James-
We have used the AccQT method quite a bit, but have not seen the problem you describe. Here are a couple of thoughts:
1. What kind of samples do you run? Are they unhydrolyzed free amino acids, or the result of complete acid hydrolysis of a protein? If unhydrolyzed, are there any materials around that may build up on the column? If acid hydrolyzed, is it a glycoprotein, producing degraded material that might bind to the column? Any lipids that may build up? The question is whether you can do any sample cleanup before you inject.
Here's a good test: do you see this degradation of chromatography if you just repeatedly run standards?
2. How about incorporating a higher concentration of acetonitrile into your wash step, after every chromatogram? You might have to equilibrate for a longer time with Buffer A, but that's why we have automation!
Vernon
Vernon A. Shoup
Regeneron Pharmaceuticals
Rensselaer, NY 12144
(518)488-6012
vernon.shoup@regpha.com
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Date: 10/15/1999 11:28 AM
To: VERNON SHOUP
From: Freedy, James [OBI]
Good Day,
I have been successfully using the AccQTag chemistry and system, for
amino acid analysis. I am monitoring by both UV and fluorescence and have
determined LOD and LOQ for my specific application. As well I have
determined a dynamic range of linearity for this application and both
precision and accuracy have been demonstrated.
My question is; why after approximately 18 chromatographic runs do I
lose my retention times for the amino acids?(They slide off the back end of
the chromatogram.) Is it strictly a column conditioning phenomenon or is
there more to it than that? I have been able to bring the chromatography
back to the original optimized state twice by just re-starting the system as
described in the manual(5min, 60%acn/40%water, 9min, 100%buffer, AccQT
eluent A). My third attempt at a re-start, as in the manual, was not
successful, I had to wash the column more extensively with acn and water
before I could generate the original chromatography. I would appreciate any
input, because of the time savings in not having to work it all out on my
own. Either on-line or off will do, but on-line may benefit others as well.
Thanks in advance.
James G. Freedy Ortho Biotech, Inc.
jfreedy@OBIUS.JNJ.com