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(b) selection of appropriate fluorescence wavelengths for the best discrimination
between the 2-MV Pchl(ide)
b
and 2-MV Pchl(ide)
a
fluorescence signals,
(c) adaptation of previously derived, general purpose simultaneous equations, for
the calculation of the net fluorescence signal generated by 2-MV Pchl(ide)
a
and
b
at 77
K (3),
(d) calculation of the molar ratio of 2-MV Pchl(ide)
a
/2-MV Pchl(ide)
b
at 77
K,
from the net fluorescence signals,
(e) calculation of the amount of 2-MV Pchl(ide)
b
from the total amount of 2-MV
Pchl(ide)
a,
which is determined at 293
K and from the calculated 2-MV Pchl
(ide)
a
/2-MV Pchl(ide)
b
molar ratio.
3.8.1.1 Selection of Appropriate Wavelengths for the Calculation
of the Net 2-MV Pchl(ide) a and b Fluorescence Signals
in a Mixture of the Two Compounds
Selection of appropriate wavelengths was based on two criteria: (a) reasonably
pronounced fluorescence signals with a high signal to noise ratio and (b) minimal
contribution of any other fluorescence signal to the 2-MV Pchl(ide)
a
and
b
signals
at the selected wavelengths. On the basis of the aforementioned criteria, fluores-
cence emission wavelengths at 635 and 643 nm, elicited by excitation at 440 and
463 nm respectively were selected for the calculation of the net 2-MV Pchl(ide)
b
and
a
fluorescence signals in a mixture of both compounds. Fluorescence emis-
sion spectra of 2-MV Pchl(ide)
a
and
b
mixtures in diethyl ether at 77
K, elicited by
excitation at 463 nm [the Soret excitation maximum of 2-MVPchl(ide)
b
in ether at
77
K], exhibit a pronounced 2-MV Pchl(ide)
b
fluorescence emission spectrum
with a maximum at 643 nm, while 2-MV Pchlide
a
, exhibits much weaker emission
signals. On the other hand, fluorescence emission spectra elicited by excitation at
440 nm [close to the Soret excitation maximum of 2-MV Pchl(ide)
a
in diethyl ether
at 77
K], exhibit a pronounced 2-MV Pchl(ide)
a
fluorescence emission spectrum
with a maximum at 625 nm, and a less pronounced Pchl(ide)
b
emission band
between at 635 and 648 nm [see Chap.
12
and (Shedbalkar et al.
1991
)].
3.8.1.2 Calculation of the 2-MV Pchl(ide) a/2-MV Pchl(ide) b Molar
Ratio in a Mixture of Both Compounds
The procedure involved:
(a) Calculation of the net fluorescence emission amplitudes at 635 and 643 nm of
2 MV Pchl(ide)
a
and
b
respectively in a mixture of both compounds, and
(b) Calculation of the 2-MV Pchl(ide)
a
/2-MV Pchl(ide)
b
molar ratio from the net
fluorescence amplitudes.
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