Development of Analytical and Preparatory Techniques - Chlorophyll Biosynthesis and Technological Applications - page 91

3.5.1 General Equations for the Determination of Net
Monovinyl and Divinyl Fluorescence Signals
in the Absence of Interference by Other Monovinyl
and Divinyl Signals

We have reported in this chapter that when two fluorescent compounds, X and Y,
having different but overlapping fluorescence excitation and emission properties,
occur together in a particular sample, the net fluorescence signals generated by
compound X can be separated from the fluorescence signals generated by com-
pound Y by calculation via two unknown simultaneous equations (see above) and
(Tripathy and Rebeiz 1985 ).
For example, let a and b represent any two fluorescence excitation or emission
wavelengths of compound X. Likewise let c and d represent any fluorescence
excitation or emission wavelengths of compound Y. We have demonstrated earlier
in this chapter that the deconvoluted net fluorescence signals, X(Ea Fb) and Y
(EC Fd), generated by compounds X and Y, respectively, at the designated
wavelengths a, b, c, d, can be determined as described by Eq. ( 3.4 ), from

X ð E a F b Þ¼½ð F a F b Þð E c F d Þ= k 2 ð l = K 1 Þ (3.37)

and

Y ð E C F d Þ¼½ð F c F d Þð E a F b Þ= k 4 ð l = K 2 Þ (3.38)

where ( E a F b) and (Ec F d) represent the fluorescence excitation ( E ) and emission
( F ) amplitudes of the X + Y mixture, at the a, b, and c, d wavelengths, respectively,
and where

K 1 ¼ 1 ð k 1 = k 2 Þ

(3.39)

K 2 ¼ 1 ð k 3 = k 4 Þ

and

k 1 ¼ X ð E c F d Þ= X ð E a F b Þ; k 2 ¼ Y ð E c F d Þ= Y ð E a F b Þ

(3.40)

k 3 ¼ Y ð E a F b Þ= Y ð E a F d Þ; k 4 ¼ X ð E a F b Þ= X ð E a F d Þ

In what follows we will illustrate the use of these generalized equations for the
calculation of the net fluorescence excitation signals of mixtures of MV and DV
MPE and of mixtures of MV and DV Pchl(ides).