Chromatography Forum

I'm having trouble separating two peptides- both > 80 aa and 97% identical.

One has S, the other RY (near the N term), but are otherwise identical.
These peptides are very well retained on RP columns, and are have a basic pI, 2 lys + 7, 8 Arg.

I can sort of resolve these on a C4 column using HFBA + MeCN, but the peaks are extremely broad due to a very shallow gradient (0.14% MeCN/min). It is hard for me to tell if they are completely resolved or not due to this.

Since these peptides differ in number of + charges (+9, +10), would SCX be a better choice? Will I run into the same problem (broad, poorly shaped peaks) from having to use a shallow gradient?

I suppose you have tried TFA and/or different pH's (triethylamine acetate)?

I have tried TFA.

I went with HFBA as ion pairing agent in hopes I could exploit the difference in Arg content. You expect increased retention for the peptide containing the extra arg, However, I suppose a neutral pH or something like phosphoric acid- could invert order of elution. Perhaps this is worth trying.

I guess my main question could be re-phrased as:

I can't make the gradient any more shallow in my C4 HFBA RP method, where the center of each peak is < 2 minutes apart, but they are so broad, I fear they over-lap. Is the charge difference between a +9 and a +10 peptide enough of a difference to be able to resolve these peaks by > a few minutes on SCX?

Your concern is understandable about the ability of SCX to tell the difference between a peptide with, say, 9 (+) charged residues and one with 10. That said, a chromatography column can distinguish between a single residue variation in favorable cases provided the column can see it. In this case, since your variation is near the N-terminus, then select a mode of chromatography where the N-terminus is a preferred binding site. That means SCX, per this link to a recent article on peptide orientation effects on selectivity: http://pubs.acs.org/doi/pdf/10.1021/ac100651k
Since your peptide is so basic, then you have the option of using a WCX column, not just an SCX one, for cation-exchange. You can bind the peptides in something like 15 mM ammonium acetate, pH 5, and then run a gradient to 15% unbuffered acetic acid or else 2% unbuffered formic acid. The drop in pH will uncharge the carboxyl- groups of the WCX material, causing your peptides to elute in a convenient volatile solvent. Of course, you won't be able to monitor absorbance unless your peptide has an aromatic residue, permitting you to use longer wavelengths. Here's a link to a poster of mine from 1997 on the subject, involving our PolyCAT A columns: http://www.polylc.com/downloads/ISPPP_1 ... poster.pdf

I bet the case could easily be handled by HIC.
I've used HIC for separation of extremely similar peptides/proteins (typically degradants) and it never failed me.

Best Regards