Chromatography Forum

Hello,
I am trying to analyse a very polar small peptide (MW: 230) by HPLC using silica column (normal phase). The problem is that we are having variation in the results due to the presence of NaCl (MW: 58) in the extract, probably because of the interaction between the salt and the column. I would like to know if there is any solution to avoid/remove salt interference during the HPLC analysis? Is there any other column that I can use to analyse the peptide considering its polarity and its molecular weight (close to the molecular Weight of NaCl)?
Thanks a lot

You didn't specify your mobile phase, but I'm guessing that it's a mixture of a buffer and acetonitrile, which would make it HILIC. Possibly increasing the buffer concentration, or adding NaCl to your mobile phase, or switching to a different HILIC column might help. Depending on your peptide, doing SPE on a mixed-bed ion-exchange cartridge might work.

The 60-A pore version of our PolyHYDROXYETHYL A material has a fractionation range in SEC between 20-600 Da when eluted with a mildly denaturing mobile phase such as 50 mM formic acid. It can separate amino acids from dipeptides, dipeptides from tetrapeptides, etc. I don't see why it wouldn't separate your small peptide (3 residues?) from a salt such as NaCl. It's routinely used for analysis of peptides this size in protein hydrolyzates.

I’m far from convinced that NaCl would interfere with any peptide in that sense.
Unless you’re running IEx., which obviously isn’t the case.
If you describe the method in more details and tell us exactly what you observe, we might come up with a solution – not necessarily NaCl removal.

Best Regards