Development of Analytical and Preparatory Techniques - Chlorophyll Biosynthesis and Technological Applications - page 90

3.5 Quantitative Determination of Monovinyl
(MV) and Divinyl (DV) Mg-Protoporphyrins
(Mg-Protos) by Spectrofluorometry at 77 K
At 77 K electronic spectroscopic methods of analysis are not suited for direct
quantitative determinations. Indeed, at low temperatures solutions do not freeze
evenly and generate macroscopically non-homogeneous glasses. As a conse-
quence, low-temperature glasses prepared from the same fluorescent solution
exhibit wide variations in the magnitude of their fluorescence emission and
excitation signals, which in turn depend on the condition of the glass. However,
it has been our experience that, in frozen samples of a solution containing MV and
DV tetrapyrroles, the ratio of MV to DV fluorescence signals is independent of the
condition of the glass. This is probably due to the random microscopic distribu-
tion of the MV and DV molecules before and after freezing. It was therefore
conjectured that if one determines, by 293 K spectrofluorometry, the total amount
of MV + DV tetrapyrroles in a mixture of the two compounds, and determines
the MV/DV ratio in the mixture by 77 K spectrofluorometry, then the calculation
of the amounts of MV and DV tetrapyrroles in the mixture reduces to simple
arithmetic.
The methodology for determining small amounts of tetrapyrroles at 293 K
has already been described in Sects. 3.2 and 3.3 (see above). Therefore, what
remains to be done is to develop the methodology for determining by 77 K
spectrofluorometry the ratio of MV and DV components in various tetrapyrrole
mixtures. This in turn reduces to deriving equations that permit the determina-
tion, at 77 K, of the net fluorescence signal generated by any MV tetrapyrrole in
a MV + DV tetrapyrrole mixture, and of that generated by its DV analog. The
ratio of MV to DV fluorescence signals is then computed and that ratio can be
readily converted to a ratio of MV/DV tetrapyrrole concentrations by reference
to a standard calibration curve. The latter would relate various MV/DV tetra-
pyrrole concentration ratios to the ratio of their net MV and DV fluorescence
signals. The amount of MV and DV tetrapyrroles in the sample is then computed
(a) from the total tetrapyrrole concentration which has been determined from the
sample at 293 K and (b) from the authentic MV/DV tetrapyrrole ratio for that
particular sample, which has been determined from the 77 K spectra.
The derivation of generalized equations for calculating net MV and DV
tetrapyrrole fluorescence signals from low-temperature fluorescence spectra, in
the absence and presence of other interfering MV and DV tetrapyrroles, is
described below.
Next, the quantitative determinations of MV and DV Mg-Protos and MV and
DV Pchl(ides) in mixtures of MV and DV Mg-tetrapyrroles, similar to those
encountered in extracts of etiolated and greening tissues will then be described.