Dear Group
With due respect to Alex, and without any angst over pH limits of columns,
I would caution all who do operate at higher pH that corrosion of stainless
steel is something to worry about, although I suspect it is more an issue
of inorganic cations. Comments from the hardware crowd would be
appreciated to clarify this.
Never the less, a flushing of the systems with some sort of acid each day
would be recommended to reduce the pitting of the stainless steel hardware
that could occur with time.
All the best.
Amos Heckendorf
The Nest Group, Inc.
--------
> 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) 496-2310fax: 1 (301) 496-0599
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