SUBJECT: DCPIP and pH
DATE: 10/95
Hi Biolabbers!
We are using the Dickey-Morgan cellular respiration lab this semester that
was
presented at a Benjamin-Cummings workshop several years ago and is in Jean's
manual. Several groups tried to do the aerobic respiration part of the
experiment (which involves DCPIP as an indicator) and found that the color
of
the solution changed depending on the solution's pH - pink to purple to
blue at
pH7 and up. The problem was found with acidic pH's using a citric acid sodium
phosphate buffer system. Anyone out there ever have this problem? Any ideas?
I would appreciate any help or suggestions.
Take care and thanks,
Rosemary Boone
Dept. Biological Sciences
Univ. of Pittsburgh
rmboone@vms.cis.pitt.edu
(412) 624-9325
I think the problem here is that the citric acid is reducing the DCPIP.
This can easily happen with electron acceptors that are used in
experiments on respiration and photosynthesis. Another thing to be sure
to avoid is ascorbic acid. You might check the redox potentials to see if
this is an accurate assessment of the problem.
Marvin Fawley
From: Marvin W Fawley <fawley@plains.nodak.edu>
Rosemary,
We use DCPIP as an indiator in a redox experiment with
succinic dehydrogenase. We use a crude wheat germ
homogenate which we spin down so that the activity is
concentrated in the supernatant. We add a phosphate
buffer at pH 7.4.
We routinely see several color changes, although because
of the buffer I never thought to double check the pH. What
we see is reproducible: the supernatant + DCPIP + extra
succinate turns clear, indicating reduction. Preparations
with the supernatant and DCPIP but no succinate turn a
greenish, I figure either from mild reduction from the crude
extract or some other interaction with the supernatant- either
way the color follows the supernatant. Preps with DCPIP
and succinate and either no extract or the pellet turn purple.
Again, I didn't attribute this to a pH change but it could be;
my guess was possible metal ion interactions.
In any event, we find the colors reproducible and use this
as a way to illustrate the importance of controls: we have
the students try various mixtures and compare the colors.
Since some combinations clearly lack substrate or enzyme,
they can associate those colors with non-enzymatic side
reactions and focus on the one combination in which
enzyme, substrate, and dye are present and lead to
reduction.
John Dickerman
Northern Illinois University
DeKalb, IL 60115
T80JWD1@WPO.CSO.NIU.EDU
We ran a photosynthesis lab a few weeks ago and also had a problem using
acidic solutions with DPIP as the indicator. The students experimented
with factors effecting the rate of photosynthesis. They tested pH and
found that they were unable to measure absorbance over time. Apparently
the acid added to the chloroplast solution was reducing the DPIP too
quickly for the absorbance to be measured. I'm not sure if using another
acid would take care of the problem.
Barbara
**********************************
Barbara D. Stegenga
208A Coker Hall
Biology Department
UNC-Chapel Hill, NC 27599
Davidsoh.Coker@mhs.unc.edu
(919)966-3180
FAX:(919)962-1625
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