Chlorophyll extraction from harvested plant material Mgr inż. Krystian Miazek Supervisor: Prof. dr hab. inż. Stanisław Ledakowicz Abstract In this work, extraction of chlorophyll with methanol, ethanol and acetone from harvested plant material was evaluated. Total chlorophyll concentration in extracts of fresh birch leaves, pine needles and sow thistle leaves was determined using spectrophotometric methods. Chlorophyll content was higher in birch (0.32 %) and sow thistle leaves (0.28 %) than in pine needles (0.23 %). Keywords: chlorophyll, extraction, leaves, needles 1. Introduction Chlorophyll is a green pigment found in several varieties in plants and algae [1]. Chlorophyll structure consists of tetrapyrrole ring with a central magnesium ion and a long hydrophobic phytol chain. Two types of chlorophyll, a and b are present in green algae and terrestrial plants. The difference between these two chlorophylls is a methyl moiety in chlorophyll a replaced by a formyl group in chlorophyll b. The ratio of chlorophyll a to chlorophyll b in higher plants is approximately 3:1. bsorbs light mainly in the red (650 700 nm) and the blue - violet (400 500 nm) regions of the visible spectrum (Figure 1). Green light (~550 nm) is not absorbed but reflected giving chlorophyll its characteristic color. possess a green-blue color, and chlorophyll b possess a green-yellow color [2], [3]. The structural difference between chlorophyll a and b molecules increases the range of sun light captured by plants in the process called photosynthesis. Phytol chain Figure 1. Chemical structure and absorption spectrum of chlorophyll a and b [4].
Photosynthesis is the process of using light energy to fix carbon dioxide into carbohydrates and discharge oxygen as a byproduct. Photosynthesis occurs in photoautotrophic organisms like higher plants or algae and consists of light and dark reactions. Light reactions take place in thylakoid membranes of chloroplasts. These thylakoid membranes contain photosystems which are basic units of photosynthetic apparatus. Photons with wavelengths between 400 and 700 nm are absorbed by chlorophylls in photosystems. One molecule of chlorophyll absorbs one photon and loses one electron. From chlorophylls, electrons are carried via proteins to electron transport chain where they are reduced and NADPH is generated (1). Chlorophylls regain lost electron via photolysis of water to oxygen and protons (2). As a result, a proton gradient across the thylakoid membrane is created and this gradient is used to generate ATP (3). Dark reactions takes place in the stroma of chloroplasts and comprise a process called Calvin Benson cycle. In the Calvin cycle (4), a 5 carbon sugar, ribulose-biphosphate (RuBP) reacts with a molecule of CO 2 to form phosphoglycerate (PGA). PGA is further reduced with the use of ATP and NADPH to produce a three carbon sugar, phosphoglyceraldehyde (PGAL). During regenerative phase, five PGAL molecules are converted to three RuBP molecules. A primary product of photosynthesis is phosphoglyceraldehyde that serves as a precursor for building carbohydrates (Figure 2) [5]. NADP + + 2H + + 2e - NADPH + H + (1) H 2 O ½ O 2 + 2H + + 2e - (2) ADP + Pi ATP (3) Light energy H 2 O Photosystems O 2 Light reactions Dark reactions NADPH + H + ATP Thylakoid membranes Stroma Chloroplasts PGA CO 2 RuBP (4) Calvin Benson Cycle PGAL Carbohydrates Figure 2. General mechanism of photosynthesis.
