Chromatography Forum

Hi,

I have a basic peptide pI ~11, Mw ~1500 Da that I want to buffer exchange using SEC column. I currently have G15 (MWCO>1500Da) and G10 sephadex (MWCO >700 Da). I am wondering if anyone can suggest me a type of buffer that would successfully elute this peptide? I try to stay with aqueous based buffer since this is for clinical application. I have tried phosphate, tris buffer, pH ranges from 5.5 to 8.5, and NaCl concentration up to 0.5 M, but this peptide seems to stick in the column. I am running isocratic mode. Another thing, this is my first experience with basic peptide. Any suggestions will be appreciated. Thank you.

Does your peptide also have any aromatic residues (Phe; Tyr; Trp)? The more highly crosslinked grades of Sephadex will adsorb aromatic compounds actively, causing them to elute after the Vt peak. Either use an SEC material that has no such problems (which means one based on silica, not on a polymer or polysaccharide) or try a different mode of chromatography. I would recommend using a weak cation-exchange (WCX) material. You can bind your peptide in something like 15 mM ammonium acetate, pH 5, and then elute it with a step to 10% acetic acid, cf. the following link: http://www.polylc.com/downloads/ISPPP_1 ... poster.pdf
There are a number of papers in the literature that have taken advantage of this method. In your case, you would recover your peptide in the acetate salt form, which I would think would be desirable for a clinical application. This assumes that you don't mind drying down the collected fraction to get rid of the excess acetic acid.

Andy Alpert

Andy,

Thank you for the advice. However, if I were to use the highly crosslinked sephadex,what would be the best solvent to use to elute the peptide isocratically?

Regarding the peptide, I dont know the sequence since our client does not provide that info.

Thank you

If the peptide is sticking to the matrix through interaction with the aromatic residues, then using high salt concentrations could conceivably strengthen this interaction if it's a form of hydrophobic interaction. Such being the case, disrupt the interaction by including some organic solvent; say, 10-20%. That will perturb the SEC fractionation range, of course. You'd be better off using an SEC material other than Sephadex.

Andy,

Sorry for coming back here after a month. Could you give a recommendation on some SEC materials other than polymeric based? Our peptide is basic pI ~11, Mw ~3500 Da (the previous 1500 Da was a typo). It has two Tyr and 1 Phe. The peptide is not compatible with phosphate buffer and will precipitate in the presence of NaCl. It will tolerate NaCl 50-100 mM in certain buffers like glycine. Once it percipitates out of solution, it cant be re-dissolved.

Thank you

If the peptide is sticking to the matrix through interaction with the aromatic residues, then using high salt concentrations could conceivably strengthen this interaction if it's a form of hydrophobic interaction. Such being the case, disrupt the interaction by including some organic solvent; say, 10-20%. That will perturb the SEC fractionation range, of course. You'd be better off using an SEC material other than Sephadex.

Better read my book chapter on the subject. Go to the Literature section of the PolyLC Web site (http://www.polylc.com/Literature.v.2.htm) and click on the link at the bottom of the list. It documents your kind of problem and offers a solution with a silica-based material that PolyLC manufactures called PolyHYDROXYETHYL A.

If you have more detailed questions, feel free to contact me offlist.

Andy Alpert
aalpert@polylc.com