Has anyone else experienced this? We have been running 10mM ammonium acetate in both water and methanol phases, with a gradient 100% aqueous to 100% organic in 3.5min,and back to 100% aqueous to equilibrate . The columns failed alot so we run with pre-columns and in line filters. We have to change them regularly to prevent column blockages. We have tried filtering, doping the organic with aqueous and vice versa, but there has been no great improvement. (its an LC-MS application.)
Any suggestions? much appreciated
By Benjamin on Monday, October 14, 2002 - 08:49 am:
I believe your problem is buffer precipitation in 100% organic phase. The salt you are using is expected to be very soluble in water, but it may have limited solubility in 100% methanol at 0.01M level.
See if you stop the gradient at a point lower than 100% methanol, lets say 75% methanol. This may help.
By Anonymous on Monday, October 14, 2002 - 10:24 am:
If you're using C18 (which I'm assuming you are), you may have a phase wetting phenomenon. (hydrocarbons don't much like 100% aqueous, they may even start sticking to one another, dependong on the composition). Perhaps you might want to try 95% aqueous to 5 % aqueous. Again I'm not sure what your s.p. is, so this is just a guess.
By Anonymous on Monday, October 14, 2002 - 05:45 pm:
What are you injecting?
By Anonymous on Tuesday, October 15, 2002 - 12:12 am:
Jane, I remember that when I used to work with TSP LC-MS I could dissolve ammonium acetate 0.1M in pure methanol, so I think the problem could be on the sample or column side (just an idea!).
By Jane on Tuesday, October 22, 2002 - 06:27 am:
Thanks for all of the advice. I suspect it is the buffer precipitation problem and will tray finishing at a lower percentage. I'll let you know how it goes.
PS I am injecting neat Methanol or 50/50 water methanol.
By ScottF on Wednesday, December 4, 2002 - 12:30 pm:
So, if it isn't a buffer problem (Anon 12:12), and it probably isn't a 'phase wetting' problem since that takes longer than a few minutes at 100% water to occur, and the symptoms would occur almost immediately, it only leaves a couple other common problems, assuming the failure is due to plugging.
Since you are running a very short run time, I assume this is either a short column, and/or a very small bore column. The column may have very fine frits, perhaps finer (0.5) than the frits in the filters (2.0?) you are using.
If you are changing these filters regularly, you are removing all the particles from the pumping system and sample. But maybe you are removing particles bigger than 2 um, and letting smaller ones through to the column frit.
Ron Majors gave a seminar in our lab a couple weeks ago and mentioned this. It is a common problem when folks move to these tiny columns for LC/MS work.
Have you tried to backflush the columns to see if the inlet frit can be unplugged?
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