Isolation of Chlorophyll a from Spinach Leaves and ...

Jurnal Matematika & Sains, Agustus 2011, Vol. 16 Nomor 2

Isolation of Chlorophyll a from Spinach Leaves and Modification of Center Ion with Zn2+: Study on its Optical Stability Nurhayati† and Veinardi Suendo* Inorganic and Physical Chemistry Research Division, Faculty of Mathematics and Natural Sciences, Institut Teknologi Bandung, Bandung, Indonesia *Corresponding author, e-mail: [email protected] Received 21 December 2010, accepted for publication 29 March 2011 Abstract It is well-known that the pure chlorophyll a, which has a magnesium central ion, is not stable against high intensity light exposure. It is observed that it degrades with a lifetime of 12.8 seconds based on the evolution of fluorescence signal intensity under the irradiation of 60 mW diode laser at 405 nm that resonates with its Soret band. In order to be applied as an optical material, the stability of chlorophyll a needs to be improved. One way to enhance the photostability of chlorophyll a is by changing the center ion, where in this study Mg2+ in chlorophyll a is replaced by Zn2+ to form Zn-pheophytin a. The results show a significant increase in the decay lifetime from 12.8 to 49.5 seconds by introducing the Zn2+ ion into porphyrin ring. It is observed that the absorption maximum of Soret band is red shifted from 413 nm to 424 nm after the introduction of Zn2+ into porphyrin ring, while the Q band maximum is blue shifted from 667 nm to 658 nm. The mass spectroscopy result confirmed that Mg2+ ions have been replaced by Zn2+. The results show the presence of m/z 933.5 and m/z 971.5 species, which are typical for Zn-pheophytin a and Zn-pheophytin a dihydrate, respectively. Keywords: Chlorophyll a, Photodegradation, Optical materials, Optical stability, Porphyrin, Zn-pheophytin a. Abstrak Telah diketahui secara umum bahwa klorofil a murni, dengan ion pusat magnesium, bersifat tidak stabil terhadap penyinaran berintensitas tinggi. Telah diamati pada penelitian ini bahwa klorofil a mengalami degradasi dengan waktu hidup 12.8 detik yang diperoleh dari evolusi intensitas sinyal fluoresensi pada penyinaran dengan laser dioda berdaya 60 mW pada 405 nm yang beresonansi dengan pita Soret. Agar dapat diaplikasikan sebagai material optik, kestabilan klorofil a perlu ditingkatkan. Salah satu cara untuk meningkatkan kestabilan klorofil a adalah mengganti ion pusatnya. Dalam penelitian ini kation yang digunakan adalah Zn2+ yang akan membentuk Zn-pheophytin a. Hasil penelitian menunjukkan peningkatan nilai waktu hidup yang signifikan dari 12.8 detik menjadi 49.5 detik dengan masuknya ion Zn2+ ke dalam cincin porfirin pada klorofil a. Diamati bahwa penyerapan maksimum pita Soret mengalami pergeseran merah dari 413 nm ke 424 nm setelah pergantian ion pusat dengan Zn2+, sedangkan pita Q maksimum mengalami pergeseran biru dari 667 nm menjadi 658 nm. Hasil spektroskopi massa memastikan bahwa ion Mg2+ telah tergantikan oleh ion Zn2+. Ditemukan keberadaan spesi dengan m/z 933,5 dan m/z 971,5 yang merupakan m/z spesifik untuk Zn-pheophytin a dan Zn-pheophytin a dihidrat. Kata kunci: Kestabilan optik, Klorofil a, Fotodegradasi, Material optik, Porfirin, Zn-pheophytin a. organic and inorganic materials. Organic semiconductor materials such as conjugated organic compounds are developed for optical and electronic applications. These compounds have a flexible chemical structure that can be designed based on requirement. In addition, some of organic materials are available in nature and easy to obtain. One of the organic semiconductors is chlorophyll. Chlorophyll is a green pigment that is usually found in many plants, algae and cyanobacteria which consists of several types: chlorophyll a, chlorophyll b, chlorophyll c and chlorophyll d. Chlorophyll derived from Greek, chloros means green and phyllon means leaf. Chlorophyll can absorb purple and red lights. Special physiological properties owned by plant due to the presence of chlorophyll are the ability to convert solar radiation energy into chemical energy. This

