Composition and Biomass of Phytoplankton
where
etc., as above.
Spectrophotometric Determination of Chlorophyll Pigments
and Their Degradation Products by Other Methods
159
A large number of comparative methodological studies have emerged during the past
decade for the spectrophotometric and fluorometric estimation of pigments. Other
organic solvents, methanol and ethanol in particular, have been used for the extraction
of pigments and compared to the removal efficiencies of acetone (Marker et aI., 1980;
Nusch, 1980; Riemann, 1980, 1982: many others). Acetone is the most straightforward
to use as a solvent but is less efficient than methanol or ethanol for the extraction
of chlorophyll. Acidification of methanol extracts for evaluation of phaeopigments
is extremely sensitive; slight excesses of acidity can introduce large experimental errors
(Marker et aI., 1980; Marker and Jinks, 1982). Such acidification problems are less
serious with ethanol [see Nusch (1980)] and least serious for acetone (Marker, et aI.,
1980, Marker and Jinks, 1982). It is clear that, for precise research purposes, chromatography (HPLC) is the preferred method for pigment analyses [e.g., Jacobsen (1982)].
However, HPLC is expensive to perform and not conducive to rapid, multiple pigment
analyses. Comparative analyses of spectrophotometric, fluorometric, and chromatograpic (HPLC) determinations of chlorophyll a found differences to be less than 10%
in most cases [e.g., Schanz and Rai (1988)]. Because determinations of phaeopigment
concentrations are of major importance, often exceeding 50% of the total pigment
concentrations, pigment degradation products must be measured, and acetone is
usually the preferred solvent for extraction of chlorophylls and phaeopigments.
Because of the modest extraction efficiency of acetone, however, it is important that
an effective grinding process in subdued light always be performed.
Fluorometric Determination of Chlorophyll a and Phaeopigments
The fluorometric assay is based on the fact that pigments fluoresce after they are
extracted in an organic solvent and then excited by light of specific wavelengths. The
fluoresced light emitted by the sample then is filtered selectively to obtain the peak,
which is detected by a sensitive photomultiplier tube. Chlorophyll a, when excited
by light between 430 to 450 nm, gives a maximum fluorescent emission between 650
and 675 nm. Extraction procedures are identical to those described earlier for the
spectrophotometric methods, except that glass fiber filters (Whatman GF IF, 0.6 to
0.7-Jlm pore size) must be used. In the fluorometer, excitation is provided around
440 nm, and emission is detected at about 660 nm. The output of the fluorometer is
in arbitrary units; therefore it must be calibrated using a chlorophyll a solution of
known concentration. A brief description of procedures for doing this is given below.
Details of the methodology are given in Yentsch and Menzel (1963), Holm-Hansen
et al. (1965), Strickland and Parsons (1972), Stainton et ai. (1977), and Sterman (1988).
Standardization with Chlorophyll Solution
1. Using 90% alkaline acetone solution, extract from about 5 cm 2 of a fresh green
lettuce leaf to obtain 25 ml of chlorophyll solution that is deep green in color.
Filter and store briefly in darkness.
where
etc., as above.
Spectrophotometric Determination of Chlorophyll Pigments
and Their Degradation Products by Other Methods
159
A large number of comparative methodological studies have emerged during the past
decade for the spectrophotometric and fluorometric estimation of pigments. Other
organic solvents, methanol and ethanol in particular, have been used for the extraction
of pigments and compared to the removal efficiencies of acetone (Marker et aI., 1980;
Nusch, 1980; Riemann, 1980, 1982: many others). Acetone is the most straightforward
to use as a solvent but is less efficient than methanol or ethanol for the extraction
of chlorophyll. Acidification of methanol extracts for evaluation of phaeopigments
is extremely sensitive; slight excesses of acidity can introduce large experimental errors
(Marker et aI., 1980; Marker and Jinks, 1982). Such acidification problems are less
serious with ethanol [see Nusch (1980)] and least serious for acetone (Marker, et aI.,
1980, Marker and Jinks, 1982). It is clear that, for precise research purposes, chromatography (HPLC) is the preferred method for pigment analyses [e.g., Jacobsen (1982)].
However, HPLC is expensive to perform and not conducive to rapid, multiple pigment
analyses. Comparative analyses of spectrophotometric, fluorometric, and chromatograpic (HPLC) determinations of chlorophyll a found differences to be less than 10%
in most cases [e.g., Schanz and Rai (1988)]. Because determinations of phaeopigment
concentrations are of major importance, often exceeding 50% of the total pigment
concentrations, pigment degradation products must be measured, and acetone is
usually the preferred solvent for extraction of chlorophylls and phaeopigments.
Because of the modest extraction efficiency of acetone, however, it is important that
an effective grinding process in subdued light always be performed.
Fluorometric Determination of Chlorophyll a and Phaeopigments
The fluorometric assay is based on the fact that pigments fluoresce after they are
extracted in an organic solvent and then excited by light of specific wavelengths. The
fluoresced light emitted by the sample then is filtered selectively to obtain the peak,
which is detected by a sensitive photomultiplier tube. Chlorophyll a, when excited
by light between 430 to 450 nm, gives a maximum fluorescent emission between 650
and 675 nm. Extraction procedures are identical to those described earlier for the
spectrophotometric methods, except that glass fiber filters (Whatman GF IF, 0.6 to
0.7-Jlm pore size) must be used. In the fluorometer, excitation is provided around
440 nm, and emission is detected at about 660 nm. The output of the fluorometer is
in arbitrary units; therefore it must be calibrated using a chlorophyll a solution of
known concentration. A brief description of procedures for doing this is given below.
Details of the methodology are given in Yentsch and Menzel (1963), Holm-Hansen
et al. (1965), Strickland and Parsons (1972), Stainton et ai. (1977), and Sterman (1988).
Standardization with Chlorophyll Solution
1. Using 90% alkaline acetone solution, extract from about 5 cm 2 of a fresh green
lettuce leaf to obtain 25 ml of chlorophyll solution that is deep green in color.
Filter and store briefly in darkness.