We have been experiencing problems with a mysterious peak that shows up approximately 6 minutes into 13min gradient run.

A- water,0.1%TFA
B-ACN, 0.1%TFA
wash between injections, meoh/thf

so far the column was replaced, along with the rotor on the Gilson 215 liquid handler, along with all tubing, sample loop between injector and pump, the gold seal/cap was replaced on the purge valve and the peak at 6minutes is still there, the same size.

the solvents were also remade.

Accordingly it seems to be on the pump side.
Not sure what would cause this peak to appear at such consistency even when the gradient conditions were changed.

Any thoughts ?
Thanks.

This is another one of those "need more information" problems!

- Does the peak appear on blank gradients (i.e. no injection made)? If yes, you have (mostly) exonerated the injection system and should focus on the pump and/or mobile phase chemistry.

- Is the peak comparable in width to other peaks near the same retention time? If it's much wider, this suggests a late-eluter from a previous injection.

- What wavelength? The lower the wavelength, the more likely you are to be seeing a "system peak" from the injection process.

- You mentioned that the peak has the same retention time when the gradient conditions were changed -- what specific changes did you look at?

Regards
-- Tom Jupille / LC Resources

Does the peak get larger with longer equilibration?

Does it change at all with any modification or elimination of the wash?

How long have you run this method?

Did you develop the method or was it transferred from another source?

Did you get a new vendor or lot of materials for any of your mobile phase components?

What's the mode of detection?

Inquiring minds want to know...