160
Exercise 10
2. Dilute this stock, if necessary, so that E6650 (I-cm path length) would be
approximately 0.6 ( = ca. 6 mg chi a/I).
3. Measure absorbances (l-cm cuvettes) at 750, 665, and 645 nm against a reference
cell using 90% acetone.
4. Assuming that only chlorophylls a and b occur in lettuce, calculate the chlorophyll
a concentration:
Chi a (mg/l) = 11.57 E6650 - 1.31 E6450
where
E6650 = A6650 - A 750o' etc., as above.
5. Prepare accurate dilutions of the standard solution with 90% alkaline acetone to
obtain at least three readings on each of the sensitivity settings of the fluorometer
(I x, 3 x, 10 x, and 30 x) in such a way that the readings approximate 25, 50,
and 75 divisions on the 0 to 100 unit scale of the fluorometer.
6. Plot the concentrations of chlorophyll a (,ug/l) against fluorometer readings for
each sensitivity setting. The slope of these lines should provide linear calibration
factors (F) for the fluorometer reading for each sensitivity setting:
Chi a (,ug/l)
F(I x.3 x .10 x .30 X) = scale reading
Analyses
1. Zero the fluorometer using 90% acetone as a reference solution.
2. Using the alkaline acetone extracts of known volume as prepared for spectrophotometric analyses discussed earlier, measure the fluorescence to the nearest 0.2
unit with an appropriate sensitivity setting.
3.
3
(F)(fluorometer reading)(v)
Chi a (,ug/I) or mg/m ) = . - - - - - - . - -
(V)
where v = volume of extract in ml; and V = volume of water filtered in m!.
4. After the first reading in alkaline acetone (Rb) is taken, remove the tube and add
two drops of 4N HCI. Mix the contents of the tube by inversion and allow to sit
for 2 min.
5. Remeasure the fluorescence of the acidified sample (Ra) when a stable value has
been reached.
6. Using serial dilutions of chlorophyll extracts from lettuce, as discussed earlier,
determine the fluorescence in basic 90';{) acetone (R bst ,,) and in the extracts after
they have been acidified (RastJ Then the ratio, r, is obtained:
These ratios r are determined at different instrument sensitivities.
Then,
Chi a (,ug/I or mg/m 3 ) = (F)(- ~--)(Rb - Ra)
r-1
Phaeopigment (,ug/I or mg/m 3 ) = (F)( - ~ )(rRa - Rb)
r-l
Exercise 10
2. Dilute this stock, if necessary, so that E6650 (I-cm path length) would be
approximately 0.6 ( = ca. 6 mg chi a/I).
3. Measure absorbances (l-cm cuvettes) at 750, 665, and 645 nm against a reference
cell using 90% acetone.
4. Assuming that only chlorophylls a and b occur in lettuce, calculate the chlorophyll
a concentration:
Chi a (mg/l) = 11.57 E6650 - 1.31 E6450
where
E6650 = A6650 - A 750o' etc., as above.
5. Prepare accurate dilutions of the standard solution with 90% alkaline acetone to
obtain at least three readings on each of the sensitivity settings of the fluorometer
(I x, 3 x, 10 x, and 30 x) in such a way that the readings approximate 25, 50,
and 75 divisions on the 0 to 100 unit scale of the fluorometer.
6. Plot the concentrations of chlorophyll a (,ug/l) against fluorometer readings for
each sensitivity setting. The slope of these lines should provide linear calibration
factors (F) for the fluorometer reading for each sensitivity setting:
Chi a (,ug/l)
F(I x.3 x .10 x .30 X) = scale reading
Analyses
1. Zero the fluorometer using 90% acetone as a reference solution.
2. Using the alkaline acetone extracts of known volume as prepared for spectrophotometric analyses discussed earlier, measure the fluorescence to the nearest 0.2
unit with an appropriate sensitivity setting.
3.
3
(F)(fluorometer reading)(v)
Chi a (,ug/I) or mg/m ) = . - - - - - - . - -
(V)
where v = volume of extract in ml; and V = volume of water filtered in m!.
4. After the first reading in alkaline acetone (Rb) is taken, remove the tube and add
two drops of 4N HCI. Mix the contents of the tube by inversion and allow to sit
for 2 min.
5. Remeasure the fluorescence of the acidified sample (Ra) when a stable value has
been reached.
6. Using serial dilutions of chlorophyll extracts from lettuce, as discussed earlier,
determine the fluorescence in basic 90';{) acetone (R bst ,,) and in the extracts after
they have been acidified (RastJ Then the ratio, r, is obtained:
These ratios r are determined at different instrument sensitivities.
Then,
Chi a (,ug/I or mg/m 3 ) = (F)(- ~--)(Rb - Ra)
r-1
Phaeopigment (,ug/I or mg/m 3 ) = (F)( - ~ )(rRa - Rb)
r-l