At 06:50 PM 7/21/2000 -0400, John.Holt@aventis.com wrote:
>What is the best solution to the problem that everyone's favorite ion
>pairing agent for reverse phase HPLC, trifluoroacetic acid, is not very
>compatible with electrospray MS??
>
John,
Several companies market C18 columns with evidently decent performance at
much lower TFA concentrations. But I still think the available literature
supports the conclusion that TFA gives superior chromatographic results
and, often, better solubility of the proteins or peptides -- as pointed out
in the original report (W. C. Mahoney and M. A. Hermodson. Separation of
large denatured peptides by reverse phase high performance liquid
chromatography. Trifluoroacetic acid as a peptide solvent. J.Biol.Chem. 255
(23):11199-11203, 1980).
So, I prefer the "solution" or "fix" proposed by Alex Apffel and colleagues
(A. Apffel, S. Fischer, G. Goldberg, P. C. Goodley, and F. E. Kuhlmann.
Enhanced sensitivity for peptide mapping with electrospray liquid
chromatography-mass spectrometry in the presence of signal suppression due
to trifluoroacetic acid-containing mobile phases. J.Chromatogr.A. 712
(1):177-190, 1995).
They showed that suppression could be alleviated by mixing the column
effluent with a short chain acid and alcohol. Since then, we found
slightly better performance by simply using neat acetic acid, mixed in a
tee and then pumped onto to the MS. Thus, you need another pump to
implement this solution. The acetic acid is added at half the flow rate
coming from the LC. So, for a "typical" narrow bore column with the LC
pumping at 200 ul/min, the post-column addition of acetic acid would be at
100 ul/min.
Rod Levine
NIH
Bldg 3, Room 106 MSC 0320
Bethesda, MD 20892-0320
email: rlevine@nih.gov
voice: 1 (301) 496-2310
fax: 1 (301) 496-0599
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