Chromatography Forum

Dear All,
A polypeptide I am working with seems to be incompatible with my HPLC methodology. The polypeptide is 6 kDa and the C-terminus is a thioesterified. It is insoluble but is soluble in 5M urea or guanidinium chloride so this is what I use as sample loading solvent. The mobile phase I use is standard H2O/MeCN 0.1% TFA and I run linear gradients, typically 10-60%. The problems start when I try and analyse the polypeptide by HPLC. I get a very weak peak relative to the amount I injected. I know how much I injected as I have analysed the sample by SDS-PAGE and it is 95% pure. The peak I obtain has a very messy tail to it and it is also messy leading up the peak. I have attached a chromatogram and the peak I am talking about has a retention time of 25 min. To me it seems as though the polypeptide is not very soluble in the movile phase. The 0.1% TFA makes the pH of the mobile phase very similar to the pI of the polypeptide and was thinking this may be causing the problems with solubility.
Has anyone had similar problems with peptides and would trying different mobile phase such as propanol seem like a sensible idea? I suppose I could try lowering the TFA concentration as well.

Thanks,
Sat

Hi chichibabin,

There are several things in this topic that make me wonder.
You say the peptide pI is similar to the mobile phase pH. But how similar? According to the info you give, the pH in the mobile phase is about 2. Naturally I can’t dismiss the possibility of a peptide having pI = 2, but it would be very, very unusual, if not unheard of.
Another thing I noticed is the un- retained peak at the start of the run (the void volume). Are you sure your peptide is not in there?
Finally I’ll draw your attention to the very start of the run – it is clear to me that the column is not in equilibrium with the start conditions (was it 10 % B?). The MeCN concentration is probably much higher than 10 % at the time of the sample injection.

So here are some suggestions for you:

1. Make sure your peptide is soluble in the mobile phase (I’m almost sure it is, but investigate it by mixing sample solution and mobile phase and observe the mixture visually)

2. Equilibrate your column with the start conditions before injecting the sample.

3. Inject also your solvent (urea or guanidinium) without sample in it and see if the peaks you observe are similar to the peaks you see when you inject the actual sample (the peak at 25 min might just be some junk from the mobile phase or the solvent)

God luck

Maybe it’s the sample solubility as well.
I know that having very different chemical environments in your sample Vs. mobile phase could create similar effect.

If so, try to make the 2 environments as relate as possible.