Hello,
My lab recently acquired a combination ORP electrode, with the refrence being Ag/AgCl and the working electrode being platinum.
We are hoping to use this electrode to measure the reduction potential of buffers prepared using biologically active compounds, such as GSH and GSSG and probe redox active systems by artificially setting the potential.
Recently, I was trying to validate the electrode by preparing 1 mM total concentration solutions of varying ratios of GSH and GSSG. This was done as, to my understanding regardless of concentration the ratio of Ox vs Red determines the potential value of the solution via the Nernst Equation. However the readings I got were all positive, and nowhere close to the expected potential, even when correcting for the electrode difference between Ag/AgCl and SHE.
Secondly, in 1x PBS pH 7.4, I added increasing amounts of BME up to 1 M and got an exponential decay like curve asymptotically approaching ~-120 (SHE) mV.
I am having trouble making sense of these results, namely the GSH vs GSSG ratio, and why the readings would not follow the nersnt equation.
Can anyone explain how to use these ORP electrodes, and where I may be going wrong in these experiments? All the information I can find online are referring to waste water treatment.
Dear Darius Chernitsky,
I see two serious mistakes in your reasoning concerning the Nernst equation as applied to the measurement of the redox potential, specifically, in the glutathione (GSH/GSSG) system.
First. The statement that “regardless of concentration the ratio of Ox vs Red determines the potential value…” is wrong, since the Nernst equation is defined in terms of activity, not concentration. (In simple terms, intermolecular interactions reduce the effective concentration of the particles in solution so that from the "viewpoint of an electrode", the perceived concentration is smaller than the actual concentration.) This means that activity coefficients both of the components of the redox couple should be taken into account. The data on activity coefficients in solutions of GSH/GSSG may not be available, especially in buffer systems in which you are going to measure the potentials.
Second. The Nernst equation is applicable for equilibrium conditions, both “inside” the redox system and between the electrode and the redox components in solution. From the fact that the equilibrium between GSH and GSSG in biochemical systems is established rapidly (due to the involvement of enzymes) does not follow that this will be so in model solutions. It is natural to expect the appearance of kinetic limitations. Thus, the simple solution to the problem that you hoped for is hardly possible.
Regards,
Rouvim Kadis
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