As a simpler solution to peptide mapping by LC/MS, we have recently found
that the use of Ammonium Hydroxide (20-50mM, pH 9.5) can be used in positive
ion mode to give separations which are comparable in resolution to TFA based
systems with 5-10X better Signal to Noise. The selectivity is somewhat
different depending on the peptide. The application requires a stationary
phase that is base-stable. We have been using the Zorbax Extend packings
without any problems. An attractive feature that we haven't exploited yet
is that this mobile phase system should work for both positive and negative
ion modes, so mode switching could be used for some advantage. I have a
PDF copy of a poster from ASMS if anybody is interested.
Alex Apffel, Ph.D.
Chemical and Biological Systems Department
Agilent Laboratories
3500 Deer Creek Road
Palo Alto, CA 94304
Tel: 650-485-6090
Fax: 650-485-8502
Email: alex_apffel@agilent.com
-----Original Message-----
From: Rod Levine [mailto:rlevine@nih.gov]
Sent: Monday, July 24, 2000 6:11 AM
To: Recipients of ABRF List
Subject: Re: LC-MS
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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