Composition and Biomass of Phytoplankton
157
Degradation products of the chlorophylls, phaeophytins, are similar structurally
to chlorophyll except that the magnesium is lost from the ring structure. Phaeophytins
absorb light at the same wavelengths as the chlorophylls but less strongly. Since both
chlorophylls and phaeophytin pigments occur in fresh waters in variable amounts,
depending on environmental conditions, concentrations of both must be estimated
for each sample. The total amount of pigment is determined in alkaline acetone
(chlorophyll plus phaeophytin). The same sample is then acidified, during which time
the chlorophyll is degraded to phaeophytin, so that the chlorophyll concentration
can be determined by the difference in absorbance.
Procedures
1. Measure the extinction coefficient of your pigment extracts in 90% alkaline acetone
solution at 750 (for turbidity blank), 665, 664, 663, 647, 630, and 480 nm. Absorption
at 750 nm is subtracted from each of the other values to correct for the presence
of turbidity and colored materials. The pigments and phaeophytins absorb very
little light at 750 nm. The spectrophotometer should be brought to 100% transmission at each wavelength by referencing against a matched cell containing only
90% acetone. When the cells are exactly matched, fill both cells with 90% acetone
and determine the "blank" correction at each wavelength. Correct all extinction
coefficient values by this amount. Record extinction values to the nearest 0.001
in the range of 0 to 0.04 and to the nearest 0.005 for extinctions of coefficients
exceeding 0.04.
2. Add 0.1 ml of 1 N HCl per ml of extract directly to the spectrophotometric cell,
reseal, and mix thoroughly. Allow to rest in the cuvette holder for 5 min.
3. Remeasure the extinction coefficient values at 750, 665, and 663 nm.
4. Rinse the cells copiously with alkaline acetone and shake dry prior to use with
the next sample.
Calculations
Monochromatic Method (Lorenzen, 1967)
ChI a( gil or mg/m 3 ) = (k)(F)(E6650 - E665 a)(V)
p
(V)(Z)
where
E6650 = turbidity-corrected absorption at 665 nm before acidification
= A6650 - A 7500' where A = the absorption value
E665 a = turbidity-corrected absorption at 665 nm after acidification = A665 n - A750 a
k = absorption coefficient of chlorophyll a, = 11.0
F = factor to equate the reduction in absorbancy to initial
chlorophyll concentration, 1.7:0.7, or =2.43
R = maximum ratio of E6650:E665a in the absence of pheopigments, = 1.7
v = volume of extract in ml
V = volume of water filtered in liters
Z = length of light path through cuvette or cell in cm.
.
3
(k)(F) [R(E665J - E665J(V)
Phaeoplgments (pg/l or mg/m ) = - - - - - -
. - - - -
(V)(Z)
157
Degradation products of the chlorophylls, phaeophytins, are similar structurally
to chlorophyll except that the magnesium is lost from the ring structure. Phaeophytins
absorb light at the same wavelengths as the chlorophylls but less strongly. Since both
chlorophylls and phaeophytin pigments occur in fresh waters in variable amounts,
depending on environmental conditions, concentrations of both must be estimated
for each sample. The total amount of pigment is determined in alkaline acetone
(chlorophyll plus phaeophytin). The same sample is then acidified, during which time
the chlorophyll is degraded to phaeophytin, so that the chlorophyll concentration
can be determined by the difference in absorbance.
Procedures
1. Measure the extinction coefficient of your pigment extracts in 90% alkaline acetone
solution at 750 (for turbidity blank), 665, 664, 663, 647, 630, and 480 nm. Absorption
at 750 nm is subtracted from each of the other values to correct for the presence
of turbidity and colored materials. The pigments and phaeophytins absorb very
little light at 750 nm. The spectrophotometer should be brought to 100% transmission at each wavelength by referencing against a matched cell containing only
90% acetone. When the cells are exactly matched, fill both cells with 90% acetone
and determine the "blank" correction at each wavelength. Correct all extinction
coefficient values by this amount. Record extinction values to the nearest 0.001
in the range of 0 to 0.04 and to the nearest 0.005 for extinctions of coefficients
exceeding 0.04.
2. Add 0.1 ml of 1 N HCl per ml of extract directly to the spectrophotometric cell,
reseal, and mix thoroughly. Allow to rest in the cuvette holder for 5 min.
3. Remeasure the extinction coefficient values at 750, 665, and 663 nm.
4. Rinse the cells copiously with alkaline acetone and shake dry prior to use with
the next sample.
Calculations
Monochromatic Method (Lorenzen, 1967)
ChI a( gil or mg/m 3 ) = (k)(F)(E6650 - E665 a)(V)
p
(V)(Z)
where
E6650 = turbidity-corrected absorption at 665 nm before acidification
= A6650 - A 7500' where A = the absorption value
E665 a = turbidity-corrected absorption at 665 nm after acidification = A665 n - A750 a
k = absorption coefficient of chlorophyll a, = 11.0
F = factor to equate the reduction in absorbancy to initial
chlorophyll concentration, 1.7:0.7, or =2.43
R = maximum ratio of E6650:E665a in the absence of pheopigments, = 1.7
v = volume of extract in ml
V = volume of water filtered in liters
Z = length of light path through cuvette or cell in cm.
.
3
(k)(F) [R(E665J - E665J(V)
Phaeoplgments (pg/l or mg/m ) = - - - - - -
. - - - -
(V)(Z)