Radhika:
Proline can interconvert between an endo- or exo- conformation. Normally the
interconversion is fast on the time scale of HPLC and you get a single peak
which is the time-weighted average of the two conformations. With some
sequences, though, the interconversion is slow and the two forms can be
resolved. Collecting and rerunning the peaks affords the same two-peak
pattern if you wait long enough for each form to reform the equilibrium
distribution. The interconversion is accelerated by high temperatures and
retarded by low ones. Accordingly, try the HPLC run at an elevated
temperature if you have a column oven. If the two forms converge, then this
is probably the explanation.
Regards,
Andy Alpert
PolyLC Inc.
Columbia, MD
(410) 992-5400
*******************************************************
<< Subj: Pepsyn:Peculiar problem
Date: 1/20/2000 5:18:06 PM Eastern Standard Time
From: rkrishnan@smtplink.Coh.ORG (Radhika Krishnan)
Sender: abrf-request@aecom.yu.edu (Association of Biomolecular Resource
Facilities)
To: abrf@aecom.yu.edu (Recipients of ABRF List)
Hi Colleagues,
I have benefitted a lot form the discussions that go on in this group ,
and
would like to avail of your collective wisdom.
I am faced with a peculiar problem with the synthesis of two peptides
that
are designed to identify a immunological epitope. The sequences are as
follows,
TTVYPPSSTAK and TVYPPSSTAK.
I have synthesized these two sequences twice on the Pioneer with two
different
batches of the acid Lys resin. My HPLC shows two peaks almost on the top of
each
other. We tried purifying the major peak on a prep column, but analysis of
the
"pure" peaks seem to indicate that nothing has changed. They look identical
to
the crude HPLC. The mystery is that the MALDI of both the samples do not
indicate anything untoward. The only peaks are that of the peptide.
Is it possible that one of the amino acids might be a racemic mixture?
All
my reagents are form PE Biosystems.
Will AAA tell me anything if there were isomers.
I have calibrated both my analytical and prep c18 columns.
Thanks.
Radhika.
City of Hope, Duarte CA.
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