6 Analytical Protocols in Chlorophyll Analysis
139
stationary phases, has resolved completely the separation between all the known
MV and DV chlorophylls. In addition, there is a continuous reduction on the particle
size of the columns, from the initial 5 μm, through the 3.5 μm to the present sub-2 μm
particles. This reduction obeys to an increasing necessity to shorten the analysis time
and increase the data throughput. Decreasing particle size implies increase in backpressure, substituting the HPLC by UHPLC (ultra-HPLC), able to deliver pressures
above 40 MPa, and generally preferred when coupled with mass spectrometry.
In relation with the mobile phases, the use of an ion pair agent is required to
improve the separation between acidic chlorophylls (very polar dephytylated chlorophylls). Between the different ionic pairs, ammonium acetate and more recently,
tetrabutylammonium acetate are the most popular reagents included in the different
gradients (Table 6.3).
Additionally, advancements have been developed over the classical HPLC
methods. As an example, Latasa (2014) has increased the sensitivity in reverse phase
HPLC (RP-HPLC) by adding an extra loop to the HPLC system that should be
equipped with an autosampler able of mixing and repeat injections. The idea is to
pre-load the extra loop with alternative injections of sample and water and ejected
into seat, repeating the process as many times required until the complete volume
is injected (around 2400 μL). Applied in several investigations, authors advised
increasing in the elution gradient and the importance of the mixing in the loop and
not in separate vials. Other alternative to resolve pairs of chlorophylls that co-eluted
in one peak is to use the first derivative spectrum technique (Suzuki et al. 2015). This
method is based on the differences in pigment absorption spectra and it has been
successfully applied to differentiate chlorophyll c 2 from MgDVP and chlorophyll b
from DV chlorophyll b.
As certain chlorophylls exhibit the same absorption spectra, other chlorophylls are
difficult to separate, and chlorophyll standards are scarce, the combination of HPLC
with MS is a helpful tool that has been used for many years during the analysis of
chlorophylls. Airs and Garrido (2011) compiled the main MS techniques employed
to obtain the MS and MS
2 of the main chlorophylls in phytoplankton. Recently, this
information has been updated in a special thematic issue of mass spectrometry of
chlorophylls derivatives, specifically devoted to chlorophylls c (Gavalás-Olea et al.
2018), chlorophylls of phototrophic prokaryotes (Airs 2018), and chlorophyll a and
b and its derivatives (Hynninen 2018; Viera et al. 2018).
For MS chlorophyll analysis different ionization techniques have been applied.
Initially, fast atom bombardment (FAB) was a breakthrough methodology during
the 1980s–90s, providing the first insights in the MS characterization of phytoplankton chlorophylls (Garrido and Zapata 1996). However, this technique has been
overcome with softer ionization modes, with better signal performance. Although
the matrix-assisted laser desorption (MALDI) spectrometry was employed during a
long period of time, the nature of the matrix influenced the results. Recently, electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) are the
139
stationary phases, has resolved completely the separation between all the known
MV and DV chlorophylls. In addition, there is a continuous reduction on the particle
size of the columns, from the initial 5 μm, through the 3.5 μm to the present sub-2 μm
particles. This reduction obeys to an increasing necessity to shorten the analysis time
and increase the data throughput. Decreasing particle size implies increase in backpressure, substituting the HPLC by UHPLC (ultra-HPLC), able to deliver pressures
above 40 MPa, and generally preferred when coupled with mass spectrometry.
In relation with the mobile phases, the use of an ion pair agent is required to
improve the separation between acidic chlorophylls (very polar dephytylated chlorophylls). Between the different ionic pairs, ammonium acetate and more recently,
tetrabutylammonium acetate are the most popular reagents included in the different
gradients (Table 6.3).
Additionally, advancements have been developed over the classical HPLC
methods. As an example, Latasa (2014) has increased the sensitivity in reverse phase
HPLC (RP-HPLC) by adding an extra loop to the HPLC system that should be
equipped with an autosampler able of mixing and repeat injections. The idea is to
pre-load the extra loop with alternative injections of sample and water and ejected
into seat, repeating the process as many times required until the complete volume
is injected (around 2400 μL). Applied in several investigations, authors advised
increasing in the elution gradient and the importance of the mixing in the loop and
not in separate vials. Other alternative to resolve pairs of chlorophylls that co-eluted
in one peak is to use the first derivative spectrum technique (Suzuki et al. 2015). This
method is based on the differences in pigment absorption spectra and it has been
successfully applied to differentiate chlorophyll c 2 from MgDVP and chlorophyll b
from DV chlorophyll b.
As certain chlorophylls exhibit the same absorption spectra, other chlorophylls are
difficult to separate, and chlorophyll standards are scarce, the combination of HPLC
with MS is a helpful tool that has been used for many years during the analysis of
chlorophylls. Airs and Garrido (2011) compiled the main MS techniques employed
to obtain the MS and MS
2 of the main chlorophylls in phytoplankton. Recently, this
information has been updated in a special thematic issue of mass spectrometry of
chlorophylls derivatives, specifically devoted to chlorophylls c (Gavalás-Olea et al.
2018), chlorophylls of phototrophic prokaryotes (Airs 2018), and chlorophyll a and
b and its derivatives (Hynninen 2018; Viera et al. 2018).
For MS chlorophyll analysis different ionization techniques have been applied.
Initially, fast atom bombardment (FAB) was a breakthrough methodology during
the 1980s–90s, providing the first insights in the MS characterization of phytoplankton chlorophylls (Garrido and Zapata 1996). However, this technique has been
overcome with softer ionization modes, with better signal performance. Although
the matrix-assisted laser desorption (MALDI) spectrometry was employed during a
long period of time, the nature of the matrix influenced the results. Recently, electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) are the