1. Introduction Energy is very important for human life because all of human activities require energy. Energy sources based on the fossil energy are limited and non-renewable. Therefore, in order to anticipate the scarcity of energy in the future, the government of Indonesia began to promote the usage of alternative energy sources. The Presidential decre of Republic of Indonesia also encourages researchers to develop alternative energy sources with high potential and can be renewed. One of the alternative energy sources which are being developed is solar cells that can absorb light and the convert it into electricity. Based on its constituent, the semiconductor materials can be classified into two types, namely

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process is called photosynthesis. Carbon dioxide and water serve as the raw materials that are converted into glucose and oxygen with the help of sunlight and chlorophyll. The net reaction that occurs in photosynthesis process is as follows : 6 CO2 + 6 H2O  C6H12O6 + 6 O2 (1) The light energy is absorbed by chlorophyll in accordance with the energy required to excite electrons in the chlorophyll molecule to the higher energy levels. These electrons are captured by plant proteins, then to be used in the reduction reaction of photosynthesis. Chlorophyll a, which is one of porphyrin derivatives, has been developed as an organic material for solar cells-like process of photosynthesis (Barazzouk and Hotchandani, 2004). However, several studies described that the pure chlorophyll a which has been isolated from the leaves is not stable because of the absent of other leaf pigments, such as carotenoids, which normally stabilize them (Anderson and Robertson, 1960). One way to enhance the photostability of chlorophyll a is by changing the center ion that modifies the electronic environment in the phorpyrin ring, thus this study focused on the replacement Mg2+ in chlorophyll a by Zn2+ to form Zn-pheophytin a. In photosynthesis, light energy is converted into chemical bond energy after being captured by pigment molecules, called chlorophyll, a substituted tetrapyrrole. The four nitrogen atoms of the pyrroles are coordinated to a magnesium ion. Aside from being a major component of photosynthesis in plants, chlorophyll or chlorophyll derivatives can be utilized in human life are photodynamic agent in tumor or cancer therapy (Brandis et al., 2006; DrzewieckaMatuszek et al., 2005; Ulatowska-Jarza et al., 2005), developed in geology for the analysis of sediment and oil (Keely, 2006; Woolley et al., 1998), as photosensitizer on photoenergy conversion systems such as photovoltaic cells (Amao and Yamada, 2007). Because their many benefits, chlorophyll or chlorophyll derivatives are studied, modified and synthesized in many different disciplines, such as chemistry and physics for different applications, i.e.: electronic, optoelectronic, electrochemistry, catalytic and photophysics. Based on the location and type of functional groups on the porphyrin ring, chlorophyll can be divided into chlorophyll a, b, and d. Chlorophyll a and b are mostly discovered in higher plants, while chlorophyll d on cyanobateria. This study is focused on chlorophylls a because in the photodynamic therapy and analysis in geology, chlorophyll a or its derivates are more widely used (Brandis et al., 2006; Keely, 2006). However, chlorophyll a is less thermally stable than chlorophyll b (Tan and Francis, 1962; Canjura et al., 1991). Therefore, a study on stabilizing chlorophyll a was conducted here using the most simple approximation, by replacing its central ion with Zn2+ ion. Zinc atom was chosen

