Proceedings of the Third International Conference on Mathematics and Natural Sciences

(ICMNS 2010)

ISOLATION OF CHLOROPHYLL a FROM SPINACH AND

ITS MODIFICATION USING Fe2+ IN PHOTOSTABILITY
STUDY

Rachma Ditya Sandiningtyas and Veinardi Suendo*

Inorganic and Physical Chemistry Division, Department of Chemistry, Faculty of
Mathematics and Natural Sciences, Institut Teknologi Bandung,
Jalan Ganesha 10, Bandung 40132, Jawa Barat, Indonesia
*Corresponding author. Tel.: +62 222502103, Fax: +62 222504154, email:
vsuendo@chem.itb.ac.id

Abstract. Chlorophyll is a natural pigment that is usually found in many green

plants. Because of its structure that have conjugated double bonds, the chlorophyll
can be explored its potential as optical materials. In this research, chlorophyll was
isolated from the leaves of spinach (Amaranthus Spec div.). The separation of
chlorophyll a was conducted by using the sucrose column chromatography
technique. The pure chlorophyll a then further modified by replacing the central
ion of chlorophyll a, which is Mg2+, to become Fe2+ to form Fe-pheophytin a. The
optical characteristics and the purity of chlorophyll a, pheophytin a, and Fe-
pheophytin a were investigated using spectroscopic methods. The different
characteristics of chlorophyll a and Fe-pheophytin a were distinguished from each
other by the examination of their absorption and emission spectra. The absorbance
measurements revealed the presence of a shift in the absorption peak of Fe-
pheophytin a with respect to chlorophyll a. The maxima of absorption of Fe-
pheophytin a were observed at 393 nm and 627 nm, while the chlorophyll a gives
413 nm and 668 nm that represent the Soret and Q bands, respectively. Based on
fluorescence measurements, it was observed that Fe-pheophytin a gives a red shift
relatives to chlorophyll at 702.5 nm. The photostability of chlorophyll a and Fe-
pheophytin a were investigated by comparing the decay lifetime based on the
fluoresescence measurement. Here, the decay lifetime of Fe-pheophytin a is 43.88
seconds, which is much longer than 12.79 seconds of chlorophyll a. This result
suggests that Fe-pheophytin a is more stable intrisically than chlorophyll a.

Keywords: chlorophyll a, decay lifetime, Fe-pheophytin a, optical material, optical

stability.

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1 Introduction
Chlorophyll is a natural pigment found in every green plant. Chlorophyll is an
important photosynthetic pigment in plants that absorb the sunlight at wavelength
of red, blue, and purple. The absorption of light by chlorophyll occurs due to the
presence of porphyrin structure that coordinated with magnesium ion. Chlorophyll
is used as an absorber of the sunlight for the process of photosynthesis in plants.
Because of its ability to absorb light, chlorophyll may be explored its potential as
optical materials. The example of its applications, which is now widely studied, is
for the application of solar cell. In many researches of solar cell, the researchers
tried to fabricate the solar cells through many ways. For example by adding the
chlorophyll from the plant into a photovoltaic device where the chlorophyll acts as
an active material, then the device can generate the electron-hole pairs to produce
the electricity. Porphyrin itself is an interesting organic molecule familiy that is
known as organic optoelectronic materials, which can interact with photon through
both absorption and emission processes. Many of researchers were interested in
chlorophyll due to its porphyrin-metal-complex structures which has a high
molecular symmetry of D4h. Porphyrin has conjugated double bonds structure that
allows the electron to be easily excited through absorption process of
electromagnetic waves. Based on the structure and its interaction with light, the
chlorophyll has a good potential as optical materials for solar cells and light
emitting diode (LED) applications. The recent studies report that the fabrication of
the solar cells based on chlorophyll directly from plants were not successful due to
its low stability, even in the form of complete devices. Many previous works
mentioned that the the low stability of chlorophyll directly isolated from the leaves
is due to the lack of other pigments that usually present in the leaf. Thus, the
chlorophyll has to be stabilized prior to any device fabrications. In this study, the
stability of chlorophyll after isolation from the leaves was studied and compared
with its derivate after modification in the form of Fe-pheophytin a. The modification
was carried out by replacing the central atom of chlorophyll with Fe2+. The main
objective of this research is to isolate and purify the chlorophyll a from the spinach
leaves, and then carry out the chemical modification using Fe2+ ions to improve its
stability against light exposure.