2. Materials and methods 2.1 Plant material preparation Birch leaves (Figure 3), pine needles (Figure 5) and sow thistle leaves (Figure 7) were harvested in a central region of Poland (51 51' 0'' N, 19 25' 0'' E). Harvested plant material was cut with scissors into small pieces. 2.2 Plant material extraction Fresh material samples were extracted with methanol (pure), ethanol (95% v/v) and acetone (80% v/v) in sealed tubes kept at room temperature in dark. Extraction time was up to 3 h for birch leaves and up to 48 h for pine needles and sow thistle leaves. Independent samples in the number of 5 (birch leaves), 6 (pine needles) and 3 (sow thistle leaves) were used in this project. 2.3 Chlorophyll measurement Concentration of chlorophyll a and b in obtained extracts was measured spectrophotometrically (T80+ UV/VIS Spectrometer PG Instruments Ltd) with equations presented below (Table 1). According to these measurements, chlorophyll a and b content in fresh materials of birch leaves, pine needles and sow thistle leaves was determined. Table 1. Equations used to measure chlorophyll a and b in solvent extracts of tested plant materials [6]. Solvent Equations Methanol (pure) Chl a = 16.72. A 665 9.16. A 652 Chl b = 34.09. A 652 15.28. A 665 Ethanol (95% v/v) Chl a = 13.36. A 664 5.19. A 648 Chl b = 27.43. A 648 8.12. A 664 Acetone (80% v/v) Chl a = 12.25. A 663 2.79. A 646 Chl b = 21.50. A 646 5.10. A 663 Where: Chl a chlorophyll a [µg/ml] Chl b chlorophyll b [µg/ml] A 665 absorbance at wavelength 665 nm [-] A 652 absorbance at wavelength 652 nm [-] A 664 absorbance at wavelength 664 nm [-] A 648 absorbance at wavelength 648 nm [-] A 663 absorbance at wavelength 663 nm [-] A 646 absorbance at wavelength 646 nm [-]
Figure 3. Birch leaves (Betulaceae). Birch leaves 0,3 Concentration [%] 0,2 0,1 0 Methanol pure Ethanol 95 % Acetone 80 % Solvent type Figure 4. Concentration of chlorophyll a and b in fresh material of birch leaves.
Figure 5. Pine needles (Pinaceae). Pine needles 0,3 Concentration [%] 0,2 0,1 0 Methanol pure Ethanol 95 % Acetone 80 % Solvent type Figure 6. Concentration of chlorophyll a and b in fresh material of pine needles.
Figure 7. Sow thistle leaves (Asteraceae). Sow thistle leaves 0,3 Concentration [%] 0,2 0,1 0 Methanol pure Ethanol 95 % Acetone 80 % Solvent type Figure 8. Concentration of chlorophyll a and b in fresh material of sow thistle leaves.
Table 2. Total chlorophyll content in fresh materials of birch leaves, pine needles and sow thistle leaves. Solvent Total chlorophyll (a + b) content Birch leaves Pine needles Sow thistle leaves Methanol (pure) 0.32 0.21 0.27 Ethanol (95% v/v) 0.31 0.22 0.28 Acetone (80% v/v) 0.31 0.25 0.28 Average 0.32 0.23 0.28 3. Results and discussion In this study, three different solvents were used to extract chlorophyll from fresh materials of birch leaves, pine needles and sow thistle leaves. and b concentrations in selected materials extracted with pure methanol, 95 % v/v ethanol and 80 % v/v acetone were at approximate level with a constant chlorophyll a : b ratio equal to 3:1 (Figure 4, 6, 8), indicating that all tested solvents can be interchangeably harnessed for chlorophyll extraction. On the other hand, concentration of chlorophyll a and b showed variations according to material type used (Figure 4, 6, 8). The highest total chlorophyll content (a + b) was detected in birch leaves (0.32 %) followed by sow thistle leaves (0.28 %) and pine needles (0.23 %) (Table 2). 4. Literature [1] Aminot A and Rey F. Standard procedure for the determination of chlorophyll a by spectroscopic methods. March 2000. International Council for the Exploration of the Sea. ISSN 0903-2606. [2] Ćwiczenia z biochemii. Wydawnictwo Naukowe PWN SA Warszawa 1999. ISBN 83-01- 13944-7. [3] Młodzińska E (2009). Survey of Plant Pigments: Molecular and Environmental Determinants of Plant Colors. Acta Biologica Cracoviensia Series Botanica 51/1: 7 16. [4] http://www.chm.bris.ac.uk/motm/chlorophyll/chlorophyll_h.htm [5] Berg JM, Tymoczko JL, Stryer L. Biochemistry-5 th edition. 2002 by W. H. Freeman and Company New York. [6] Lichtenthaler HK, Buschmann C (2001). Chlorophylls and Carotenoids: Measurement and Characterization by UV-VIS Spectroscopy. Current Protocols in Food Analytical Chemistry F4.3.1-F4.3.8.