Re: peptide HPLC questions
Tim_Slattery@berlex.com
Tue, 7 Apr 1998 16:17:39 -0700
Chris
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Normally for a peptide of that length I'd say that a C-18 would be=20
fine, but if the problem is hydrophobicity causing it to stick to th=
e=20
column, going to C-4 would help.=20
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I assume you are using 100 A pore material now. That should work for=
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this, but I prefer the larger pore size.
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Before assuming that your peptide is simply sticking to the column=20
however, I would consider two things. First with three arginine=20
residues there could be strong interaction with any silanols dependi=
ng=20
on pH and ion pairing agents in the buffer.If you are already using=20
0.1% TFA in your buffers I'd try a polymeric column (like a=20
PolymerLabs PL-RPS). Also, any of the newer base-deactivated columns=
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could give an improvement.
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Second if you are having solubility problems, is your peptide fully =
in=20
solution, or is it precipitating on the column? Start by making sure=
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the peptide is completely solublized. If the peptide comes off the=20
column at 30% ACN, dissolve your sample in 20% (and start the gradie=
nt=20
at 20%). If TFA helps try dissolving in 2% TFA. DMSO is also a good=20
solvent that is compatible with HPLC. If you do have a polymeric=20
column you could try dissolving your sample in a solution of 5M urea=
,=20
5M guanidine, 0.2% TFA, 10% ACN, 1% Zwittergent 3-08. Dilute 1:1=20
before injecting. This stuff seems to shorten the life of silica=20
columns though.
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Raise the temperature of the column. Most poorly behaved peaks will=20
improve at increased temperature. Try 45 - 55C.
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Try a B buffer using isopropanol instead of acetonitrile. IPA is a=20
stronger solvent than ACN and may work better if hydorphobicity is a=
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problem.
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If after all that you are still having problems you could dig up J.=20
Chromatography 443, (1988) 363-397 which might give you some ideas.
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Good luck.
--tks
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Tim Slattery
Protein Biochemistry and Biophysics Department=20
Berlex Biosciences
tim_slattery@berlex.com
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_____________________________ Reply Separator ___________________________=
______=20
Subject: peptide HPLC questions
Author: chris halkides <halkidesc@UNCWIL.EDU> at Internet=20
Date: 4/6/98 10:04 PM
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To All,
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I have a student who is new to peptide HPLC. The sequence of the peptide=
is=20
LARHYINLITRQRY. The solubility of the peptide is low in water, somewhat=20
better in TFA. The peptide seems not to want to come off in acetonitrile=
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gradients, at least not as a sharp peak. So far, TEA has not helped.
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1. What type of column is likely to give the best results: C-4, C-8, or=
C-18?
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2. Is there an advantage to going to 300 =C5 pore size?
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3. Are there any modifiers we should be trying? Is there a normal serie=
s=20
of modifiers that one typically runs through.
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4. Is there anything special about this sequence that is likely to lead =
to=20
problems.
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Thanks very much for any imput.
=20
Chris Halkides
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Christopher Halkides
Dept. of Chemistry, UNCW
601 S. College Road
Wilmington, NC 28403-3297
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