Chromatography Forum

Can anyone answer the following?

If I have three HPLCs (they are the exact same system) and I use the same column, buffer, settings, etc., why do I get three very different looking chromatograms?

System #1 produces a chromatogram with no rise in baseline.
System #2 produces a rise in baseline of 140mAU by the end of the run and a sharp hump at approximately 15 minutes.
System #3 produces a rise in baseline of approximately 50mAu and a rounded hump at approximately 15 minutes.

This is reversed phase and the mobile phase is HPLC water/0.1%TFA and ACN/ 0.9%TFA. The column is C4, detection is at 218.

When you say the same buffer, do you mean you moved the bottles from one system to another, or do you mean prepared the same way? TFA mobile phases are very touchy about precision of the measurements. Try switching bottles among the systems and see if the problem follows one bottle.

That said, 140 mAU is extreme even for TFA, so you should also check for contamination. The most probable sources are the mobile phase filters (replace), the degasser (switch channels around), the proportioning valve (switch channels), the pump seals (pull the heads and inspect). Also a leaky proportioning valve can cause this by dribbling some UV active buffer from an unused channel.

Also, I assume you meant to write "ACN/0.09% TFA" since 0.9% would cause a bad baseline indeed.