Development of Analytical and Preparatory Techniques - Chlorophyll Biosynthesis and Technological Applications - page 92

3.5.2 Calculation of the Net Fluorescence Amplitudes at
the Monovinyl and Divinyl Soret Excitation Maxima
of Monovinyl and Divinyl Mg-Protoporphyrins
in a Mixture of the Two Tetrapyrroles
The various involved procedures will be discussed below.
3.5.2.1 Choice of the Excitation and Emission Wavelengths That Give
the Best Distinction Between Monovinyl and Divinyl signals
in a Mixture of Monovinyl and Divinyl Mg-Protoporphyrins
Dicarboxylic, monocarboxylic and fully esterified Mg-Protos, i.e., Mg-Proto, Mpe,
and Mg-Proto diester, exhibit nearly identical fluorescence emission and excitation
properties. Therefore the equations derived for any MV and DV Mg-Protos pair are
valid for the other two MV and DV Mg-Proto pairs.
The adaptation of Eqs. ( 3.37 )and( 3.38 ) to the deconvolution of the MV and
DV Mg-Proto signals consists essentially in choosing appropriate a, c excitation
wavelengths and b, d emission wavelengths for Eqs. ( 3.37 )and( 3.38 ). An
appropriate choice is one that gives the most precise determination of the net
MV and DV Mg-Proto Soret excitation signals. Indeed we have previously
demonstrated that MV and DV Mg-Protos exhibit readily distinguishable fluores-
cence excitation maxima in glasses of diethyl ether at 77 K (Belanger and Rebeiz
1982 ).Under these conditions, the DV and MV Mg-Proto species occur mainly
(95 %) in the pentacoordinated state (Belanger and Rebeiz 1984 ;Rebeizand
Belanger 1984 ). The rest occurs in the hexacoordinated state (Belanger and
Rebeiz 1984 ; Rebeiz and Belanger 1984 ). The pentacoordinated DV Mg-Protos
species exhibit a Soret excitation maximum at 424-425 nm and a fluorescence
emission maximum at 591 nm (Belanger and Rebeiz 1982 ). Thus their designa-
tion as DV Mg-Protos (E424 F591) On the other hand, the pentacoordinated MV
Mg-Proto species exhibit Soret excitation and fluorescence emission maxima at
417 and 589 nm, respectively (Belanger and Rebeiz 1982 ). Thus their designation
as MV Mg-Protos (E417 F589) (Belanger and Rebeiz 1982 ). An examination of
the aforementioned DV and MV Mg-Proto fluorescence parameters ties readily
indicated that the most convenient way of differentiating between the MV and DV
Mg-Proto signals is by their Soret excitation maxima, which are about 7 nm apart
in ether at 77 K. Furthermore, as a consequence of the overlap of the DV and MV
Mg-Proto fluorescence bands, we have observed that the best resolution of the
MV and DV Soret excitation signals in mixtures of MV and DV Mg-Protos is
achieved as follows: (a) by recording the DV excitation spectrum at the emission
maximum of DVMg-Proto, i.e., at 591 nm and (b) by recording the MV excitation
spectrum at the short wavelength tail of the emission band, between 587 and
584 nm. In what follow the adaptation of Eqs. ( 3.37 )and( 3.38 )tothe