2 Methods
2.1 Preparation of crude Chlorophyll extraction from spinach

All chlorophyll extractions were performed under dim green light to minimize
photo-oxidative reactions.2 Fresh spinach leaves were purchased from local
market. Spinach leaves were separated from the stem and then weighed about 100
gram and put into a blender. Cold acetone was added as much as 500 mL. Then
the spinach leaves were grinded in a blender for about 3 minutes. The filtrate of
crude chlorophyll was separated using a Buchner funnel with a Whatman filter
paper No.1. The residual solids that are not filtered then washed with 100 mL of
acetone. Then the filtrate was added with dioxane approximately one seventh of its
volume. After that, the mixture was added with deionized water as much as one
seventh of the volume of the mixture by dropwise and then stirred using a
magnetic stirrer. The mixture was precipitated by stored in the freezer at -20 C

860
for 1 hour until dark green precipitate obtained at the bottom of the solution and a
yellow liquid on top. The precipitate of crude chlorophyll was separated by filtration
using two layers of Whatman filter paper No. 1 with a Buchner funnel. The solution
then were added with dioxane as much as one seventh of the volume of the
solution and also deionized water as much as one seventh of the volume of the
solution while slowly stirred. The precipitation was then performed for the second
time in the same way at -20 C for 1 hour. After that the precipitate was filtered
using a Buchner funnel and two layers of Whatman paper No.1. The crude
chlorophyll solids were filtered and diluted with acetone until colorless paper
return. Then a solution of crude chlorophyll is evaporated from the solvent so that
only the remaining solid green crude chlorophyll left. Thus, the chlorophyll were
dissolved using the small volume of diethyl ether in order to obtain the chlorophyll
solution prio to any further separation using column chromatography.

2.2 Sucrose Column Preparation

Sucrose was dried in the oven with a temperature of 80C for several hours until
the sucrose is completely dry and free from water. The dry sucrose was removed
from the oven and cooled for a while in a dessicator. After that it was sieved to
obtained sucrose with homogeneous particles size. Finally, the sucrose was
conserved in a sealed bottle prior to any column preparation.

To make a sucrose column, the most important thing that has to be noted here is
that the column should be completely dry and free from water, and also the
column must be connected to a vacuum pump. The column was filled with
previously prepared dried sucrose by slowly inserting with the help of the vacuum
pump and followed by compaction through pressing the sucrose from the top of the
column. After the column has been filled with sucrose, then the petroleum ether
was introduced slowly into the column.

2.3 Separation of Chlorophyll a Using Sucrose Column

Chromatography

Chlorophyll solution in diethyl ether that had been prepared earlier was introduced
into the sucrose column that has been filled with petroleum ether. After that, the
elution was performed by adding the petroleum ether until the yellow band is
appeared. The elution was then continued with different eluent, which is diethyl
ether in petroleum ether until yellow green, blue green, and gray band were
appeared and separated. Furthermore, the elution was continued by changing the
eluent with 2-propanol in petroleum ether until all three bands are completely
separated. Then both bands were taken using a spatula in order to obtain
chlorophyll a (blue green), chlorophyll b (yellow green), and pheophytin a (gray).

2.4 Modification of Chlorophyll a Using Fe2+

Pure chlorophyll a was dissolved in petroleum ether then added by glacial acetic
acid solution until its color changed to gray, indicating that chlorophyll a has
become pheophytin a.1 Then the solution of pheophytin a was added with FeCl2
which was dissolved in glacial acetic acid and stabilized with small amount of
ascorbic acid. After that sodium acetate solution was added, then the solution was

861
heated in 80C for 30 minutes until the solution appeared in different color,
indicating that the Fe-Pheophytin a has been succesfully formed. To separate the
Fe-pheophytin a in petroleum ether, the solution was inserted into the separating
funnel and added with petroleum ether and excess of water until two separated
phases were obtained. The Fe-pheophytin a may be obtained by recovering the
petroleum ether phase at the top of the funnel.

