I am purifying my peptides, easy linear 6 to 11 AA peptides, on a semi-prep column ( C18, 130A, 5um, 10 mmx250mm) but I am having trouble because my first injection is OK, but starting on the second one, the peptide elutes in the front. Any ideas? I have checked all the solvents and the HPLC, the pressure is stable, etc.
You did not provide your actual method or conditions. Please post these when inquiring about HPLC methods as we need the information to provide constructive suggestions.
What you describe could be a simple case of not using a strong enough solvent to elute EVERYTHING off the column in each run (or not waiting long enough). The column should be flushed and washed after the run. This makes the most sense as you state that your first injection is fine, but the second one is not. The peak seen in inj #2 was still on the column when you finished your first run and is just now eluting.
The most common explanations for this:
- Method is inadequate and needs a stronger solvent to elute all samples.
- Failure to properly wash and re-equilibrate the column in between runs.
- Contamination from: Fouled column being used or carryover.
You need to equilibrate the column for at last 10 column volume. That means, if you use 25 ml/min you will need 6 min at the initial condition before the new injection.
- Badges
- Science method