Hi Anyone
I have puzzling phenomenon occuring when I try to do chlorophyll
contents of leaves. Can anyone suggest a reason for this?
I have extracted my leaf as per Porra 2002 (Porra 1989 seems to be
offline somehow).
ie. 80% acetone buffered with HEPES at pH 7.8.
I have tested the procedure by creating a dilution series and
measuring the aborbances of these solutions.
The shock was to find that there was an initial linear increase of
absorbance with dilution passing through the origin, but at a specific
dilution the relationship shifted up by the value of A750!
So the advice of Porra (2002) to minus A750 from A646.6 to correct for
turbidity was only necassary above a dilution threshold.
Other absorbances 480, 654 show the same phenomenon, but each has a
different dilution threshold at which the turbidity correction becomes
necassary. While it is necassary to correct for turbidity for A664 at
all dilutions.
Why does turbidity change stepwise in a wavelength and dilution
specific manner?
Incidentally, if I correct for turbidity on the absorbances that are
artificially high, all points fall on the same relationship for each
absorbance.
Discussion of turbidity seems to be lacking from the chlorophyll
literature...
Below is some of the data
Dilution A750 A664 A647 A480
0.016 0.6 0.654 0.024 0.04
0.029 0.601 0.717 0.052 0.083
0.032 0.6 0.711 0.052 0.08
0.040 0.602 0.746 0.678 0.112
0.048 0.602 0.792 0.694 0.139
0.056 0.599 0.795 0.695 0.141
0.063 0.599 0.825 0.71 0.163
0.083 0.603 0.886 0.731 0.207
0.091 0.604 0.909 0.754 0.227
0.108 0.598 0.982 0.783 0.878
0.167 0.6 1.204 0.89 1.049
0.231 0.602 1.445 1.013 1.213
0.333 0.601 1.799 1.19 1.477
Thanks Matthew Gilbert