2.5 Characterization

Chlorophyll a, pheophytin a, and Fe-pheophytin a were characterized using UV-Vis

spectrophotometer and compared with literature ones to investigate their purity. To
prove that Fe2+ has been entered into pheophytin a, Fe-pheophytin a was
characterized by ESI-MS. The emission characterization was also conducted to
chlorophyll a and Fe-pheophytin a. The stability test was carried out with the laser
radiation at a wavelength of 405 nm. This test was performed in the solution of
transparent polymer PMMA (polymethylmethacrylate) in chloroform. The sample
solution then was inserted into a capillary tube. This Capillary tube then was
tested for its stability by laser illuminated method as illustrated schematically in
Figure 1. The presence of PMMA is necessary to reduce the evaporation rate of the
solvent during measurement, while the capillary tube method improves the signal
detection due to the waveguide effect.

Capillary
tube
Laser

Spectrometer
Fluorescence
signal

Fiber optic

Figure 1 Experimental set up for optical stability measurement

862
3 Results and Discussion
3.1 Isolation of Chlorophyll from Spinach Leaves

In this study, spinach leaves were choosen as the source of chlorophyll because it
is not only containing chlorophyll in abundance, but also easily to be found in the
local market. In this isolation method, cold acetone is added on the leaf to break
out cells, thus the chlorophyll molecule can be extracted easily. To increase the
efficiency of the extraction process, the leaves were ground using a blender, which
also increases the leaf surface. The result of this process is dark green colored
liquid containing chlorophyll.

Then the next process is to precipitate the chlorophyll by adding the dioxane and
the excess of deionized water into the crude chlorophyll extract. The function of the
addition is to precipitate chlorophyll from extract solution by the formation of
dioxane-chlorophyll-water complex. This precipitation process should be kept in
the refrigerator for 10 hours at -12C. After that, the dark green precipitate
containing chlorophyll is filtered to separate from the clear yellow colored liquid.
The green precipitate obtained from the filtration process was dissolved again by
using acetone. This solution which is contain chlorophyll then was added with
more dioxane and excess of water, thus the second precipitation occurs in the
refrigerator to obtain more chlorophyll with higher purity.

3.2 Sucrose Column Preparation and Separation

The separation method of chlorophyll a and b was carried out using sucrose
column chromatography technique.1 In this method, the sucrose must be free of
water. Therefore, to ensure this condition, the sucrose should be kept in the oven
first for about six hours at80 C to remove all waters. Before preparing the sucrose
column, it must be ensured that the column is completely dry. Then the prepared
dry sucrose was inserted into the column which has been connected to a vacuum
pump to make the sucrose completely compact inside the column. After the
sucrose is almost filled the column, then the column should be filled with
petroleum ether until all the sucrose is submerged. This must be done immediately
in order not to let the sucrose to interact with the water in the air.

In the separation process of chlorophyll a and b, all solvent that used as eluent
must be also free from water, thus they must be distillated before used. After that,
the chlorophyll solution could be introduced into the column of sucrose, and then
eluted using petroleum ether until the yellow band appeared at the bottom and the
dark green band stayed still on the top of the column. The yellow band was
possibly carotenoid that has been separated from chlorophyll. Then separation was
continued by adding the eluent until the yellow carotene band goes out of the
column. After that, the eluent was replaced using 10% of diethyl ether in
petroleum ether. This eluent is used to dissolve some of the chlorophyll that has
been stuck at the top of the sucrose column. This is because the chlorophyll is very
soluble in diethyl ether. The process was continued until the blue-green band and

863
the yellow green bands appeared, but were not separated yet. The separation then
continued with the elution using 0.5% of 2-propanol in petroleum ether until those
bands well separated. Variations eluent is used because of the polarity of each
eluent are different, with the following order: petroleum ether < diethyl ether < 2-
propanol. The difference in polarity of the eluent makes the chlorophyll a and b
can be separated in the sucrose column. In this work, 0.092 gram of pure
chlorophyll a and 0.101 gram of pure chlorophyll b were obtained from 100 gram
spinach leaves.

