Re: IE HPLC

Amos Heckendorf (nestgrp@world.std.com)
Wed, 1 Dec 1999 09:09:16 -0500

>It's easy, I thought, just get an anion-exchange column (DEAE) and get a
>salt gradient going. Hah!
>
>I have a highly charged peptide (30aa, 4 Glu's, no positive charges) which
>is refusing to budge from the column. I've been running 10mM Tris (pH 8.5
>- I want to seperate this guy from another with pI about 7, and didn't
>want to do a pH gradient) and shoving loads of NaCl in, but nothing comes
>off. I just ran 2M NaCl for 1.5h (this is how Vydac say I should clean the
>column of any highly charged stuff) and still nothing.
>
>Is there anything I can do to avoid doing a pH gradient? (Kosmotropic
>salts?) I don't want to fill the entire HPLC system with salt crystals.
>This negative peptide has a pI of about 3, so what buffer systems would be
>good for going from pH8 to pH3? Is it okay doing a pH gradient with
>DEAE? (I read somewhere that it's much better to do salt gradients(?))

Jenny

Thank you for a question I think I can answer after months of waiting!

You were wise to be about two pH units from the pI of your peptide,
however, it sounds like you are at a pH too high for what the column was
designed for. Most DEAE separations can be done at a pH range of 5.5-7.0,
which is low enough to keep the column charged and high enough to keep the
peptide from being uncharged (Some DEAE marketing pieces say the columns
are still charged up where your pH is, but I would be careful up there, it
would be safer with a QAE chemistry.). Check to see if your peptide is not
sticking to the column. If it is not, then drop the pH to 5.5, use
phosphate buffer and go get your separation.

If it is being retained, then you should be asking what else the peptide is
asking you to think about. In order of most to least likely:
1. Is it soluble?
2. Is it so hydrophobic that it is aggregating at just about the time it is
electrostatically releasing from the column because of the salt?
3. Is it hydrophobicly sticking to the column backbone chemistry?
4. Is it chelating metal sites on the frits?

If you want to see if it is a hydrophobicity problem you can either treat
the column with ammonia (pH gradient for anion exchangers) to desorb the
molecule without increasing the salt, or you can add small amounts of
organic (ca. 5-10% EtOH, MeCN, etc.) to promote solubility or reduce
hydrophobic associations.

Low amounts of organic may not denature your peptide, and they will not
risk salt precipitation either, so this seems like a safe course to follow.

If it is chealting metals, try 40mM EDTA di-sodium salt treatment of the
column overnight and periodically in the future. Sometimes frits can cause
this problem, but this treatment takes care of the problem most times.

As a side bar: Have any of you noticed an oxidation of your column fittings
with the use of formic acid? You would have seen a rust color on all of
the fittings when you take them apart.

Best of luck, and let us know how you solved it.

Regards,
Amos

Amos Heckendorf (nestgrp@world.std.com)

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