72
measurements in acetone extracts are usually made at 665 nm before and after acidification
(Lorenzen, 1967). Chlorophyll band c have absorption spectra that overlap with the spectrum
of chi a. Trichromatic equations, such as those designed by Richard and Thompson (1952)
for calculation of the concentration of Chlorophyll a, band c separately after absorption
readings at 665, 645 and 630 nm, can hardly be reconciled with Beer's law and have been
criticized heavily by Rott (1980). Improved trichromatic equations have been given by Jeffrey
and Humphrey (1975), but even these cannot always be used without caution (Rott, 1980;
Lorenzen and Jeffrey, 1980).
The specific absorption coefficients of the major phaeopigments (phaeophorbide a and
phaeophytin a) are known, and so are their absorption spectra. However, unknown
phaeopigments may also be present (Gieskes and Kraay, 1986a, b), perhaps with different
spectral characteristics, and a choice of a fixed acid factor (l.7: Lorenzen, 1967b) for all
natural phytoplankton populations may therefore occasionally give questionable results. The
acidification step in the method described by Lorenzen is quite critical (Moed and Hallegraeff,
1978): too much acid causes shifts in the absorption maximum to wavelengths below 660 nm
and increases the 750 nm blank reading, e.g. when fucoxanthin breaks down to products
absorbing in that spectral region (Riemann, 1978).
ABSORPTION SPECTRA OF FRESHLY COLLECTED SUSPENDED MATTER
In recent years, interest has been renewed in an old, very simple method for the measurement
of phytoplankton pigments, and this time not only chi a (foregoing sections) but also other
pigments. The procedure typically involves filtration of suspended matter onto a glassfiber
filter (Whatman GF/C or GF/F) which is then placed, while still wet, in the light path of a
spectrophotometer to obtain an absorption spectrum of any wavelength range between 220 and
720 nm (Shibata e£ al., 1954; Triiper and Yentsch, 1967; Shibata, 1969; Gieskes and Kraay,
1990). When the spectrum is recorded quickly one doesn't even need a glass support for the
filter. Second and higher derivative analysis of such spectra (Fig. 7) provides much
information on the chlorophylls and phaeopigments in the sample, and also of different
carotenoids (Faust and Norris, 1985; Stolte, 1988; Bidigare e£ al., 1989). An advantage of
absorption spectra made of material that is not extracted in acetone is also that the absorption
by water-soluble phycobilin pigments is visible (Steenbergen and Korthals, 1982; Zevenboom,
1986). These pigments are present in Cryptophyceae and Cyanobacteria, algal classes that may
dominate natural phytoplankton in tropical seas (Waterbury e£ aZ., 1979) but are also present
in temperate waters (Gieskes and Kraay, 1983a).
measurements in acetone extracts are usually made at 665 nm before and after acidification
(Lorenzen, 1967). Chlorophyll band c have absorption spectra that overlap with the spectrum
of chi a. Trichromatic equations, such as those designed by Richard and Thompson (1952)
for calculation of the concentration of Chlorophyll a, band c separately after absorption
readings at 665, 645 and 630 nm, can hardly be reconciled with Beer's law and have been
criticized heavily by Rott (1980). Improved trichromatic equations have been given by Jeffrey
and Humphrey (1975), but even these cannot always be used without caution (Rott, 1980;
Lorenzen and Jeffrey, 1980).
The specific absorption coefficients of the major phaeopigments (phaeophorbide a and
phaeophytin a) are known, and so are their absorption spectra. However, unknown
phaeopigments may also be present (Gieskes and Kraay, 1986a, b), perhaps with different
spectral characteristics, and a choice of a fixed acid factor (l.7: Lorenzen, 1967b) for all
natural phytoplankton populations may therefore occasionally give questionable results. The
acidification step in the method described by Lorenzen is quite critical (Moed and Hallegraeff,
1978): too much acid causes shifts in the absorption maximum to wavelengths below 660 nm
and increases the 750 nm blank reading, e.g. when fucoxanthin breaks down to products
absorbing in that spectral region (Riemann, 1978).
ABSORPTION SPECTRA OF FRESHLY COLLECTED SUSPENDED MATTER
In recent years, interest has been renewed in an old, very simple method for the measurement
of phytoplankton pigments, and this time not only chi a (foregoing sections) but also other
pigments. The procedure typically involves filtration of suspended matter onto a glassfiber
filter (Whatman GF/C or GF/F) which is then placed, while still wet, in the light path of a
spectrophotometer to obtain an absorption spectrum of any wavelength range between 220 and
720 nm (Shibata e£ al., 1954; Triiper and Yentsch, 1967; Shibata, 1969; Gieskes and Kraay,
1990). When the spectrum is recorded quickly one doesn't even need a glass support for the
filter. Second and higher derivative analysis of such spectra (Fig. 7) provides much
information on the chlorophylls and phaeopigments in the sample, and also of different
carotenoids (Faust and Norris, 1985; Stolte, 1988; Bidigare e£ al., 1989). An advantage of
absorption spectra made of material that is not extracted in acetone is also that the absorption
by water-soluble phycobilin pigments is visible (Steenbergen and Korthals, 1982; Zevenboom,
1986). These pigments are present in Cryptophyceae and Cyanobacteria, algal classes that may
dominate natural phytoplankton in tropical seas (Waterbury e£ aZ., 1979) but are also present
in temperate waters (Gieskes and Kraay, 1983a).