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- Posts: 7
- Joined: Fri May 06, 2011 8:11 pm
First, I am using a Beckman P/ACE MDQ, my capillary is effective length 50cm, 50um i.d.x375um o.d. fused silica capillary. My running buffer is 40mM SDS with 20mM Phophate buffer ph 7.0 and the separation Voltage is 25kV.
I have spoken with Beckman's MEKC expert, and they have given us a new rinse procedure to try, but I still see my current crash. I can provide that if people find it is necessary to correcting my problem, but it's fairly standard 0.1M NaOH, Water, buffer, etc..... I have also varied my rinse times, and nothing fixes this. This includes the new column rinses as well as the daily and in between injection rinses.
I am injecting small organic molecules (anisole, cyanophenol, etc.) and they are all diluted in pure methanol, so I am doing a 0.4psi injection for 5 seconds. I can track my current with no injection and see that it is fairly stable, although for the first 6 minutes or so, it will increase from 20-25 uA before it stabilizes. Once we inject even pure methanol, the current drops within a few minutes (time varies) from ~20 to ~3-4 uA. This type of work is performed throughout the literature, so I'm confused as to why the methanol seems to be contributing to this issue. We've injected Phosphate buffer, with 1% methanol and it will run ok for an injection or two, then just crash inexplicably.
I agree this sounds like a current leakage issue, but I have checked my electrodes, installed new columns, had other people install the columns to make sure it isn't specific user error, etc. One of my colleagues suggested increasing my buffer concentration, so I ran 100mM Phosphate alone (no SDS, so effectively running CZE), and I achieved a decent current profile for a couple of injections, but on the third injection of 1% methanol it crashed again.
Every "expert" I have spoke to about this says my methods are sound. I'm completely dumbfounded. I almost forgot, I also tried using other stocks of buffer to make sure I didn't have an isolated contamination of my phosphate bottle or SDS bottle, and that made no difference either.
Anyone have any other insight into what I could be overlooking? I know this is a long post. If you want more info, please ask! Thanks for any help you can provide!