Polyhydroxyethyl artifact

[pipettemonkey]

I have a HILIC column that has a massive artifact peak showing up in the middle of a gradient. After > 10 blank gradients, the peak remains. It is active 220 and 280 nm, and a different HILIC column (with the same mobile phase, TEAP pH=2) has no such artifact.

In HILIC, starting conditions sometimes are > 85% MeCN. Possible some protein/peptide precipitated in or around the column frit, and is bleeding off?

How can I get rid of this? At what point is it a good idea to reverse the flow on the column?

[Uwe Neue]

Not clear what the origin of the problem is. It could be column bleed, then it won't go away. It could be adsorbed stuff, and than it should decline with repeated runs. Do you want to talk about the column chemistry?

[pipettemonkey]

Can you elaborate on "column bleed?" Interesting you mention this. The manufacturers mention something about this, and that this loss could be accelerated by formic acid. I just never imagined it would absorb at 280 nm. I am trying an over-night treatment with 50 mM HCOOH at low flow rate. We'll see what happens.

[Uwe Neue]

The ligand that is attached to the surface can come off slowly, which looks like something that changes very slowly with time. Since you know what the ligand is, you should be able to guess if the spectrum that you see fits to this possibility.