Chromatography Forum

Can someone please let me know if I can load 1mg/ml peptide dissolved in 100% DMSO on a Waters C18 solide phase extraction column? I am trying to separate a linker of 250 Da from a peptide which is 600 Da??

Do I have to modify the peptide in DMSO so it can bind efficiently to the column?

Thank you

Gop

DMSO is a solvent with a high elution strength. A peptide is not likely to bind to a C18 from DMSO.

You have several options: a strong dilution of the DMSO with water, maybe 10 to 1. If the peptide is not too polar, it should bind to the C18.

Alternatively, you could bind it to a cation exchanger, Oasis MCX. Do you know what the ionic form of the peptide is? If it is in the cationic form, you can bind it directly to the cation exchanger. If not, you need to add acid. You elute it again, by changing the pH to a high pH, best with ammonia.

Thank you, Uwe.

My peptide is cyclic RGDK and this is soluble in water or buffer. Do you think a 10:1 ratio of PBS:DMSO (100 mM phosphate, 150 nM NaCl, 0.5% Na-azide) will be useful on a C18 column?

I do not have an Oasis column. I have to order it.

Gop

Unfortunately I think that it is too polar to be retained on a C18 column. The second path, retention on a cation exchanger, is the way to go.

Considering that you have two basic amino acids and one acidic amino acid, an even better approach might be to use a weak cation exchanger (Oasis WAX) at neutral pH for enrichment, with elution unter acidic conditions.

You need to decide based on the nature of the linker from which you want to separate your peptide.

It also occurred to me, that you do not necessarily need to think about retaining the peptide, if the linker is more hydrophobic than the peptide. You could retain the linker, while the peptide moves through the SPE cartridge unretained. What is the linker?

Thanks again. The linker is succinidimyl-PEG-4-benzaldehyde. with a molecular weight of 247 Da.

The last idea - retaining the linker on the Sep-Pak C18, while the peptide breaks through - might work, if you dilute your DMSO sample at least 10:1 with water.