichlorophenyl)-1,1-dimethylurea) served as the
inhibitor of electron transport. It blocks passage of
electrons from primary acceptor of photosystem II
called plastoquinone and it also prevents DCPIP
from being reduced. It is also observed that the
degree of inhibition depends on its concentration.
Higher concentration of DCMU makes the electrons
to be blocked from passing the plastoquinone and
there is little reduction in DCPIP. While Low
concentration of DCMU leads the electrons to be
moderately inhibited from passing plastoquinone
and the DCPIP is continuously reduced.

4. Hall,
David
Oakley
(1981). Photosynthesis (3rd ed.). University
of London: Edward Arnold. pp. 14, 79, 84.
Conclusion
It can be concluded that chloroplast were
successfully isolated from oregano leaves in the
experiment. Photosynthetic pigments of chlorophyll
a, chlorophyll b, carotenes and xanthophylls were
also successfully isolated and eluted in the
experiment. These photosynthetic pigment are
responsible for the absorption of light energy
required in Photosystem II. It can be deduced from
hills reaction that light and chloroplast are the most
important component required for photosystem II to
occur in green plants. Without the presence of light
water cannot be split to release electrons which are
taken up by DCPIP and measured using
spectrophotometer. Thus the greater the light
intensity, the greater the amount of electrons
produced and the greater the NADPH or reduced
DCPIP formed.

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