Chromatography Forum

Hello

We are experiencing a baseline dip directly after our peak of interest that is about 4X noise. It does not seem to be effected by the size of the peak of interest itself.

The analysis is for inositol utilizing an RI detector under conditions that are fairly close to the USP monograph for this compound.

Agilent had us do an extended rinse of the reference cell and this did not help. I was wondering if there are some general possible reasons for this phenomenon when working with an RI detector that others have come across.

Thanks

Do you see the same dip if you just inject diluent (no sample)?
Are both the diluent and the mobile phase degassed?

Yes - I just checked and we do see a dip in the baseline at the same retention time, about the same size with just diluent (water) - water is also the MP here as well. We are degassing the mobile phase through a degasser and have degassed the samples by sonication.

Perhaps our samples are not appropriately degassed vs the mobile phase? Could that cause such a dip?

We have narrowed this down to the injector I think. Doubling the volume of the injection doubles the size of the negative peak when we reinject a blank of our mobile phase. To rule out systemic contamination we did a benchtop RI reading of our MP effluent from the machine (waste) vs MP input bottle vs degassed sample solvent (also water). All were exactly the same.

We are going to scour The Google for information on what to do when our injector is providing us with a contaminant that lowers the RI reading below baseline (air bubble seems like a possibility here) - but if anyone has some ideas to throw our way I would appreciate it.

Perhaps our samples are not appropriately degassed vs the mobile phase? Could that cause such a dip?

Yes it can. The problem with sonication/aspirator is that it's a "one-shot" procedure; re-gassing (did I just make up a new word?) begins as soon as you take off the vacuum.

One way to test is to degas some of your mobile phase, then immediately make a series of injections with it over a half-hour or so. If the size of the system peak changes, then it's dissolved air. If it doesn't , it's not.

Two quick comments. (1) You may be due for injector maintenance. A worn injector valve rotary seal could cause the exact problem you are describing (as could improper column washing too, but the fact that the peak increases when you increase the injection tells us it is a worn seal). 2) Degas in a continuous manner. Do not use sonication. It stops "degassing" as soon as you remove the bottle from the sonicator resulting in drift. Use a properly maintained in-line vacuum degasser or continuous helium sparging.