3.3 Modification of Chlorophyll a with Fe2+

At this step, the chlorophyll a was modified by replacing the central ion with Fe2+.
The process was carried out by removing Mg2+ from chlorophyll a in petroleum
ether with boiling point fraction of 60-80C by acidification to form pheophytin a as
shown its molecular structure in Figure 2.

Figure 2 Molecular structure of Pheophytin1

With the loss of Mg2+ as its center ion, the chlorophyll will turned into gray colored
substance called pheophytin a. In order to introduce the Fe2+ ion into the
pheophytin a, the FeCl2 solution in glacial acetic acid was prepared and stabilized
with ascorbic acid, and then mixed with the pheophytin a solution in petroleum
ether. The ascorbic acid is used as an antioxidant to prevent the Fe2+ to be oxidized
to become Fe3+. The process takes place at a temperature of 80C for 30 minutes.
Reaction temperature must be kept constant in order not to damage the
chlorophyll. The method that was used in this reaction is the common reflux
method under nitrogen atmosphere. The reflux method is used to prevent the
excess evaporation of solvent during the reaction, while the nitrogen atmosphere
prevents the oxidation of Fe2+ due to oxygen gas in the air. The color of refluxed
solution changes during the reflux from the blue-green color of chlorophyll a, then
turns into the gray color indicates the formation of pheophytin a, which finally
becomes bright green when the Fe-pheophytin a is formed.

864
3.4 UV-Vis Absorption Analysis

Chlorophyll a, chlorophyll b, pheophytin a, and Fe-pheophytin a are obtained, then

characterized to determine the absorbance in the visible light region. The results of
characterization using UV-Vis absorption spectrophometry, can also be used to
predict the purity of chlorophyll a, chlorophyll b, pheophytin a, and Fe-pheophytin
a by comparing their spectrum with literature ones. Figure 3 shows the
comparison between the absorption spectrum of chlorophyll a measured in this
study and that obtained from literature.

1.6
417,75 result
1.4 literature
1.2

1.0
Absorbance

0.8
413
0.6
659,25
0.4
668
0.2

0.0

300 400 500 600 700

Wavelength (nm)

Figure 3 Absorption spectra of chlorophyll a

Based on the absorption spectrum of chlorophyll a, can be understood that the
chlorophyll a has two absorption regions that similar to other porphyrin derivatives
in the wavelength ranges of 380-480 nm and 560-680 nm, referred to as the Soret
and Q bands, respectively. The UV-Vis absorption transitions usually present in
the form of absorption bands. This is due the contribution of vibration and rotation
on the electronic transitions. The absorption spectrum width due to electronic
transitions from one state to another may include several vibrational states. This is
due to the energy difference between two adjacent levels of vibrational state is
much smaller than of electronic levels. This feature leads to the peak broadening of
the electronic transition that consists of spectral peaks that are located very close
each other. Each of these peaks cannot be observed clearly in the the absorption
spectrum due to the lack of the equipment resolution. A group of peaks with
certain width will eventually coincide each other, thus can only be observed as the
overlaps of many peaks in a low resolution spectrophotometer. Therefore, the
absorption spectra generally have the broad shape or observed as a band instead of
the group of single absorption peaks.3
The absorption spectrum of chlorophyll a in Figure 2 shows that maximum of
Soret band is at 413 nm, while the Q band is at 668 nm. If we compared with the
absorption spectrum of chlorophyll a in PhotochemCAD data base, there a shift in
absorbance about 4 nm. However, from the contour of the spectrum, it can be seen
that obtained chlorophyll a was pure. This is because there is no widening of the

865
absorbance spectrum around 410-500 nm, indicating that there are no other
pigments presence in the chlorophyll a solution. Then the absorbance of
pheophytin a and Fe-pheophytin a were compared with chlorophyll a.

