RE: ABD 140c HPLC

Manfred Raida (Manfred_Raida@CompuServe.com)
Tue, 21 Apr 1998 10:03:43 -0400

Douplas Lamont wrote:
Dear ABRFers,

Would also appreciate any ideas you may have on the following problem...?

Have following system setup for tryptic mapping with online LC/MS

140c HPLC - 10ml syringe pumps, 75ul dynamic mixer
ABD UV Detector 785A - 2.4ul flowcell
Valco low volume rheodyne - 20ul loop (PEEK)
PEEK tubing to connect everything together - 005"
TurboChrom Professional S/W to control the pump and collect the data.

Column - Phenomenex 150x1mm
Buffers - low TFA (0.02%) in both A and B

I ran the above system with a tryptic digest sample and compared it with a
chromatogram generated using the same buffers, column, gradient and sample
on a Pharmacia SMART system. The Smart system gave a much better
chromatogram with sharp well resolved peaks. The 140 system gave a poor
profile with broad peaks and less detail.

Any help would be great

thanks in advance

Dougie

Douglas Lamont

We run a 140B at a API III with 1 mm columns and we compared the
chromatograms with SMART chromatograms and they are very similar, hard to
tell which one is better.
We changed our system a little, maybe this will help you:
1. remove the dynamic mixer and replace it after a T-connector by a 1 x 10
mm C18 column as a static mixer, you can also use two columns in series.
This works fine for flow rates up to 50 uL and tested down to 5 uL (0.5 mm
x 100 mm C18 columns, we run them in routine) on our 140D system, you
didnot write whats your flowrate is. Furthermore we replaced the flow cell
by the (very expensive) LC Packing U-cell with some nL volumes. Using the
normal cell from ABI we got no usable chroamtograms, the peaks tailed due
to the large volume of the cell. A cheaper but less sensitive alernative is
the linear capillary cell which measured just through the fused silica
capillary, we used this in the beginning but with some money from a friend
we could chnage to the U-cell. The column we use is either a YMC ODS AQ 3
um, 100 A which turned out to be very nice for peptide mapping or a
Reprosil C18 material (I donot know the producer) with identical dimensions
which was packed by a small geman company. The peaks are very sharp. If you
go down to lower flow rates we have good experience in replacing the peek
capillaries by fused silica like 50 um ID and 375 um OD (Polymicron
Technologies), Applied offers a nice system which allows the connection to
normal HPLC fittings but you can also look into Upchurchs catalogue. One
last point is that we never stop our system, even during holidays we run
gradient after gradient (without injection and mass spec). This works very
good and we have no problems.
Please contact me if you have further questions. I hope I could help you.
Manfred Raida
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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
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