because the Zn chlorophyll complex is also found in nature. For example purple bacteria (Acidiphilium sp.) contains Zn-Bacteriochlorophyll a complex and Acidophilic organisms (Rhodophyte galdieria sulphuria (Cyanidium caldarium) has Zn-Proto as a precursor of biliprotein (Csatorday et al., 1981). In addition there are similarities between the properties of chlorophyll to replace the Zn-Mg-chlorophyll complex that they have the same redox potential (Geskes et al., 1995). 2. Materials and Methods A hundred grams of spinach leaves was blended with 500 mL of cold acetone (-10 oC) and then filtered off using a Buchner funnel. The solid was washed with 100 mL of acetone and the filtrate was added by 1/7 volume dioxane of the total volume. Then the filtrate mixture was added by 1/4 volume of distilled water of the total volume while kept stirred using a magnetic stirrer. This mixture was precipitated by cooling at -20 oC for 10 days and then filtered off using 2 layers of Whatman filter paper No.1. The obtained green precipitate was dissolved with 100 mL of acetone, and then the same procedure as described above was repeated again, which followed by the evaporation to remove all solvents (Shioi, 2006). Mixture of chlorophyll a, b, and carotene in diethyl ether were introduced into a sucrose column. Column that contains chlorophyll was firstly eluted with petroleum ether, then followed by the 10% diethyl ether in petroleum ether until the blue-green and yellow-green bands were obtained. Then the elution was continued with the mixture of 0.5% 2-propanol in petroleum ether until the two bands were separated (Shioi, 2006). Into 150 mL of a acetone solution of chlorophyll a (2.2 × 10-4 M) was added 10 mL HCl (0.1 M) and mixed at 40 oC for 30 minutes to form pheophytin a. Pheophytin a was extracted from the acetone-water solution by adding hexane. Into 20 mL of a chloroform solution of pheophytin a (2.2 × 10-4 M) was mixed with 4 mL acetate buffer pH 5 and 21 mg of Zn(II)acetate dihydrate in 40 mL of methanol. This mixture was kept stirring at 40 °C under nitrogen gas atmosphere for 1 hour. Then the chloroform-methanol solution was evaporated to obtain green solid of Znpheophytin a (Inoue et al., 1993). The purity of chlorophyll a, pheophytin a, and Zn-pheophytin a was analyzed using UV-visible light spectrophotometer and the measurement of emission spectrum with a StellarNet mini-spectrometer. The measured spectra were compared to the literature ones. Moreover, to verify that the Zn2+ ion has been introduced successfully into the porphyrin ring, the sample was also characterized by using ESI-MS (Electro Spray Ionization-Mass Spectroscopy). The stability test carried out by irradiation using a 60 mW diode laser at a wavelength of 405 nm. In this test, the chlorophyll solution in chloroform was mixed with PMMA (polymethylmethacrylate) to form 10%

Nurhayati and Suendo, Isolation of chlorophyll a from spinach leaves and modification……… 67 (w/v) PMMA solution. The solution then was inserted into capillary tubes prior to any stability measurement. The capillary tubes were tested by laser irradiation method with the set up schematically shown in Figure 1. Capillary tube filled with sample

Fluorescence spot

Beam absorber

Laser beam Diode laser (405 nm)

Propagated fluorescence signal Waveguide

Mini-spectrometer Optical fiber

Figure 1. Schematic of experimental setup for photostability measurements. The capillary tube was used to increase the fluorescence signal intensity through multi-reflection during the wave propagation along the tube. The advantage of this technique is using only a small amount of sample to get a large number of nearly clean spectra without using long wave pass filter that usually used to cut the excitation/laser signal in common fluorescence measurement setup. Here, the PMMA acts to increase the solution viscosity, thus the pigment molecules are trapped spatially during measurement.

