Dear Plant Ed Folks,
I have a student who is interested in measuring chlorophyll content in a
chrysophyte alga, which has chlorophyll a and c, but no chlorophyll b.
We
are using Arnon's calculations to determine total chlorophyll and
chlorophyll a, but the numbers don't jive. Maybe someone can tell me
what
I've missed.
1. She does an extraction with 100% acetone, then splits the extract
into
two equal portions. One portion she uses to measure total chlorophyll
by
adjusting the solution to 80% acetone with water, measuring the
absorbances
at 645 and 663nm, and using Arnon's calculation works up how much total
chlorophyll is present.
2. She can also use the same absorbances to calculate how much chl a is
present, since Arnon has a formula for calculating just chl a. If she
does
that she gets numbers something like this: 130 ug/ml total chlorophyll,
50
ug/ml chlorophyl a.
3. The second portion of the initial extract she extracts into a small
volume of hexane which concentrates the chlorophyll, and from this she
spots a known volume onto a TLC plate. The pigments are separated on
the
TLC plate, and the chlorophyll a band is scraped off and re-extracted
into
80% acetone.
When she measures the chlorophyll in this sample, she again uses
absorbance
at 645 and 663, but uses Arnon's formula for chlorophyll a only, since
that
is the only pigment in this part of the extract. The value that she
gets
for just chlorophyll a, and we know it is just chl a because it was
separated on from all other pigments on the TLC, is close to 130 ug/ml,
suggesting that virtually all the chlorophyll in the extract is chl a.
That could be true, because there is very little chlorophyll c present,
but
if so, why does calculating how much chl a there is in step 2 tell us
there
is a very different amount of chl a (130 ug/ml vs. 50 ug/ml.)? The
student's calculations all check out when I do them, so I don't think it
is
a calculating error. I wondered if Arnon's formulas are only valid for
tissue that has significant amounts of both chl a and chl b. Is it
possible that his formulas can't be used for non-green algae chlorophyll
extracts?
Thanks for your help,
Kathleen Archer
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Kathleen Archer
Dept. Biology
Trinity College
Hartford, CT 06106
Ph: (860)297-2226
kathleen.archer at mail.trincoll.edu
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