I've pH'd too far. - (Feb/24/2009 )
This is a daft question, but whilst making up TBS I got a bit acid-happy and have mananged to pH it to 4.5. Do I have to start again, or can I bring it back up with something else? I'm not sure of the rules of this, as I haven't been silly enough to do it before
Any help would be fab,
ps, is there any way of getting access to old accounts? Mine got wiped :-(
depending on the sensitivity of your procedure to salt, you can just adjust the pH to where you need it with naoh.
you can add more tris and other components to make it to a larger final volume and finish the adjustment.
most of us lost our accounts in the big crash of 2009. we just have to continue on...
I have done that before. As suggested, adjust the pH with either NaOH or with a Tris base and it should be good to go.
The only problem with adding NaOH is that the salt content goes up. That is, if you can't have NaCl in the buffer, once you add the NaOH you have it.
Thanks all - most helpful! I'm only using the buffer for western blot washing etc so I assume the salt content won't be a big problem anyway. I didn't actually think of adding more Tris and increasing the volume - I'll bear that in mind.
Whilst I was pH-ing, one drop of HCl took the 1 litre of buffer from 8.3 to 4.5, when it had only been dropping a couple of 0.1's per drop before that. I was using concentrated HCl granted, but I've done this enough times to know this is really unusual. Out of interest, any ideas why it happened this time?
It's just media. You're introducing a variable that is probably of no significance but certainly unneccessary. Start over.
scoob00 on Feb 26 2009, 11:42 AM said:
are you preparing the buffer at use concentration or a concentrated stock?
the pK of tris is about 8.3 (some say 8.1). when you were adjusting from above the tris was resisting change towards the pK. when you pass the pK the buffer won't resist change away from the pK as well especially with dilute buffer. hence the big change from a single drop of concentrated hcl.
we always prepare concentrated stocks to final pH then make small adjustments to the diluted stock, with dilute acid or base, as necessary.