Download Chlorophyll extraction from harvested plant material

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. Chlorophyll absorbs 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. Chlorophyll a 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.
Chlorophyll a
Chlorophyll b
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 CO2 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+ + 2eH2O
ADP + Pi
NADPH + H+
(1)
½ O2 + 2H+ + 2e- (2)
ATP
(3)
Light energy
H 2O
Photosystems
Light reactions
O2
Thylakoid membranes
NADPH + H+
ATP
Dark reactions
Stroma
Chloroplasts
PGA
CO2
(4)
RuBP
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)
Chla = 16.72.A665 – 9.16.A652
Chlb = 34.09.A652 – 15.28.A665
Ethanol (95% v/v)
Chla = 13.36.A664 – 5.19.A648
Chlb = 27.43.A648 – 8.12.A664
Acetone (80% v/v) Chla = 12.25.A663 – 2.79.A646
Chlb = 21.50.A646 – 5.10.A663
Where:
Chla
Chlb
A665
A652
A664
A648
A663
A646
chlorophyll a
chlorophyll b
absorbance at wavelength 665 nm
absorbance at wavelength 652 nm
absorbance at wavelength 664 nm
absorbance at wavelength 648 nm
absorbance at wavelength 663 nm
absorbance at wavelength 646 nm
[µg/ml]
[µg/ml]
[-]
[-]
[-]
[-]
[-]
[-]
Figure 3. Birch leaves (Betulaceae).
Birch leaves
Concentration [%]
0,3
0,2
Chlorophyll a
Chlorophyll b
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
Concentration [%]
0,3
0,2
Chlorophyll a
Chlorophyll b
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
Concentration [%]
0,3
0,2
Chlorophyll a
Chlorophyll b
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. Chlorophyll a 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 83-0113944-7.
[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-5th 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.