HPLC Troubleshooting Tutorial: Problems with the UV Baseline

I was using a Cogent Bidentate C8 2.o™ column in my HPLC method recently when I observed a strange phenomenon. Instead of a normal, flat baseline in the UV monitoring readout, there was a highly erratic pattern; the signal would descend and jump back to its starting point in a recurring “sawtooth” like waveform. Meanwhile, the pressure was stable and all other instrument parameters indicated normal operation. My isocratic mobile phase was fairly simple (50% DI water/ 50% acetonitrile/ 0.1% formic acid) and other columns I tried did not have the issue. No amount of equilibrating seemed to remedy the situation.

                I really wasn’t sure if the column had somehow been damaged but had no way of running a standard QC test if I could not observe analyte peaks amongst the unstable UV baseline. Next I thought maybe there was some contaminant on the column and it needed to be washed out. Initial trials using solvent systems such as 1:1 methanol: DI water proved unsuccessful, and then I had the idea that perhaps there was some trace immiscible solvent in the column and use of an aqueous-based mobile phase would not be appropriate.

                So I switched to pure isopropanol and gave it a try. Miscible with both reversed phase and normal phase solvents, isopropanol can often be an effective choice in general column cleaning. Soon after introducing the new solvent to the column, the UV baseline resolved into a typical, flat signal. Now the real test would be to see how my original solvent system behaved. Upon reintroducing the original mobile phase of 50% DI water/ 50% acetonitrile/ 0.1% formic acid, I was pleased to observe that the baseline had returned to normal.

                How can we account for this behavior? The outcome of the experiment seemed to indicate to me that the column did indeed have some minor amounts of a nonpolar solvent in it, which would create immiscibility issues with the reversed phase solvents I was using. These could have been residual packing solvents and will pose no problems once completely removed.

This just shows you if you think a column may be defective, try to investigate all your possibilities before prematurely concluding that it has been damaged. You can save your laboratory money by getting the most out of each valuable HPLC column instead of replacing it. I find issues like this can crop up in one form or another quite often; a column is suspected to be defective when in reality, there is a simple solution to resolve the issue. So the next time your HPLC column exhibits some unexpected behavior, try to ask yourself what might be causing it and whether it can be fixed.