6 Analytical Protocols in Chlorophyll Analysis
135
discussed in the work of Hynninen and Elfolk (1973): transesterification by chlorophyllase, epimerization reactions to form chlorophyll a, and if large amounts of algae
are used, rapid formation (up to 40%) of allomeric forms and probably dichloric forms
of chlorophyll a are produced.
Finally, several studies have explored the use of dimethylformamide (DMF) as an
extraction solvent superior to methanol, ethanol, or acetone (Neveux 1988; Suzuki
and Ishimaru 1990; Jeffrey et al. 1997). In this sense, DMF has been shown to be
an efficient extractant for both phytoplankton (Neveux 1988; Suzuki and Ishimaru
1990; Suzuki et al. 1993; Furuya et al. 1998) and intertidal sediments (Honeywill et al.
2002). Specifically, DMF is recommended for cyanobacteria and coccoid green algae
that are recalcitrant (Neveux 1988). Another advantage of DMF is that chlorophylls
are highly stable in this solvent for weeks, allowing appropriate storage in the dark
(Heyward 1991; Jeffrey et al. 1997; Schumann et al. 2005).
Independent of the solvent selection, the mechanical disruption of the cells significantly enhances the extraction in comparison with the simple soaking or immersion for several hours. Different cell disruption methods have been employed such
as grinding (Schumann et al. 2005; Simon and Helliwell 1998), homogenization
(Sartory and Grobbelaas 1984), or sonication (Simon and Helliwell 1998; Sartory
and Grobbelaas 1984). Additional studies assess the convenience of freeze drying
(lyophyllization) prior to solvent extraction (Hagerthey 2006). However, probably the
conventional method prior to chlorophyll extraction is filtering the biomass followed
by freezing. Although multiple types of filters can be used, the glass filter is most
commonly used. Selection of pore size and quality is dependent on the phytoplankton
characteristics.
Use of organic solvents for lipophilic extraction poses a serious threat to the
environment. For example, the Montreal Protocol in 1987 proposed to restrict or
to eliminate the manufacture and the use of ozone depleting solvents such as chlorofluorocarbons (CFCs). Since then, an increasing trend with high impact in the
past few years is in the application of “green extraction techniques” that avoid the
use of solvents (Table 6.2): supercritical fluid extraction (SFE), microwave assisted
extraction (MAE), ultrasound-assisted extraction (UAE), assisted by pulsed electric field extraction (PEF), or extraction assisted by enzymes (EAE). Although these
techniques have been applied preferentially for carotenoid extractions, several assays
have been developed for chlorophyll extraction. SFE is a popular method with several
advantages as the high purity of the extracting, the extraction requires less processing
steps, is significantly safer than extraction with organic solvents and it can be operated
at moderate temperatures to minimize the extract degradation. However, the chlorophyll extraction from microalgae using SFE depends on the fluid density which is a
function of the operating pressure and temperature. Consequently, prior to its application it is necessary to optimize the conditions for a specific sample (Nobre et al.
2013). For example, the optimum conditions for Nannochloropsis gaditana (MacíasSánchez et al. 2005) were 60 °C and 400 bar while for Synechococcus sp. (MacíasSánchez et al. 2007) are 60 °C and 500 bar. MAE is an efficient method that takes
advantage of microwave irradiation to accelerate the elimination of a variety of natural
matrix compounds, causes direct generation of heat within the matrix, by friction
135
discussed in the work of Hynninen and Elfolk (1973): transesterification by chlorophyllase, epimerization reactions to form chlorophyll a, and if large amounts of algae
are used, rapid formation (up to 40%) of allomeric forms and probably dichloric forms
of chlorophyll a are produced.
Finally, several studies have explored the use of dimethylformamide (DMF) as an
extraction solvent superior to methanol, ethanol, or acetone (Neveux 1988; Suzuki
and Ishimaru 1990; Jeffrey et al. 1997). In this sense, DMF has been shown to be
an efficient extractant for both phytoplankton (Neveux 1988; Suzuki and Ishimaru
1990; Suzuki et al. 1993; Furuya et al. 1998) and intertidal sediments (Honeywill et al.
2002). Specifically, DMF is recommended for cyanobacteria and coccoid green algae
that are recalcitrant (Neveux 1988). Another advantage of DMF is that chlorophylls
are highly stable in this solvent for weeks, allowing appropriate storage in the dark
(Heyward 1991; Jeffrey et al. 1997; Schumann et al. 2005).
Independent of the solvent selection, the mechanical disruption of the cells significantly enhances the extraction in comparison with the simple soaking or immersion for several hours. Different cell disruption methods have been employed such
as grinding (Schumann et al. 2005; Simon and Helliwell 1998), homogenization
(Sartory and Grobbelaas 1984), or sonication (Simon and Helliwell 1998; Sartory
and Grobbelaas 1984). Additional studies assess the convenience of freeze drying
(lyophyllization) prior to solvent extraction (Hagerthey 2006). However, probably the
conventional method prior to chlorophyll extraction is filtering the biomass followed
by freezing. Although multiple types of filters can be used, the glass filter is most
commonly used. Selection of pore size and quality is dependent on the phytoplankton
characteristics.
Use of organic solvents for lipophilic extraction poses a serious threat to the
environment. For example, the Montreal Protocol in 1987 proposed to restrict or
to eliminate the manufacture and the use of ozone depleting solvents such as chlorofluorocarbons (CFCs). Since then, an increasing trend with high impact in the
past few years is in the application of “green extraction techniques” that avoid the
use of solvents (Table 6.2): supercritical fluid extraction (SFE), microwave assisted
extraction (MAE), ultrasound-assisted extraction (UAE), assisted by pulsed electric field extraction (PEF), or extraction assisted by enzymes (EAE). Although these
techniques have been applied preferentially for carotenoid extractions, several assays
have been developed for chlorophyll extraction. SFE is a popular method with several
advantages as the high purity of the extracting, the extraction requires less processing
steps, is significantly safer than extraction with organic solvents and it can be operated
at moderate temperatures to minimize the extract degradation. However, the chlorophyll extraction from microalgae using SFE depends on the fluid density which is a
function of the operating pressure and temperature. Consequently, prior to its application it is necessary to optimize the conditions for a specific sample (Nobre et al.
2013). For example, the optimum conditions for Nannochloropsis gaditana (MacíasSánchez et al. 2005) were 60 °C and 400 bar while for Synechococcus sp. (MacíasSánchez et al. 2007) are 60 °C and 500 bar. MAE is an efficient method that takes
advantage of microwave irradiation to accelerate the elimination of a variety of natural
matrix compounds, causes direct generation of heat within the matrix, by friction