6 Analytical Protocols in Chlorophyll Analysis
133
(determination of photosynthetic pigments in seawater) which published in 1997 a
monograph compiling the state of art, on determination of phytoplankton pigments
(Jeffrey et al. 1997). In the recent times, the working group at SCOR (156) related
to chlorophylls is dealing with the active chlorophyll fluorescence for autonomous
measurements of global marine primary productivity (SCOR Working Group 156).
Since then, different reviews or books have been published, compiling the knowledge
on chlorophyll analysis in phytoplankton (Roy et al. 2011a, b).
6.2 Chlorophyll Extraction Methods from Microalgae
and Cyanobacteria
Multiple methods have been proposed to extract and quantify chlorophyll pigments
in phytoplankton, as no specific protocol is applicable to all the species known
due to lack of applicability of a common protocol to all known species (Bowles
et al. 1985; Cartaxana and Brotas 2003). The extraction efficiency depends on the
resistance of the cell wall, the penetration power of the solvent and the solvation
properties, the duration of the extraction, and the type of mechanical disruption used
(Wright et al. 1997). Classically, the main and more generalized method used to
extract intracellular chlorophyll from microalgae and cyanobacteria is the application
of organic solvents. The organic solution penetrates through the cell membrane,
dissolves lipids and lipoproteins of the chloroplast membranes, and facilitates the
pigment extraction. Arnon (1949) was the first researcher to use acetone to extract
chlorophyll a from higher plants for spectrophotometric measurements. Since then,
as shown in Table 6.1, a variety of methods have been developed, each one for specific
taxonomic groups.
Richards and Thompson (1952) successfully applied acetone to phytoplankton and
provided the first equations for the spectroscopic quantification of this pigment from
algae. These authors based their studies on the extraction of pigments in submerged
algae and the excellent quantitative data obtained led to the recommendation of the
method as a standard protocol by SCOR (UNESCO 1966). Since then acetone has
become probably the most common solvent, although it is important to optimize the
proportion of acetone:water (Mantoura and Llewellyn 1983). High amount of water
during the extraction facilitates chlorophyllase activity (Wright et al. 1997), forming
in vitro large proportions of chlorophyllides. This precaution should be taken with
species with high chlorophyllase activity levels as in diatoms (Barrett and Jeffries
1971). In general, acetone is a better solvent to extract nonpolar chlorophylls.
133
(determination of photosynthetic pigments in seawater) which published in 1997 a
monograph compiling the state of art, on determination of phytoplankton pigments
(Jeffrey et al. 1997). In the recent times, the working group at SCOR (156) related
to chlorophylls is dealing with the active chlorophyll fluorescence for autonomous
measurements of global marine primary productivity (SCOR Working Group 156).
Since then, different reviews or books have been published, compiling the knowledge
on chlorophyll analysis in phytoplankton (Roy et al. 2011a, b).
6.2 Chlorophyll Extraction Methods from Microalgae
and Cyanobacteria
Multiple methods have been proposed to extract and quantify chlorophyll pigments
in phytoplankton, as no specific protocol is applicable to all the species known
due to lack of applicability of a common protocol to all known species (Bowles
et al. 1985; Cartaxana and Brotas 2003). The extraction efficiency depends on the
resistance of the cell wall, the penetration power of the solvent and the solvation
properties, the duration of the extraction, and the type of mechanical disruption used
(Wright et al. 1997). Classically, the main and more generalized method used to
extract intracellular chlorophyll from microalgae and cyanobacteria is the application
of organic solvents. The organic solution penetrates through the cell membrane,
dissolves lipids and lipoproteins of the chloroplast membranes, and facilitates the
pigment extraction. Arnon (1949) was the first researcher to use acetone to extract
chlorophyll a from higher plants for spectrophotometric measurements. Since then,
as shown in Table 6.1, a variety of methods have been developed, each one for specific
taxonomic groups.
Richards and Thompson (1952) successfully applied acetone to phytoplankton and
provided the first equations for the spectroscopic quantification of this pigment from
algae. These authors based their studies on the extraction of pigments in submerged
algae and the excellent quantitative data obtained led to the recommendation of the
method as a standard protocol by SCOR (UNESCO 1966). Since then acetone has
become probably the most common solvent, although it is important to optimize the
proportion of acetone:water (Mantoura and Llewellyn 1983). High amount of water
during the extraction facilitates chlorophyllase activity (Wright et al. 1997), forming
in vitro large proportions of chlorophyllides. This precaution should be taken with
species with high chlorophyllase activity levels as in diatoms (Barrett and Jeffries
1971). In general, acetone is a better solvent to extract nonpolar chlorophylls.