226
Phytoplankton
method with natural samples for two reasons. In some samples, other degradation
products of chlorophyll a and band phaeophytin a and b (apparently chlorophylides
and phaeophorbides, i. e., chlorophylls or phaeophytins missing part or all of the phytol
tail) do not migrate, but remain at the origin with chlorophyll c and phaeophytin c, each
of which also lacks a phytol tail. Chlorophyll c is only sparingly soluble in diethylether
or acetone, so that total recovery is not possible. Chlorophyll a and b are accurately
determined by this method without interference from degradation products.
Hiqh Performance Liquid Chromatography. More recently, the applicability has been
examined of high performance liquid chromatography to the determination of various chlorophylls [643-658].
Chlorophyll in natural waters is frequently estimated by trichromatic spectrophotometry of algal extracts. When no interfering compounds are present, these trichromatic equations are good estimates. The major criticism is that, in natural plankton
extracts, spectrally similar chlorophyll breakdown products are frequently present.
Thus, chlorophyll cannot be accurately determined by this method. One way to avoid
the problem is to separate chromatographically the breakdown products from the
chlorophylls before measurement. Separation of the spectrally similar chlorophylls a
and b (as well as degradation products) will result in a more accurate determination of
the chlorophylls.
Tests carried out with the high performance liquid chromatographic technique endorsed the claim that it causes negligible degradation of both the chlorophylls and the
xanthophylls [650]. A more efficient separation of plant pigments could be achieved on a
silica stationary phase than on a CIS reversed-phase medium. A 30 em column packed
with Partisil 10 gave an efficient separation of the individual carotenoids, chlorophylls a
and b, and many of the degradation products of the latter pair. The solvent consists of
light petroleum (b. p. 60-80 0c), acetone, dimethyl sulphoxide, and diethylamine in the
ratio 75:23.25:1.5:0.25 by volume. Unfortunately, this solvent is not sufficiently polar to
elute phaeophorbide and chlorophyll c. With samples containing these pigments, it is
necessary to carry out an additional, stepwise, elution with a more polar solvent. Further
tests showed that excellent resolution of these compounds could be achieved by means of
a mixture containing light petroleum (b. p. 60-80 0c), acetone, methanol, and dimethyl
sulphoxide in the ratio 30:40:27:3 by volume, respectively.
In order to identify the various peaks on the chromatograms, extracts of a range of
algae from various classes were injected repeatedly onto the Partisil 10 column
operated at a flow rate of2 ml min-I. The eluates corresponding with individual peaks
of known retention times were collected and identified from their absorption spectra.
Chlorophylls and their degradation products were characterized by their spectra in
ether and in (1 + 9) water-acetone, based on data from Strickland [651] . Carotenoids
were identified by comparison of their wavelengths of maximum absorption in hexane, ethanol, and carbon disulphide with the tabulated values published by Davies
[652]. Identifications were confirmed by thin-layer chromatography on silica gel G
[653]. The retention times of various pigments are shown in Table 9.8. The retention
times are extremely reproducible.
Since the high performance liquid chromatographic method provides pigments
with a high degree of purity, it is relatively easy to standardize the technique.
Phytoplankton
method with natural samples for two reasons. In some samples, other degradation
products of chlorophyll a and band phaeophytin a and b (apparently chlorophylides
and phaeophorbides, i. e., chlorophylls or phaeophytins missing part or all of the phytol
tail) do not migrate, but remain at the origin with chlorophyll c and phaeophytin c, each
of which also lacks a phytol tail. Chlorophyll c is only sparingly soluble in diethylether
or acetone, so that total recovery is not possible. Chlorophyll a and b are accurately
determined by this method without interference from degradation products.
Hiqh Performance Liquid Chromatography. More recently, the applicability has been
examined of high performance liquid chromatography to the determination of various chlorophylls [643-658].
Chlorophyll in natural waters is frequently estimated by trichromatic spectrophotometry of algal extracts. When no interfering compounds are present, these trichromatic equations are good estimates. The major criticism is that, in natural plankton
extracts, spectrally similar chlorophyll breakdown products are frequently present.
Thus, chlorophyll cannot be accurately determined by this method. One way to avoid
the problem is to separate chromatographically the breakdown products from the
chlorophylls before measurement. Separation of the spectrally similar chlorophylls a
and b (as well as degradation products) will result in a more accurate determination of
the chlorophylls.
Tests carried out with the high performance liquid chromatographic technique endorsed the claim that it causes negligible degradation of both the chlorophylls and the
xanthophylls [650]. A more efficient separation of plant pigments could be achieved on a
silica stationary phase than on a CIS reversed-phase medium. A 30 em column packed
with Partisil 10 gave an efficient separation of the individual carotenoids, chlorophylls a
and b, and many of the degradation products of the latter pair. The solvent consists of
light petroleum (b. p. 60-80 0c), acetone, dimethyl sulphoxide, and diethylamine in the
ratio 75:23.25:1.5:0.25 by volume. Unfortunately, this solvent is not sufficiently polar to
elute phaeophorbide and chlorophyll c. With samples containing these pigments, it is
necessary to carry out an additional, stepwise, elution with a more polar solvent. Further
tests showed that excellent resolution of these compounds could be achieved by means of
a mixture containing light petroleum (b. p. 60-80 0c), acetone, methanol, and dimethyl
sulphoxide in the ratio 30:40:27:3 by volume, respectively.
In order to identify the various peaks on the chromatograms, extracts of a range of
algae from various classes were injected repeatedly onto the Partisil 10 column
operated at a flow rate of2 ml min-I. The eluates corresponding with individual peaks
of known retention times were collected and identified from their absorption spectra.
Chlorophylls and their degradation products were characterized by their spectra in
ether and in (1 + 9) water-acetone, based on data from Strickland [651] . Carotenoids
were identified by comparison of their wavelengths of maximum absorption in hexane, ethanol, and carbon disulphide with the tabulated values published by Davies
[652]. Identifications were confirmed by thin-layer chromatography on silica gel G
[653]. The retention times of various pigments are shown in Table 9.8. The retention
times are extremely reproducible.
Since the high performance liquid chromatographic method provides pigments
with a high degree of purity, it is relatively easy to standardize the technique.