Chromatography Forum

Before performing a related substances analysis (gradient method) I usually wash the system with water-IPA-water with narrow bore tubing connected to create back pressure. I also connect a clean up column to the aqueous pump outlet to reduce any water effects. However over the course of a long term stability study It can sometimes be difficult to obtain a baseline that is reproducible with the initial analysis (usually the slope over the gradient section). Can anyone give any ideas for obtaining a reproducible baseline over the course of a long-term stability study or any thoughts on what may be causing these differences.

Some details would help:
How are you detecting? (if UV, what wavelength?)]
What mobile phase (what organic? any buffer? if so, what?)
Does your baseline drift up or down? How much? (linking to a couple of chromatograms might help).

You've already dealt with the most common problem (contamination), other possibilities include UV cutoff of the organic solvent (can vary from lot to lot), temperature affects (can change the RI response). Detector flow cell alignment (out of alignment can exacerbate RI effects), lamp aging, etc.

Thank you for your reply.

I'm using UV detection at 230 nm, Mobile phase A is a perchlorate buffer (pH5) and mobile phase B is acetonitrile (HPLC grade). The column is temperature controlled at 40C and the lab is also temp controlled. THe gradient goes from 97%A to 90%B over over 40 mins. The steep incline occurs at around 75%B.
I'm inclined to think this is an instrumental problem but I'm not sure what system parameters I should be monitoring that may cause such an effect.
The system would still pass the gradient and step tests but I was thinking along the lines of column pressure and lamp energies. Are there any other parameters that you could think that it might be worth observing at each analysis time point?

At 230 nm, UV-mismatch should be minimal (does anyone know the UV cutoff for perchlorate?). Refractive index mismatch is more likely, particularly if the baseline drift is "hump" shaped (increases and then turns back down slightly at the end). If it *is* and RI effect, it can be exacerbated by misalignment of the flow cell. One way to tell is to compare the same method on two instruments of the same model. If there is about the same amount of drift, then it's probably detector optics, and there is not much you can do about it. If one instrument looks worse than the other, then it needs to be serviced.

A certain amount of baseline shift is normal. Data systems generally handle baseline drift gracefully so long as it isn't excessive. Without seeing a chromatogram, it's hard to tell whether or not you need to be concerned.