Re: RE: Big peaks in pth chromatogram

Charles Felice (felicec@Centocor.com)
Wed, 06 Jan 1999 09:19:28 -0500

I concur with Chris's comments. I have seen a similar issue with TFA using a 473/477 sequencer. The problem was intermittent and it was very difficult to track down. I was in contact with ABI on a daily basis for a while. After we changed a few valve blocks and a few of the driver boards and a mother board, the problem went away. We never traced it to a specific cause but by this time we didn't care about anything except that it worked again.
Your peak is coming a little later than I would expect for TFA though. I was seeing the injection peak rise and max out which totally wiped out D and N. TFA also does a number on the column. Track the number of cycles on your column and if it dies at an early age, ABD will usually replace it.
I would suggest that you do a test. Empty, rinse and dry your injector waste bottle and run a sequence. If you have these injection peak problems, check the pH of your injector waste. If the pH is low, you will have evidence to present to PE/ABD that you are getting TFA in the flask recon step. You can work your way back from there with ABD. I have always found their technical support to be excellent.
Happy hunting,
Chuck Felice
Sr. Assoc. Scientist
Product Characterization
Centocor, Inc.

>>> "Clogston, Chris" <cclogsto@amgen.com> 01/05 6:30 PM >>>
I recall a similar problem from when I used to run the old 470A sequencers.
It could be R4 dripping into the conversion flask (the valve block is
failing) during the reconstitution with S4. This usually manifested as an
intermediate problem that eventually showed up in every injection. Even a
tiny amount of TFA in the injection will create a huge peak early in the
chromatogram.

Chris Clogston
cclogsto@amgen.com
Amgen, Inc.
Thousand Oaks, CA 91320-1789

> ----------
> From: Bryan Smith[SMTP:bsmith@celltech.co.uk]
> Sent: Tuesday, January 05, 1999 12:38 AM
> To: Recipients of ABRF List
> Subject: Big peaks in pth chromatogram
>
> Manfred,
> I have been away for Christmas and have only just read your message, and
> Deb McMillen's reply. Maybe it is coincidence, but I had similar problems
> during December - large peak around the position of pth-N, somtimes big
> enough to obliterate half the chromatogram, but not on every run. This is
> on an old (very) 470A-190A, and suspecting contamination/other problem of
> lines etc, I have replaced the easier ones, plus column, reagents, etc.
> The problem seems less bad, but I still get an occasional peak of unknown,
> but at the pth-M position. I do not think that this is an air bubble
> problem as suggested by Deb McMillen - the odd peaks do not rise and fall
> that rapidly, they look like real peaks.
>
> Is it just coincidence that we both suffer this sort of problem at the
> same time? Is everyone else okay out there in sequencerland?
>
> Bryan Smith
> Celltech Therapeutics Ltd
> 216 Bath Road
> Slough
> Berks SL1 4EN
> UK
>
> -----Original Message-----
> From: Manfred Raida [SMTP:mraida@gmx.net]
> Sent: 21 December 1998 02:29
> To: Recipients of ABRF List
> Subject: big peak in pth-chromatogram
>
> Hallo to all,
> since a few days I have a strange problem with the separation of
> pth-amino
> acids on my 494 Procise. In several of the chormoatograms a big peak
> starting directly after N and going up to the upper detection limit
> occurs. The
> peaks goes up very fast and then "elutes" over the whole
> chromatogram. We
> checked the pumps, the chemicals, column, detector, performance,
> whatever may be
> the reason. It is not every chromatogram, sometimes 10 are without
> problem,
> sometimes on 2-3. There is no rule when it happens, the amino acids
> (if
> there is enough) are on this montain and can be detetcted clearly,
> but for
> sensitive analysis it does not work at all. It occurs in the
> standard and in the
> cycles.
> I am very thankful for any idea.
> Yours
> Manfred
> *************************************************
> Manfred Raida
> Niedersaechsisches Institut fuer Peptid-Forschung
> (IPF)
> Dep. of Analytical Peptide-Chemistry
> Feodor-Lynen Strasse 31
> D-30625 Hannover
> Germany
> E-Mail Manfred_Raida@compuserve.com
> Tel.: +49 511 5466 210
> Fax: +49 511 5466 102
> **************************************************
> ---
> Sent through Global Message Exchange - http://www.gmx.net
>
> The information contained in this email is intended for the personal
> and confidential use of the addressee only. It may also be privileged
> information. If you are not the intended recipient then you are hereby
> notified that you have received this document in error and that any
> review,
> distribution or copying of this document is strictly prohibited. If you
> have received
> this communication in error, please notify Celltech immediately on
>
> (+44)(0)1753 534655, or email 'is@celltech.co.uk'
>
> Celltech Therapeutics Ltd.
> 216 Bath Road,
> Slough,
> SL1 4EN,
> Berkshire,
> UK.
>
> Registered Office as above.
> Registered in England No. 1472269
>