Chromatography Forum

Hello All,

I am running a reverse phase assay on an agilent 1100 instrument. The mobile phase is a mixture of 50mM Phosphate buffer and Propanol/Acetonitrile.

On some of my runs I have the typical profile with no problems, but it seems that every 4 or 5 runs I get a very unusual baseline. My last inject had some baseline noise every 2 minutes. I then injected again, with no problems. The injection after that had a lot of baseline noise.

Thanks for your help.

"Baseline noise" covers a lot of ground. Can you be more specific as to what's going on:
- spikes?
- random?
- cyclical?
- lumps (look like they might almost be peaks)?
- drift?

Even better, get a screen shot and show us what the problem (and good) chromatograms look like (instructions are here).

Mostly I am seeing small negative peaks throughout the run.

Really, a screen shot posted would help quite a bit.

I recently had a baseline issue traced to rapid and excessive humidity changes in the laboratory - 30% swings in RH every 15 minutes drove my PDA crazy. Once I determined that the instrument was fine and was able to quantify the environmental issue (with a Temp / RH data logger), I was able to get our facilities group to fix the problem quickly and my baseline is fine

Did you do anything between injections? or did the noise gradually taper off during a run?

Also: gradient or isocratic?
what detector? (if UV or PDA, what wavelength).
what injection volume?
what diluent in the sample?

At this point I'll hazard a (very) wild guess that you're seeing small air bubbles building up in the detector cell and then breaking loose.

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Here is a picture of the chomatogram.

This is a reverse phase assay. I have tried chaning the column and the mobile phase. Some chromatograms look more typical others have varying degrees of noise.

I am pretty sure that the humidity and temperature in our lab is very stable.

In between injections I am doing a needle wash. This is an isocratic elution. The mobile phase is an 50mM Phosphate/ Acetonitrile/Propanol mixture.

We are using a UV/VIS detector with the VIS lamp off. Detecting at 214 nm. The injection volume is 10 uL. This is an injection of protein sample in formulation buffer.

Thanks for your help