Biology Reference
In-Depth Information
Table 3.11 Determination of the reliability of Eqs. (
3.44
) and (
3.45
) used to calculate excitation
amplitudes of MV and DV Chl(ide)
a
at 447 and 458 nm respectively
Percentage error between
amount of CHL
a
added
and calcd (%)
Amount of CHL
a
added (pmol/ml)
Amount of CHL
a
calcd (pmol/ml)
Mean percentage error
SD
MV
DV
MV
DV
MV
DV
MV
DV
20.00
50.00
19.32
50.68
3.40
1.36
0.63
1.78
0.75
5.79
40.00
50.00
39.86
50.14
0.35
0.28
30.00
30.00
31.25
28.75
4.17
4.17
40.00
20.00
41.84
18.16
4.60
9.20
60.00
30.00
59.69
30.31
0.52
1.30
60.00
20.00
61.05
18.95
1.75
5.25
50.00
10.00
50.04
9.96
0.08
0.40
80.00
10.00
80.14
9.86
0.18
1.40
100.00
10.00
101.16
8.84
1.16
11.60
75.00
5.00
74.40
5.60
0.80
12.00
100.00
5.00
99.96
5.04
0.44
0.80
125.00
5.00
124.65
5.35
0.28
7.00
150.00
0.20
Note: The excitation spectra were recorded at an emission wavelength of 674 nm
5.00
150.01
4.99
0.01
3.8 Quantitative Determination of Monovinyl
Protochlorophyllide b by Spectrofluorometry at 77 K
During analysis of Pchl(ide)
b
, two different situations are encountered in:
(a) Etiolated tissues with relatively high concentrations of Pchl(ide)
a
and,
(b) Greening or green tissues with high concentrations of Chl(ide)
a
and
b
.
Since Pchlide
b
and its phytyl ester exhibit identical electronic spectroscopic
properties, including identical spectrofluorometric properties (Shedbalkar et al.
1991
),
the derived equations could be used either for the quantitative determination of Pchlide
b
or its phytyl ester.
3.8.1 Determination of the Amount of 2-MV Pchl(ide)
b in the Presence of Pchl(ide) a, Using 293 and 77
K
Spectrofluorometric Analysis: Overall Strategy
The procedure involves:
(a) determination of the amount of 2-MV Pch(lide)
a
in a 2-MV Pchl(ide)
a
+2-MV
Pchl(ide)
b
mixture by spectrofluorometry, at room temperature as described
elsewhere as described in Sect.
3.2
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