140
I. Viera and M. Roca
more successful ionization modes applied to chlorophylls. In general, APCI shows
better performance for phytylated chlorophylls, as the gas efficiency is higher for
nonpolar compounds (Chen et al. 2015a), while ESI is more appropriate for polar
(dephytylated) chlorophyll (Chen et al. 2015b). Chorophyll c is a special group that
generates low-quality mass spectra, probably due to the inferior ionization efficiency
of porphyrins compared to chlorins (Airs and Garrido 2011).
Similarly, different mass analyzers have been used successfully for chlorophyll
elucidation: quadruples are often applied for the determination of known compounds,
ion trap is highly suitable to elucidate new structures through MS
n analysis (Airs
et al. 2014), and time of flight (TOF), whose excellent resolution properties makes it
the ideal mass analyzer for accurate mass determinations in high-resolution MS
(Gavalás-Olea et al. 2018). More recently, hybrid mass spectrometers allow to
combine the advantages of two mass analyzers, as triple quadrupole (Zhang et al.
2016), QTOF (Maroneze et al. 2019), or quadrupole-orbitrap mass spectrometer
(Freitas et al. 2019).
The application of the hyphenated HPLC-MS on chlorophylls has allowed not
only the determination of the accurate mass and elemental composition of the corresponding different compounds (see below) but also to describe characteristic ion
products that facilitate a rapid identification. Table 6.4 describes the main product
ions described for chlorophylls present in phytoplanktons, associated with the corresponding structural assignment when known. A detailed description of the fragmentation during the MS and MS
2 studies can be found in the works of Gavalás-Olea
et al. (2018), Airs (2018), Hynninen (2018), Viera et al. (2018). Besides specific
fragmentations as the CO loss in chlorophyll derivatives from b series (Chen et al.
2015a), chlorophylls are commonly fragmented by the phytyl chain (or the alcohol
esterifying at C17
3 ) followed by successive fragmentations through the propionic
chain and at the carboxymethyl group at C13
2 level. Further fragmentations that
implied the opening of the macrocycle have been detailed by Chen et al. (2015a, b).
6.4 Chlorophyll Standards: Obtaining Protocols
and Commercial Suppliers
Few chlorophyll compounds are available commercially, marketed by five principal
companies (Table 6.5). Frontier Scientific offers a specific section of porphyrins with
more than 600 different compounds, while DHI lab is specialized on phytoplankton
pigments. In general, working with chlorophylls requires a special attention for the
presence or formation of chlorophyll allomers.
Alternatively, chlorophyll compounds can be isolated from natural sources, higher
plants or phytoplankton. Specifically, standards of chlorophyll can be obtained from
strains of reference algal cultures. Roy et al. (2011a, b) described an updated list
of laboratory suppliers of SCOR reference cultures besides a detailed reference of
I. Viera and M. Roca
more successful ionization modes applied to chlorophylls. In general, APCI shows
better performance for phytylated chlorophylls, as the gas efficiency is higher for
nonpolar compounds (Chen et al. 2015a), while ESI is more appropriate for polar
(dephytylated) chlorophyll (Chen et al. 2015b). Chorophyll c is a special group that
generates low-quality mass spectra, probably due to the inferior ionization efficiency
of porphyrins compared to chlorins (Airs and Garrido 2011).
Similarly, different mass analyzers have been used successfully for chlorophyll
elucidation: quadruples are often applied for the determination of known compounds,
ion trap is highly suitable to elucidate new structures through MS
n analysis (Airs
et al. 2014), and time of flight (TOF), whose excellent resolution properties makes it
the ideal mass analyzer for accurate mass determinations in high-resolution MS
(Gavalás-Olea et al. 2018). More recently, hybrid mass spectrometers allow to
combine the advantages of two mass analyzers, as triple quadrupole (Zhang et al.
2016), QTOF (Maroneze et al. 2019), or quadrupole-orbitrap mass spectrometer
(Freitas et al. 2019).
The application of the hyphenated HPLC-MS on chlorophylls has allowed not
only the determination of the accurate mass and elemental composition of the corresponding different compounds (see below) but also to describe characteristic ion
products that facilitate a rapid identification. Table 6.4 describes the main product
ions described for chlorophylls present in phytoplanktons, associated with the corresponding structural assignment when known. A detailed description of the fragmentation during the MS and MS
2 studies can be found in the works of Gavalás-Olea
et al. (2018), Airs (2018), Hynninen (2018), Viera et al. (2018). Besides specific
fragmentations as the CO loss in chlorophyll derivatives from b series (Chen et al.
2015a), chlorophylls are commonly fragmented by the phytyl chain (or the alcohol
esterifying at C17
3 ) followed by successive fragmentations through the propionic
chain and at the carboxymethyl group at C13
2 level. Further fragmentations that
implied the opening of the macrocycle have been detailed by Chen et al. (2015a, b).
6.4 Chlorophyll Standards: Obtaining Protocols
and Commercial Suppliers
Few chlorophyll compounds are available commercially, marketed by five principal
companies (Table 6.5). Frontier Scientific offers a specific section of porphyrins with
more than 600 different compounds, while DHI lab is specialized on phytoplankton
pigments. In general, working with chlorophylls requires a special attention for the
presence or formation of chlorophyll allomers.
Alternatively, chlorophyll compounds can be isolated from natural sources, higher
plants or phytoplankton. Specifically, standards of chlorophyll can be obtained from
strains of reference algal cultures. Roy et al. (2011a, b) described an updated list
of laboratory suppliers of SCOR reference cultures besides a detailed reference of