Chromatography Forum

I've been working with pegylated (5 and 10kD) peptides (4-6kD) on a C18 column, and it appears that the unreacted PEG and pegylated products are eluting together late in the run.

Because typical reversed phase (C18 column, acetonitrile/water, 0.1% TFA) has worked so nicely for the peptides (eluting at 2-5 column volumes with a gradient) I was hoping to work with that method for the products as well. Are there conditions I should try with RPLC, or is there another known method for pegylated products that allows sharper resolution (I was thinking either size exclusion or hydrophobic interaction)?

Typical run:
Vydac C18 250 x 4.6 mm column
A = 0.1% TFA in water
B = 0.1% TFA in acetonitrile
10 - 50% B over 30 minutes, 1 mL/min, ambient temp.
Peptides elute at 20-25% B, product (I think) around 40%.

Any suggestions would be appreciated!

Boaz Vilozny

PEG is rather hydrophobic. It's not retained on a HILIC column until you get close to 90% ACN. Why not separate your PEGylated peptides from unreacted PEG using a HILIC SPE cartridge? At ~ 75-80% ACN your PEGylated peptides will be retained (the HILIC material will look at the peptide portion and ignore the PEG chain) while the unreacted PEG will elute in the filtrate. A step to an aqueous medium will then elute the peptide.

I do recommend using a SPE-HILIC material with a pore diameter of at least 300 Å. A material with a pore diameter of 100 Å will be too retentive of peptides this large, even if steric hindrance isn't a concern.

Thanks Andy - that's a great suggestion. By the way, I've got some columns from PolyLC, and I'll be sure to check you guys out for SPE materials as well.

Boaz