Christer,
Our group has seen some of the problems you are observing with your
chromatography. Two obvious problems are that all your amino acids
are eluting too early and you are getting no separation of Ile/Lys
near the end of the gradient. We solved the first problem by adding
100 ul neat TFA (R3) to LC-A buffer mix. This tends to move all the
amino acids later in the chromatogram and in particular moves the Asp
away from the previous coeluting shoulder. If you are lucky, this may
also improve the separation of Ile/Lys too. If Ile/Lys still are not
separated then decreasing the %B at 18.0 minutes will help. ABI
recomends dropping %B by 2% if the resolution is around 50%. If there
is no resolution as demonstrated in your chromatogram, then a more
drastic 4% decrease is recomended. Good Luck!
Richard Thoma
______________________________ Reply Separator _________________________________
Subject: Protseq PTH separation
Author: abrf-request@aecom.yu.edu at INTERNET
Date: 05/11/1999 7:36 AM
Dear ABRF:ers
I am running a 494 Procise Sequencer and I have trouble with
the PTH separation. Please have a look at the attached chromatogram.
I would bee very happy if somebody could figure out what's wrong.
I have changed all the pump seals. Solvents A , B and Premix tested.
New column and precolumn installed. The chromatograms look the same through
the whole testing procedure; a big slope in the beginning and the
hydrophobic PTHs elute too early.I am getting desperate.
Thanks in advance.
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Christer Wernstedt
Ludwig Institute for Cancer Research
Box 595
S-75124 Uppsala
Sweden
Phone: +46-18-160424
Fax: +46-18-160420
E-mail christer.wernstedt@licr.uu.se
Home page http://www.licr.uu.se
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