Hello Everyone,

I am currently having trouble fine-tuning my HPLC gradient approach in order to achieve better separation because I am working with a complicated mixture of peptides. A C18 column (150 x 4.6 mm, 5 µm) with a 0.1% formic acid mobile phase and a conventional water/acetonitrile gradient is what I'm using. Getting decent resolution is challenging because the hydrophobicity of the peptides in my combination varies a lot in this community.

The details of my current approach are as follows:
  • Column dimensions: 150 x 4.6 mm / 5 µm
  • Phase A of mobility: Water plus 0.1% formic acid
  • Phase B mobile: 0.1% formic acid + acetonitrile
  • Gradient: 30 minutes, 5–50% B
  • One millilitre per minute of flow
  • UV light detected at 214 nm
I'm having trouble with inadequate peptide separation, especially in the gradient's midsection. I've attempted extending the run duration and changing the gradient slope, but I'm still not obtaining the resolution I require. The problem is further compounded by the fact that the peak shapes are somewhat broad.

Has anyone else worked with peptide mixes and encountered problems comparable to these? Would switching to a different column (perhaps one with a shorter or lower particle size) or modifying the pH of the mobile phase be helpful? I'm not sure if using ion-pairing reagents—which I've read some people use—would be appropriate in this situation.

I would be grateful for any guidance on gradient optimisation, choosing a mendix rapid column, or other possible tactics!

Thank you in advance.