Chromatography Forum

We've been running HPLC, and have lately been experiencing some issues. It all started when we forgot to have the DAD UV lamp on at the beginning of a run, and hastily turned in on. The trial was not a success, with no peaks being observed. We attributed this to the DAD not being on from the beginning. However, since this run, we have been unable to observe any substantial peaks that were, up until this point, consistently present. Even more issues have started in the last two runs we've done. Upon running some blanks to make sure they are flat, the baseline suddenly drops to around -2000 about 20 minutes into the run. This has not happened before. Does anyone know what might be causing this? We did a self test on the DAD, and it passed everything except the Spectral Flatness test. From what I could tell, this means there is too much noise, but as we can't seem to be getting any sizable peaks at all, I suspect this may not be an issue. We've also made sure that general setting (ie, wavelength, etc) have not been changed.

we can't seem to be getting any sizable peaks at all

That's a bit to vague to be helpful. Are you seein *no* peaks at all? Or are you seeing the expected peaks at a much reduced size?

If there are no peaks at all, start by shutting down the entire system (including the computer/controller) and then restart the modules in the standard sequence so that they run through their POST. If it throws any error codes, conduct yourself accordingly.

You have probably already done this, but just for the sake of completeness, eliminate non-detector problems:
Run a standard or check sample. If the peaks are still absent, look for the obvious suspects:
- is the pump working? is mobile phase coming out of the waste line? at the correct flow (time how long it takes to fill a graduate (cylinder, not student)?
- is the injector working? can you see the autosampler tray rotating and the needle moving? can you hear the injection valve turning? If you have a manual injector, try using that.

Assuming it *is* the detector, cobble up a luer fitting connection to the inlet then zero the detector and use a syringe to push a solution of 0.1% acetone in water into the flow cell. That should give you around 1 AU of absorbance at 265 nm. If it does, that suggests that the detector is OK and that something has happened to either your column or your mobile phase to drastically change retention of your peaks. If it doesn't, then the detector is *definitely* the culprit. Try cleaning and/or replacing the flow cell (dirty windows?). If the lamp is more than half-way through its nominal lifetime, it might be worth changing that.

If all of those fail, time for a service call.

Just out of curiosity, what brand/model of DAD are you working with?


Scott Horn

That's a bit to vague to be helpful. Are you seein *no* peaks at all? Or are you seeing the expected peaks at a much reduced size?

No peaks at all, with this newer problem of the baseline plummeting to -2000 mAU persisting.

As for the brand/model, it is an Agilent 1100 series.

Thanks for all of your help thus far.

Hi,
Primarly casue of the traouble When turning on the lamp during a run, the detector migh have zeroed itself on a peak in the chromatogram.

Have you turned the lamp off and then (after a few minutes) on during a stable BLANK run, so that the detector can zero itself on a stable baseline with just mobile phase (more or less without absorbtion) pumped through the cell?
On the Agilent 1100, you can check the ligth INTENSITY curve and check that it is a shown in the manual for the handcontroller. If you are using Chemstation, you can check it from Chemstation directly (if I remember correctly).
If the light intensisty curve is normal and you have turned on the lamp on a stable blank run, then it is unlikely that you have a detector problem. Then you propably have some other problem now, see advices above to check the rest of the LC. Hope it helps.