3.5

3.0
Fe-pheophyt in a
Pheophytin a
2.5 Chlorophyll a
Absorbance

2.0

1.5

1.0

0.5

0.0

300 40 0 500 600 700

Wavelength (nm)

Figure 4 Absorption spectra of chlorophyll a, pheophytin a, and Fe-pheophytin a

There is a shift in pheophytin a and chloropyll a in the Soret and Q bands, which
occurs at a wavelength of 405 nm and 669 nm. From the spectrum, it can be seen
that the absorption maximum soret band on Fe-pheophytin a is at 393 nm while
the maximum absorption in the region Q band occurs at a wavelength of 626 nm.
Here is the comparison of absorption maxima between chlorophyll a, pheophytin a,
and Fe-pheophytin a.

Table 1 Absorption maxima of chlorophyll derivatives

max (nm)

Soret band Q band

Chlorophyll a 413 668

Pheophytin a 405 669

Fe-Pheophytin a 393 626

From these results, it can be observed that the change in central ion affects the
absorbtion transition energy.

866
3.5 Emissions Analysis

In the emission analysis, observations were made based on the fluorescence of

substances that have been irradiated with laser at the wavelength of 405 nm.

emission of chlorophyll a
0.6
absorbance of chlorophyll a

0.4
1.83
Intensity

1.85
0.2

0.0

1.5 2.0 2.5 3.0 3.5

Energy (eV)

Figure 5 Emission and absorption spectra of chlorophyll a

From the comparison between the emission and absorption spectra, it can be seen
that there is energy gap at between absorption and emission. Emission in
chlorophyll a occurred at a smaller energy. This is in accordance with the Frank-
Condon principle.3 This phenomenon occurs because of the structural relaxation
during the electronic transition occurs in the molecule caused by vibrational and
rotational motions. It is caused the differences in energy for up and down
electronic transition, namely the absorption and the emission processes. The
energy difference that occurs in chlorophyll a is around 0.02 eV. The same thing is
also observed on the absorption and emission of Fe-pheophytin a.

867
 1.0

absorbance of Fe-pheophytin a
0.8
emission of Fe-pheophytin a

0.6
Intensity

1,83
0.4 1,98

0.2

0.0
1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4 3.6

Energy (eV)

Figure 6 Emission and absorption spectra of Fe-pheophytin a

Based on the spectrum, the energy difference obtained between emission and
absorption is 0.15 eV. The difference in the relaxation structure is higher than
chlorophyll a, thus the energy gap is higher than the energy gap that occurs in
chlorophyll a. This shows that the relaxation of the molecular structure of Fe-
pheophytin a is much more different than the structure in the ground state
compared to the chlorophyll a.

3.6 Analysis of ESI MS

To prove that Fe2+ ion was succesfully introduced and became the central ion of
pheophytin a, the sample was then characterized using mass spectroscopy by ESI-
MS method.

868
 Figure 7 Mass spec. result for Fe-pheophytin a

From the characterization results of MS, obtained mass 956.52 indicating a

molecular mass of Fe-pheophytin a molecule that bound to two H2O molecules.
This is because the octahedral structure with six coordination number in the case
of Fe2+ is more stable. Coordination number is determined by the size of the central
metal atom, the number of d electrons, and the steric effects of ligands (Saito,
1996). Fe-pheophytin a is more likely to have six coordination number, which is
more stable than four coordination. The size of the central ion also affects the
coordination complex. Fe2+ has a large size with a diameter of 152 pm. With this
large size, probably there is still free space for binding complexes to be more stable.
The other masses that appear on the results of these measurements is the mass of
disconnected fragments of Fe-pheophytin a.

3.7 Stability Analysis

Stability analysis was carried out by comparing the time evolution of the emission
intensity of chlorophyll a and Fe-pheophytin a under the light irradiation with a
diode laser at wavelength of 405 nm for 200 seconds.

