Electrophoretic (Stir) Cell Update
I used the source meter that Chris brought over and tested the voltage at points across the chamber. I set up the system with 1x PBS at 15V (~10mA) with a pinholed membrane. I tested the voltage drop at the outer edges of the well, in the center, and the inner edge of the chamber (which is not quite at the membrane surface). This image outlines the voltage drops I found.
Additionally from inside to outside walls of each well I observed about .5V.
After checking the voltages and letting the system run (and push water) for about 10min, I checked the temperatures. Room temp was 21C, the positive electrode well was 28C and the negative read 26C.
Finally I gave the pH a check in each well using pH strips (there’s not enough fluid for our regular meter). In both cases it was out of the readable range of the strips, with the positive well <4.5 and the negative well >10.
Since the temp isn’t super high, I’m wondering if the pH change is leading to the rhodamine quenching. It certainly won’t be good for proteins. A stronger electrolyte buffer may be helpful though.
Update: I tried this with .25mM Tris-HCl (the buffer commonly used in electrophoresis). The pH still changed to <4.5 and 9, but wasn’t quite as dramatic as PBS. Gels are performed in large volumes which may make pH changes at the electrode not such a big deal. Tris-HCl also had a much higher current for the same voltage: 15V – 40mA. This may be because it’s a better electrolyte (?).
The pH problem would go away if you could eliminate/reduce electrolysis. Do you know anything about Ag/AgCl electrodes? My impression is that these electrodes could help, but I know very little about the options and they are probably quite expensive. The other option is to drop the voltage to a level that does not drive the reactions, but I’m not sure if you will still have a voltage in the fluid.
Today I’ll test dropping the voltage, and checking the EO and pH.
I’m not sure about the Ag/AgCl electrodes but see if I can find anything out about them.
Jim sent around a Zydney paper a few days ago where Ag/AgCl electrodes were used. You might want to start there?