3. Results and Discussion 3.1 Isolation of chlorophyll from spinach leaves (Amaranthus tricolor L.) Isolation of chlorophyll was started form the spinach leaves mixture in cold acetone at temperature about -10 °C. The chlorophyll has almost the same polarity as acetone, thus it can be dissolved easily. After the filtration using Whatman filter paper No.1, the chlorophyll in the filtrate was reacted with dioxane and distilled water to form a complex crude chlorophyll-dioxane-water. The solution mixture was stored at the low temperatures around -20 °C in order to precipitate the chlorophyll complexes completely. In this experiment the storage process was conducted approximately for 10 days to complete the precipitation process. After 10 days, the upper yellow colored solution was obtained which are the chlorophyll impurities such as flavonoids, while the dark green suspension at the bottom of the flask is the chlorophyll a complexes. Then the green precipate was filtered by using the Whatman filter paper No.1 from its impurities. The green precipitate obtained on the filter paper was then dissolved with acetone. This solid is a mixture of chlorophyll complex with other impurities such as carotene, xantofil flavonoids, and anthocyanins (Schertz, 1928). Therefore, to obtain more pure chlorophyll, the same procedure as above

can be repeated. All purification processes were carried out in the dark room or under a green dim light in order to prevent the formation of chlorophyllid. Cholorophyllid is an unstable chlorophyll derivative that lost its phytol tail (Anderson and Robertson, 1960). 3.2 Separation of chlorophyll a from other leaves pigment Sucrose column chromatography was used to separate the mixture of chlorophyll a, b and other leaf pigments, such as carotenoids after complex precipitation process. Sucrose column chromatography is reported to be very effective to separate chlorophyll a and b in large numbers (Shioi, 2006). This is due to the lower polarity of the sucrose compared to silica or alumina that is often used as stationary phases in column chromatography. Before the sucrose is introduced into the column, it must be dried for several days in the vacuum oven at 60 oC. This process eliminates the presence of water in the sucrose, which will perturb the separation process. The water molecules would increase the polarity of sucrose that makes the stationary phase (sucrose) cannot distinguish between chlorophyll a and b. Mixture of chlorophyll a, b and other leaf pigments in the sucrose column were eluted with petroleum ether. From this elution process yellow band was appeared known as a carotene band. The elution continued until the yellow band from the carotene went out from the column. After the first elution, the mixture of chlorophyll a and b was still at the top of the column. In order to separate the band of chlorophyll a and b from the top of the column, the polarity of mobile phase should be slightly increased. Therefore, subsequent elution was continued with the mobile phase of 10% diethyl ether in petroleum ether. The content of diethyl ether in petroleum ether would increase slightly the polarity of petroleum ether. From the second elution, we obtained two bands, a green band of chlorophyll a that followed by a greenyellow-blue band of chlorophyll b. Indicating that the chlorophyll a polarity of is less than of chlorophyll b. From this elution process, a gray band also appeared, which is the band of pheophytin a. From the second elution with 10% diethyl ether in petroleum ether, the separation of chlorophyll a and b was not separated completely. Therefore, the third elution is needed to separate chlorophyll a and b. In this study, 0.5% of 2-propanol in petroleum ether was used as the eluent. The third mobile phase is more polar than the first and the second ones. From the third elution, a blue green band was obtained at the bottom, followed by yellowish-green band that separated well enough in the middle of the column. In this process the yield of chlorophyll a from 100 g of spinach leaves (Amaranthus tricolor L.) is 0.092 g, while the chlorophyll b is 0.101 g.


In order to replace the center ion of chlorophyll a with Zn2+, the pheophytin a is dissolved in chloroform and then mixed into the solution of Zn(II)acetate dihydrate in methanol. After mixing, the color of the solution was observed to change from gray to bright green, which indicates that the Zn-pheophytin a have been formed. In order the complete the ion exchange process of chlorophyll with Zn2+ ions, the mixture was heated at 40 oC for one hour under nitrogen atmosphere. 3.4 Characterization of Chlorophyll a, pheophytin a, Zn-pheophytin a The spectrum of UV-visible absorption of chlorophyll a, pheophytin a, and Zn-pheophytin a are shown in Figure 2. 413

Pheophytin a Chlorophyll a Zn-pheophytin a

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Figure 3. Comparison of emission and absorption spectra of chlorophyll a in methanol.