869
 4000

1 second 1 second
20 seconds
40 seconds
3000
60 seconds
80 seconds
Intensity (a.u) 100 seconds
2000
120 seconds
140 seconds
160 seconds
180 seconds
1000 200 seconds

200 seconds

0
600 800

Wavelength (nm)

Figure 8 Evolution of emission spectra decay of chlorophyll a in PMMA solution

detik ke-1 1 second

20 seconds
40 seconds
60 seconds
1000
80 seconds
100 seconds
Intensity (a.u)

120 seconds
140 seconds
160 seconds
500 180 seconds
200 seconds
detik ke-200

0
600 800

Wavelength (nm)

Figure 9 Evolution of emission spectra decay of Fe-pheophytin a in PMMA solution

If both spectra are compared, it can be seen that the emission intensity on
chlorophyll a decreased faster than of Fe-pheophytin a which occurs gradually.
Both lifetimes can be described by describing curve based on first-order reaction.

870
 0
Fe-pheophytin a
ln (I/I0) chlorophyll a

= 43.88
s

= 12.79
s
-2
0 50 100 150 200 250 300

t (sec.)

Figure 10 Decay of Chlorophyll a and Fe-pheophytin a

Table 2 Lifetime of chlorophyll a and Fe-pheophytin a

lifetime (s)

chlorophyll a 12.79

Fe-pheophytin a 43.88

From the data, the obtained results as expected that the stability of modified
chlorophyll a with Fe2+ is more stable than chlorophyll a.

4 Conclusion
In this study we concluded that the chlorophyll has been isolated from spinach
leaves and chlorophyll a was successfully separated by sucrose column
chromatography method. The mass of chlorophyll a obtained from 100 gram of
spinach leaves is 0.092 gram. Based on the results of UV-Vis absorption
measurements, it is known that the chlorophyll a has been completely separated.
The chlorophyll a then successfully modified by changing its center ion with Fe 2+ to
form Fe-pheophytin a. This has been proved by the results of UV-Vis absorption
and ESI-MS spectrometry characterization. From the analysis of the stability test
using laser ( = 405 nm), it was obtained that the stability of Fe-pheophytin a is
higher than chlorophyll a. Chlorophyll a has the lifetime of 12.79 seconds, while
the Fe-pheophytin a provides much higher of 43.88 seconds.

871
Acknowledgment
The authors acknowledge the full financial support for this work through the
research program Hibah Strategis Nasional 2010 DIPA ITB under contract No.
14a/SK/K01.7/KP/2010 (FMIPA ITB).

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[2] Shio, Y., (2006), Large Scale Chlorophyll Using Simple Open-Column
Chromatographi Methods, Springer, Netherlands, pp. 123-131.

[3] Valeur, B., (2002), Molecular Fluorescence Principles and Applications, Wiley-
VCH, Germany, 3-56.

[4] Barazzouk.S and S. Hotchandani,. (2004), Enhanced Charge Separation in

Chlorophyll a solar cell by Gold Nanoparticles, J. Of Applied Physics., Vol 96,
No.12.

[5] Anderson I.C., and D.S. Robertson., (1959), Role of Carotenoids in Protecting
Chlorophyll from Photodestruction, J. Filter paper of the Iowa Agriculture and
Heme economics, No.1423 and 1381.1959.

[6] Schertz. F.M., (1993), The Extraction and Separation of Chlorophyll ( +)

Carotin and Xanthophylls in Fresh Green Leaves, Preliminary to Their
Quantitative Determination, J. Plant Physiology, p:211-216.

[7] Bidwell, R.G.S., (1979), Plant Physiology, 2nd edition, Collier Mc Millan
Publisher, London, 120.

872
RACHMA DITYA SANDININGTYAS

Inorganic and Physical Chemistry Division,

Faculty of Mathematics and Natural Sciences,

Institut Teknologi Bandung, Indonesia

VEINARDI SUENDO*

Inorganic and Physical Chemistry Division,

Faculty of Mathematics and Natural Sciences,

Institut Teknologi Bandung, Indonesia

E-mail: vsuendo@chem.itb.ac.id

*Corresponding author

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