Chlorophyll is a natural pigment that is usually found in many green plants. It can be explored its potential as optical materials. The optical characteristics and the purity of chlororophyll a and Fe-pheophytin a were investigated using spectroscopic methods.
Chlorophyll is a natural pigment that is usually found in many green plants. It can be explored its potential as optical materials. The optical characteristics and the purity of chlororophyll a and Fe-pheophytin a were investigated using spectroscopic methods.
Chlorophyll is a natural pigment that is usually found in many green plants. It can be explored its potential as optical materials. The optical characteristics and the purity of chlororophyll a and Fe-pheophytin a were investigated using spectroscopic methods.
ITS MODIFICATION USING Fe 2+ 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 Mg 2+ , to become Fe 2+ 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.
Proceedings of the Third International Conference on Mathematics and Natural Sciences (ICMNS 2010)
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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 Fe 2+ . 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 Fe 2+ 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 861
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 Fe 2+
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 862
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 Fe 2+ 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. Laser Capillary tube Fiber optic Fluorescence signal Spectrometer
Figure 1 Experimental set up for optical stability measurement
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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 864
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 Fe 2+ . The process was carried out by removing Mg 2+ 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 Pheophytin 1
With the loss of Mg 2+ as its center ion, the chlorophyll will turned into gray colored substance called pheophytin a. In order to introduce the Fe 2+ 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 Fe 2+ to be oxidized to become Fe 3+ . 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 Fe 2+ 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.
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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. 300 400 500 600 700 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 668 413 659,25 417,75 result literature A b s o r b a n c e 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 866
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.
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.
300 400 500 600 700 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Fe-pheophyt in a Pheophyti n a Chlorophyll a Wavelength (nm) A b s o r b a n c e 867
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.
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. 1.5 2.0 2.5 3.0 3.5 0.0 0.2 0.4 0.6 1.83 1.85 emission of chlorophyll a absorbance of chlorophyll a Energy (eV) I n t e n s i t y 868
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 Fe 2+ ion was succesfully introduced and became the central ion of pheophytin a, the sample was then characterized using mass spectroscopy by ESI- MS method.
1.6 1.8 2. 0 2. 2 2.4 2.6 2.8 3.0 3.2 3. 4 3. 6 0.0 0.2 0.4 0.6 0.8 1.0 1,83 1,98 absorbance of Fe-pheophytin a emission of Fe-pheophytin a I n t e n s i t y Energy (eV) 869
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 Fe 2+ 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. Fe 2+ 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. 870
600 800 0 1000 2000 3000 4000 200 seconds 1 second 1 second 20 seconds 40 seconds 60 seconds 80 seconds 100 seconds 120 seconds 140 seconds 160 seconds 180 seconds 200 seconds Wavelength (nm) I n t e n s i t y
( a . u )
Figure 8 Evolution of emission spectra decay of chlorophyll a in PMMA solution
600 800 0 500 1000 detik ke-200 detik ke-1 1 second 20 seconds 40 seconds 60 seconds 80 seconds 100 seconds 120 seconds 140 seconds 160 seconds 180 seconds 200 seconds Wavelength (nm) I n t e n s i t y
( a . u )
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. 871
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 Fe 2+ 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. 0 50 100 150 200 250 300 -2 0 Fe-pheophytin a chlorophyll a t (sec.) l n ( I / I 0 ) = 43.88 s = 12.79 s 872
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). References [1] Nelson, R, Ferruzzi, M., (2008), Synthesis and Bioaccessibility of Fe- Pheophytin Derivatives from Crude Spinach Extract, J. Food Sci., 73(5), 86-91. [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, 2 nd edition, Collier Mc Millan Publisher, London, 120. 873
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