Development of Analytical and Preparatory Techniques - Chlorophyll Biosynthesis and Technological Applications - page 71

3.2.3 Development of Fluorescence Equations
for the Determination of Protoporphyrin
IX by Room Temperature Spectrofluorometry

Prior to 1975, Protoporphyrin IX (Proto) was monitored by absorption spectropho-
tometry (Granick 1948 ). Below is described the determination of Proto in a misture
of metabolic tetrapyrroles by fluorescence spectroscopy.
Let E refer to the Soret excitation maximum of Proto and F to the fluorescence
amplitude of its short-wavelength emission maximum.
Then let (E400 F622), (E400 F633), and (E440F640) represent the chlorophyllide
(Chlide) -free fluorescence amplitudes of a mixture of Chl(ide), Proto, coproporphyrin
(Copro) and protochlorophyllide a (Pchlide a) at 622, 633, and 640 nm, respectively,
which are elicited by 400- and 440-nm excitations of the hexane-extracted acetone
fraction. The following simultaneous equation can then be written:

ð E400 F633 Þ¼ Proto ð E400 F633 Þþ Copro ð E400 F633 Þ

þ Pchlide ð E400 F633 Þ; (3.20)

k 0 1 Proto k 0 2 Copro

ð E400 F622 Þ¼ ð E400 F633 Þþ ð 400 F633 Þ

k 0 3 Pchlide

þ ð 400 F633 Þ (3.21)

k 0 4 Proto k 0 5 Copro

ð E440 F640 Þ¼ ð E400 F633 Þþ ð E400 F633 Þ

k 0 6 Pchlide

þ ð E400 F633 Þ; ð 3 : 22 Þ

where

k 0 1 ¼

Proto ð E400 F622 Þ= Proto ð E400 F633 Þ

k 0 2 ¼

Copro ð E400 F622 Þ= Proto ð E400 F633 Þ

k 0 3 ¼

Pchlide ð E400 F622 Þ= Pchlide ð E400 F633 Þ

(3.23)

k 0 4 ¼

Proto ð E440 F640 Þ= Proto ð E400 F633 Þ

k 0 5 ¼

Copro ð E400 F640 Þ= Copro ð E400 F633 Þ

k 0 6 ¼

Pchlide ð E440 F640 Þ= Pchlide ð E400 F633 Þ

fluorescence emission
amplitude of Proto at 633 nm which is elicited by a 400-nm excitation of the hexane-
extracted acetone fraction. Copro (E400F633)

According to the above terminology: Proto (E400 F633) ¼

¼

fluorescence emission amplitude of
Copro at 633 nm, which is elicited by a 400-nm excitation of the hexane-extracted
acetone fraction; etc. By solving Eqs. ( 3.20 ), ( 3.21 )and( 3.22 ) for Proto (E400 F633)
the following equation is obtained: