Development of Analytical and Preparatory Techniques - Chlorophyll Biosynthesis and Technological Applications - page 76

pectrofluorometric techniques for the early detection and quantitative determination
of picomole quantities of Chlide a and b and Pheobide a and b were developed. These
techniques were based on the observation that although Chlide a exhibited similar red
emission and absorption maxima as Pheobide a and Chlide b exhibited similar
red emission and absorption maxima as Pheobide b, these four tetrapyrroles exhibited
distinct Soret excitation maxima (Bazzaz and Rebeiz 1979 ). Therefore this observa-
tion was used, to derive four simultaneous equations which permitted the determina-
tion of picomole amounts of Chlide a, Chlide b, Pheobide a, and Pheobide b in
mixtures of these tetrapyrroles without prior chromatographic segregation. The
derivation and testing of these equations is described below.

3.4.1 Calculation of Chl(ide) a

The calculation of Chl(ide) a in various pigment mixtures is described below.

3.4.1.1 Calculation of Chl(ide) a in a Mixture Containing
Chl(ide) a and b and Pheo(bide) a and b

Let E refer to the Soret excitation maximum of a fluorescent compound and F to the
fluorescence emission maximum of that same compound. Then let (F674 E433),
(F674E412), (F660 E460) and (F660 E438) represent the respective fluorescence
excitation amplitudes at 433, 412, 460 and 438 nm, of a mixture of Chl(ide) a ,
Pheobide) a , Chl(ide) b and Pheo(bide) b which were recorded either at 674 or at
660 nm. The Soret excitation amplitude contributed solely by Chl(ide) a [i.e. Chl
(ide) a (F674 E433)] may then be calculated with a formula that can be derived
from appropriate simultaneous equations. Since all the tetrapyrroles in the mixture
contribute to the Soret excitation amplitudes between 412 and 460 nm. The
following equations can be written:

ð F674 E433 Þ¼ Chl ð ide Þ a ð F674 E433 Þþ Pheo ð bide Þ a ð F674 E433 Þ

þ Chl ð ide Þ b ð F674 E433 Þþ Pheo ð bide Þ b ð F674 E433 Þ

ð F674 E4I2 Þ¼ k 1 Chl ð ide Þ a ð F674 E433 Þþ k 2 Pheo ð bide Þ a ð F674 E433 Þ

þ k 3 Chl ð ide Þ b ð F674 E433 Þþ k 4 Pheo ð bide Þ b ð F674 E433 Þ

ð F660 E460 Þ¼ k 5 Chl ð ide Þ a ð F674 E433 Þþ k 6 Pheo ð bide Þ a ð F674 E433 Þ

þ k 7 Chl ð ide Þ b ð F674 E433 Þþ k 8 Pheo ð bide Þ b ð F674 E433 Þ

ð F660 E438 Þ¼ k 9 Chl ð ide Þ a ð F674 E433 Þþ k 10 Pheo ð bide Þ a ð F674 E433 Þ

þ k 11 Chl ð ide Þ b ð F674 E433 Þþ k 12 Pheo ð bide Þ b ð F674 E433 Þ