136
I. Viera and M. Roca
between polar molecules (Kaufmann and Christen 2002). The first assays showed that
SFE is comparable with MAE, when methanol is used as a solvent (Macías-Sánchez
et al. 2009), although the use of ultrasound facilitates the penetration of DMF in the
cell membrane of microalgae, increasing the recovery of the pigments present in the
raw material (Pasquet et al. 2011). UAE significantly reduces the extraction time and
increases the extraction yields of many natural matrices, due to the production of
cavitation bubbles in the solvent (Zou et al. 2013). PEF can be used to improve mass
transfer processes, destroying cell membranes. Depending on the intensity, amplitude, duration, number, and frequency of repetition of the external in the membranes
electric pulses, reversible or irreversible pores are produced. Formation of irreversible
pores is of great importance for the extraction of bioactive compounds from natural
matrices (Zbinden et al. 2013).
6.3 Methods for Chlorophyll Analysis
Certain investigations do not require the exhaustive determination of a complete
profile or exact composition of chlorophyll derivatives. Sometimes it is necessary only to determine the amount of chlorophylls (a, b, c, and/or d). For such
purpose, rapid spectrometric methods have been developed to calculate photosynthetic, metabolically active biomass, productivity of aquatic ecosystems, or the
amount of chlorophyll per unit of protein (Ritchie 2006). Multiple algorithms and
equations based on spectrophotometric and spectrofluorimetric techniques have been
developed for routine assays based on up to three wavelengths (Jeffrey and Humphrey
1975; Porra et al. 1989, Rowan 1989), and reviewed by Porra (2006). More recently,
the application of chemometric methods have allowed to use the complete spectrum (Neveux et al. 2011) to differentiate from the di- or trichromatic methods.
This multivariate analysis enables the determination of a greater number of chlorophyll pigments. Another step is the application of artificial neural networks (Franco
et al. 2019) to analyze microalgae spectral signatures from light absorption measurements with the aim to identify the prevailing strains. Re-training this promising
methodology will reinforce the model.
However, the individual determination of chlorophylls requires the utilization of
separation techniques, reverse phase HPLC coupled with DAD detector and less
frequently with fluorescence detector, is the most commonly used application. A
complete review of the HPLC methods for pigment analysis of phytoplankton was
compiled by Garrido et al. (2011) and consequently this section mainly focuses on
the innovations developed since then. Besides the new methods (Table 6.3), we also
included the pioneering and original methods because the utility of these methods
has been assayed for decades and also because they constitute the basis of the modern
protocols. A practical protocol for the application of HPLC for characterization of
pigments in phytoplankton has been recently published (Garrido and Roy 2015).
Nevertheless, as stated before (Garrido et al. 2011) there is no perfect method of
HPLC, and it is essential to adapt the method to the properties of the sample and the
I. Viera and M. Roca
between polar molecules (Kaufmann and Christen 2002). The first assays showed that
SFE is comparable with MAE, when methanol is used as a solvent (Macías-Sánchez
et al. 2009), although the use of ultrasound facilitates the penetration of DMF in the
cell membrane of microalgae, increasing the recovery of the pigments present in the
raw material (Pasquet et al. 2011). UAE significantly reduces the extraction time and
increases the extraction yields of many natural matrices, due to the production of
cavitation bubbles in the solvent (Zou et al. 2013). PEF can be used to improve mass
transfer processes, destroying cell membranes. Depending on the intensity, amplitude, duration, number, and frequency of repetition of the external in the membranes
electric pulses, reversible or irreversible pores are produced. Formation of irreversible
pores is of great importance for the extraction of bioactive compounds from natural
matrices (Zbinden et al. 2013).
6.3 Methods for Chlorophyll Analysis
Certain investigations do not require the exhaustive determination of a complete
profile or exact composition of chlorophyll derivatives. Sometimes it is necessary only to determine the amount of chlorophylls (a, b, c, and/or d). For such
purpose, rapid spectrometric methods have been developed to calculate photosynthetic, metabolically active biomass, productivity of aquatic ecosystems, or the
amount of chlorophyll per unit of protein (Ritchie 2006). Multiple algorithms and
equations based on spectrophotometric and spectrofluorimetric techniques have been
developed for routine assays based on up to three wavelengths (Jeffrey and Humphrey
1975; Porra et al. 1989, Rowan 1989), and reviewed by Porra (2006). More recently,
the application of chemometric methods have allowed to use the complete spectrum (Neveux et al. 2011) to differentiate from the di- or trichromatic methods.
This multivariate analysis enables the determination of a greater number of chlorophyll pigments. Another step is the application of artificial neural networks (Franco
et al. 2019) to analyze microalgae spectral signatures from light absorption measurements with the aim to identify the prevailing strains. Re-training this promising
methodology will reinforce the model.
However, the individual determination of chlorophylls requires the utilization of
separation techniques, reverse phase HPLC coupled with DAD detector and less
frequently with fluorescence detector, is the most commonly used application. A
complete review of the HPLC methods for pigment analysis of phytoplankton was
compiled by Garrido et al. (2011) and consequently this section mainly focuses on
the innovations developed since then. Besides the new methods (Table 6.3), we also
included the pioneering and original methods because the utility of these methods
has been assayed for decades and also because they constitute the basis of the modern
protocols. A practical protocol for the application of HPLC for characterization of
pigments in phytoplankton has been recently published (Garrido and Roy 2015).
Nevertheless, as stated before (Garrido et al. 2011) there is no perfect method of
HPLC, and it is essential to adapt the method to the properties of the sample and the