6 Analytical Protocols in Chlorophyll Analysis
143
For chlorophylls not commercially available or present in living organism in low
amounts, it is necessary to develop specific protocols. For example, the preparation of
dephytylated chlorophylls (chlorophyllide or pheophorbide) is based on an enzymatic
de-esterification using partially purified chlorophyllase from high-activity fruits, as
orange or pepper (Chen et al. 2015a). Magnesium-free derivatives (pheophytin and
pheophorbide) are easily obtained from the counterpart chlorophylls in ethyl ether by
acidification with two or three drops of 5 M HCl (Sievers and Hynninen 1977). Allomerized chlorophylls can be formed by oxidation of the corresponding parents with
selenium dioxide (SeO 2 ) in pyridine (Laitalainen et al. 1990) at 70 °C for 3 h (Chen
et al. 2015a). The reaction of decarbomethoxylation for pyro-derivatives production has been developed in pyridine at temperatures higher than 100 °C (Pennington
et al. 1964). Although recently specific and smoother temperatures have been set up
for different chlorophyll compounds (Chen et al. 2015a). After obtaining the standard, isolation of the chlorophyll compound of interest is an essential step which is
achieved, either by TLC or chromatography, by open column or by HPLC.
6.5 Chlorophyll Identification: Chromatographic
and Spectroscopic Properties of the Chlorophylls
Present in Microalgae
The development or selection of a specific protocol for chlorophyll analysis requires
the knowledge of the chromatographic and spectroscopic properties of the different
chlorophylls. Table 6.6 shows, as example, the relative chromatographic peak elution
order and absorption maxima for the main chlorophylls in phytoplankton. The data
is an approximation and the exact information will depend on the chromatographic
characteristics: HPLC column, solvents, gradient, etc., but in any case, it will help
during the analysis.
143
For chlorophylls not commercially available or present in living organism in low
amounts, it is necessary to develop specific protocols. For example, the preparation of
dephytylated chlorophylls (chlorophyllide or pheophorbide) is based on an enzymatic
de-esterification using partially purified chlorophyllase from high-activity fruits, as
orange or pepper (Chen et al. 2015a). Magnesium-free derivatives (pheophytin and
pheophorbide) are easily obtained from the counterpart chlorophylls in ethyl ether by
acidification with two or three drops of 5 M HCl (Sievers and Hynninen 1977). Allomerized chlorophylls can be formed by oxidation of the corresponding parents with
selenium dioxide (SeO 2 ) in pyridine (Laitalainen et al. 1990) at 70 °C for 3 h (Chen
et al. 2015a). The reaction of decarbomethoxylation for pyro-derivatives production has been developed in pyridine at temperatures higher than 100 °C (Pennington
et al. 1964). Although recently specific and smoother temperatures have been set up
for different chlorophyll compounds (Chen et al. 2015a). After obtaining the standard, isolation of the chlorophyll compound of interest is an essential step which is
achieved, either by TLC or chromatography, by open column or by HPLC.
6.5 Chlorophyll Identification: Chromatographic
and Spectroscopic Properties of the Chlorophylls
Present in Microalgae
The development or selection of a specific protocol for chlorophyll analysis requires
the knowledge of the chromatographic and spectroscopic properties of the different
chlorophylls. Table 6.6 shows, as example, the relative chromatographic peak elution
order and absorption maxima for the main chlorophylls in phytoplankton. The data
is an approximation and the exact information will depend on the chromatographic
characteristics: HPLC column, solvents, gradient, etc., but in any case, it will help
during the analysis.