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Figure 2. Absorption spectra of chlorophyll a, pheophytin a, Zn-pheophytin a in methanol. From these measurements, the maximum of Soret band of chlorophyll a was observed at 413 nm, while the Q band maximum at 667 nm. According to the PhotochemCAD database (Du et al., 1998), the Soret band of chlorophyll a in methanol is at 417.15 nm and the Q band is at 659 nm. The differences in peak position of the Soret and Q band of chlorophyll a between the experiment and the literature are due to the different types of equipment used for measurements. Based on the above spectra, it can be seen that the Soret band maximum in chlorophyll a is shifted to the shorter wavelengths or to higher energy when its central atom is replaced with two hydrogen atoms. On the other hand the Q band maximum shifted to the longer wavelength or lower energy. This is because the difference in electronic structure between chlorophyll a and pheophytin a. If the spectrum absorption of chlorophyll a is compared to the Zn-pheophytin a, the Soret Band maximum is shifted from at 413 nm to 424 nm. In addition, the maximum of Q band chlorophyll a is shifted from 667 nm to 658 nm. The Soret band maximum shifted to a longer wavelength in the Zn-pheophytin a. This indicates that the energy gap between the HOMO

Based on these spectra, the difference energy absorption and emission of chlorophyll a is 0.024 eV. This Stokes shift is due to the relaxed structure of chlorophyll a in the ground state is different compared to the relaxed structure in the excited state. In the case of Zn-pheophytin a, the comparison spectra are show in Figure 4. 3

Fluorescence intensity (a.u)

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1.8422

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(Highest occupied Molecular Orbital) and LUMO +1 (Lowest Unoccupied Molecular Orbital second level) of Zn-pheophytin a smaller than of chlorophyll a. While the maximum of Q bands of Zn-pheophytin a is shifted to a shorter wavelengths or higher energy than chlorophyll a. This indicates that the energy gap between the HOMO (Highest occupied Molecular Orbital) and LUMO (Lowest Unoccupied Molecular Orbital first level) is larger than of chlorophyll a. In Figure 3, the spectrum absorption UVvisible light of the chlorophyll a were compared with its spectrum emission in order to analyse the energy shift between absorption and emission transitions, the x-axis is converted into eV.

Absorbance

3.3 The substitution of center ion of chlorophyll a with Zn2+

Fluorescence intentisy (a.u.)

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Figure 4. Comparison of emission and absorption spectra of Zn-pheophytin a in methanol. Here, the difference in energy is 0.042 eV. This difference is two times larger than of the chlorophyll a. This is because of the relaxed structure in the ground state and in excited state structure less similar than of chlorophyll a. Thus, the Znpheophytin a needs larger structure relaxation or rearrangement than of chlorophyll a that results a larger Stokes shift.

Nurhayati and Suendo, Isolation of chlorophyll a from spinach leaves and modification……… 69

Figure 5. ESI-MS spectrum of Zn-pheophytin a. Chemical structure of Zn-pheophytin a were characterized more accurately using mass spectroscopy (ESI-MS). ESI-MS measurements provide m/z 933.5, which is the characteristic of Znpheophytin a. The complete results of ESI-MS spectrum of Zn-pheophytin a is shown Figure 5. Figure 5 shows also the presence of m/z 888,6 which is characteristic to pheophytin a hydrate at functional methyl ester group (-COOCH3) and m/z 971.5 which is the characteristic of Zn-pheophytin a dihydrate. The intensity of m/z 971.5 is higher than the intensity of m/z 933.5. This is due to the complex Znpheophytin a dihydrate with six coordination number is more preferable than the Zn-pheophytin a complex with four coordination number. In this complex, the central atom still has two unoccupied orbital d at the top and bottom that likely to be filled by other ligands. In this case, the ligands that might fill these vacancies are water molecules ion. After the characterization processes are accomplished until the presence of Zn-pheophytin a have been confirmed, the study were continued with the stability test of chlorophyll a and Zn-pheophytin a with 10% w/v PMMA in chloroform under irradiation using a 60 mW diode laser at 405 nm. In this test, the integration of detector was kept the same for 500 ms and taken for 200 spectra episodically as shown in Figure 6 and 7.

