I am running 50/50 methanol/water "A" and 50/50 methanol/acetonitrile "B". Gradient 100% "A" to 95% "B" in 25 minutes.

The gradient baseline on a PE Series 200 rises too fast until it hits the 50% proportioning. It then declines back to the correct "theoretical" baseline rise and continues until the end condition.

What could cause such a discrepency in the linearity of the baseline?

Refractive index mismatch? Try checking the behavior at two different wavelengths. If the problem is worse at the longer wavelength, then that supports the RI hypothesis (red light refracts more than blue light). If it is an RI problem, try checking your cell alignment.

After a lengthy investigation, the unusual baseline was attributed to a dirty pump. The pump seals were replaced, check valves sonicated, etc...

The system was then flushed with Ammounium hydroxide and passified with nitric acid.

After bring the system back into equilibrium with the mobile phase, I noticed that the background absorption of the acetonitrile phase was considerably less than before.

There is still a small dip at the end of the gradient (95% ACN/MeOH : 5% water/MeOH)but no where near the crazy zig zag baseline that I observed previously.

Thank you Guru for offering your helpful hints.

Annonymous