Chromatography Forum

Hi!

I am running a peptide (10 residue)on a TSK Gel Amide 80 column (1mmID) at a flow rate of 30uL /min. My peptide elutes at 7 min but i see a sharp negative peak just before the elution. I am running the column in acetonitrile-TFA solvent system, going from low to high water gradient.
Can you explain the cause for the negative peak?

Solvent A: 100%acetonitrile +0.1%TFA
Solvent B: 0.1%TFA.

Thanks!

Roy.

What wavelength?
What kind of detector (variable wavelength or diode array)?

I am using a PDA detector and detection is at 213 nm.

A few possibilities come to mind:

1. What is the reference wavelength on the PDA, and is there any chance that your sample contains something which absorbs at that wavelength (and elutes just before your peptide)?

2. TFA / ACN systems are notorious for baseline problems. It may be that the process of injection is upsetting the equilibrium between the TFA and your stationary phase, with a resulting "system peak" that near coincides with your peptide.

3. Related to the previous: what's the dwell volume of your system? At that low a flow rate, you might simply be seeing the baseline upset caused by arrival of the gradient at the detector.

Thanks Tom,

I will try answering the possibilities. I can try by changing the wavelength, maybe sample preparation etc to see if the negative peak goes away.

Roy.

Is the negative peak interfering with the integration? If so the most simple thing might be to shift the peptide a bit back? If it isn´t interfering, just forget it.