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Figure 7. Emission spectrum of Zn-pheophytin a in the 10% PMMA in chloroform. The emission intensity of Zn-pheophytin a is much higher than of chlorophyll a even though the excitation wavelength (405 nm) much differs from the maximum wavelength of its Soret absorption band of 424 nm than of chlorophyll a (413 nm). The intensity emission spectra of Zn-pheophytin a at t = 200 s is at around 1800 a.u, where the chlorophyll a gives only 800 a.u. In this study, all intensities can be compared directly because of the same optical configuration in our measurement setup (Figure 1). The light luminosity of Zn-pheophytin a provides a great potential a to be used as optical materials. Based on the decay curve of the fluorescence signal intensity of chlorophyll a and Zn-pheophytin a, it is clear that Zn-pheophytin a has a higher photostability than of chlorophyll a (Figure 8). Here, we obtained the photodegradation lifetime for both pigments are 12.8 and 49.5 seconds based on the 1st order reaction mechanism approximation for chlorophyll a and Znpheophytin a, respectively. The life time of Znpheophytin a is obviously much longer, which are approximately four times longer that implies an important improvement by introducing the Zn2+ ion into porphyrin ring.

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ZnPhe a = 49.5 s

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Figure 6. Emission spectrum of chlorophyll a in the 10% PMMA in chloroform.

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Figure 8. Decay of fluorescence signal intensity of chlorophyll a and Zn-pheophytin a in the 10% PMMA in chloroform.

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4. Conclusions Chlorophyll a from spinach leaves (Amaranthus tricolor L.) was successfully isolated and purified from other leaf pigments. The center ion in chlorophyll a was replaced with Zn2+ ion successfully to form Zn-pheophytin a. It was observed from the data of UV-visible absorption that Soret band of chlorophyll a is red shifted from 413 nm to 424 nm. While the Q band maximum is shifted to the blue from 667 nm to 658 nm by replacing the center ion with Zn2+. The ESI-MS spectra of Znpheophytin a shows the presence of species m/z 933.5 and m/z 971.5, which are typical for Znpheophytin a and Zn-pheophytin a dihydrate, respectivelly. Based on the photostability test under light exposure using a 60 mW diode laser at 405 nm, it was obtained that the Zn-pheophytin a is more stable around four times than of chlorophyll a. References Amao, Y. and Y. Yamada, 2007, Photovoltaic Conversion Using Zn Chlorophyll Derivative Assembled Hydrophobic Domain Onto Nanocrystalline TiO2 Electrode, Biosens. Bioelectron., 22:7, 1561-1565. Anderson, I. C. and D. S. Robertson., 1960, Role of Carotenoids in Protecting Chlorophyll from Photodestruction, Plant Physiol., 35:4, 531– 534. Barazzouk, S. and S. Hotchandani, 2004, Enhanced Charge Separation in Chlorophyll a Solar Cell by Gold Nanoparticles, J. Appl. Phys., 96, 12. Berg, J. M., J. L. Tymoczko, and L. Stryer, 2000, Biochemistry, 4th ed., W. H. Freeman and Co., New York. Brandis, A. S., Y. Salomon, and A. Scherz, 2006, Chlorophyll Sensitizers in Photodynamic Therapy, Advances in Photosynthesis and Respiration, 25, 461–483. Canjura, F. L., S. J. Schwartz, and R. V. Nunes, 1991, Degradation Kinetics of Chlorophylls and Chlorophyllides. J. Food Sci., 56, 16391643. Csatorday, K., R. MacColl, and D. S. Berns, 1981, Accumulation of Protoporphyrin IX and Zinc Protoporphyrin IX in Cyanidium Caldarium, Proc. Nat. Acad. Sci., 78:3, 1700–1702. Drzewiecka-Matuszek, A., A. Skalna, A. Karocki, G. Stochel, and L. Fiedor, 2005, Effects of Heavy Central Metal on the Ground and

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