Chapter 6
Analytical Protocols in Chlorophyll
Analysis
Isabel Viera and María Roca
Abstract Due to the importance of chlorophyll in phytoplankton, there are a number
of analytical protocols. In this regard, the current chapter aims to be a practical
guide for scientists interested in the description of the methodologies applicable
to studying chlorophylls in phytoplankton. The complex pigment profile and the
intricacies of pigment extraction from phytoplankton are the two main challenges
encountered during chlorophyll analysis. The cell walls of certain species hinder the
solvent capacity. For instance, Cyanobacteria and certain chlorophytes are termed
recalcitrant. The current chapter will review (a) the different techniques that have
been applied to guarantee the exhaustive chlorophyll extraction from microalgae
and cyanobacteria, (b) the methods employed for chlorophyll analysis: which are
often analyzed by liquid chromatography coupled with diode array detector (DAD).
Considering the different structure of the chlorophyll derivatives present in phytoplankton, diverse chromatographic methods have been optimized. A deeper analysis
has been achieved through the mass spectrophotometric (MS) studies of chlorophyll compounds of main microalgae and cyanobacteria, including the analysis of
the fragmentation pathways to increase consistency of the identification. However,
the disposal of appropriate standards is essential in chlorophyll investigation irrespective of the employed method of analysis. Only few chlorophyll standards are
commercially available, and consequently protocols for yielding of the others have
been specifically developed. Finally, the chapter will summarize the main chromatographic, spectroscopic properties, and MS characteristics of all chlorophyll present
in phytoplankton.
Keywords Bacteriochlorophylls · Chlorophylls · Chlorophyll extraction · HPLC ·
Identification chlorophylls · Mass spectrometry
I. Viera · M. Roca (B)
Food Phytochemistry Department, Instituto de la Grasa, Consejo Superior de Investigaciones
Científicas (CSIC), University Campus, Building 46, Carretera de Utrera km. 1, 41013 Seville,
Spain
e-mail: mroca@ig.csic.es
I. Viera
e-mail: iviera@ig.csic.es
© Springer Nature Switzerland AG 2020
E. Jacob-Lopes et al. (eds.), Pigments from Microalgae Handbook,
https://doi.org/10.1007/978-3-030-50971-2_6
127
Analytical Protocols in Chlorophyll
Analysis
Isabel Viera and María Roca
Abstract Due to the importance of chlorophyll in phytoplankton, there are a number
of analytical protocols. In this regard, the current chapter aims to be a practical
guide for scientists interested in the description of the methodologies applicable
to studying chlorophylls in phytoplankton. The complex pigment profile and the
intricacies of pigment extraction from phytoplankton are the two main challenges
encountered during chlorophyll analysis. The cell walls of certain species hinder the
solvent capacity. For instance, Cyanobacteria and certain chlorophytes are termed
recalcitrant. The current chapter will review (a) the different techniques that have
been applied to guarantee the exhaustive chlorophyll extraction from microalgae
and cyanobacteria, (b) the methods employed for chlorophyll analysis: which are
often analyzed by liquid chromatography coupled with diode array detector (DAD).
Considering the different structure of the chlorophyll derivatives present in phytoplankton, diverse chromatographic methods have been optimized. A deeper analysis
has been achieved through the mass spectrophotometric (MS) studies of chlorophyll compounds of main microalgae and cyanobacteria, including the analysis of
the fragmentation pathways to increase consistency of the identification. However,
the disposal of appropriate standards is essential in chlorophyll investigation irrespective of the employed method of analysis. Only few chlorophyll standards are
commercially available, and consequently protocols for yielding of the others have
been specifically developed. Finally, the chapter will summarize the main chromatographic, spectroscopic properties, and MS characteristics of all chlorophyll present
in phytoplankton.
Keywords Bacteriochlorophylls · Chlorophylls · Chlorophyll extraction · HPLC ·
Identification chlorophylls · Mass spectrometry
I. Viera · M. Roca (B)
Food Phytochemistry Department, Instituto de la Grasa, Consejo Superior de Investigaciones
Científicas (CSIC), University Campus, Building 46, Carretera de Utrera km. 1, 41013 Seville,
Spain
e-mail: mroca@ig.csic.es
I. Viera
e-mail: iviera@ig.csic.es
© Springer Nature Switzerland AG 2020
E. Jacob-Lopes et al. (eds.), Pigments from Microalgae Handbook,
https://doi.org/10.1007/978-3-030-50971-2_6
127