Having issues attempting to configure dual detectors in series with a 10mm and a 60mm flow cell, for Aglinet HPLC
The detector with the 60mm flow cell installed will not read at the desired wavelength, 210nm. It fails the intensity test at the lowest intensity range. I tried flushing using di water, reran the intensity test, and was able to get a pass. Then, I switched back to the solvent mobile phase (75% ACN with 0.1% formic acid and 25% di water with 0.1% formic acid), and reran the intensity test, and was able to pass again.
I started running a test sequence and I could see both detectors creating peaks on the chromatogram as the sample ran. However, this morning when I went to review the result set, there was no peaks from the 60mm detector, only background noise.
I have the 60mm flow cell in the Agilent 1290 Infinity detector, and the 10mm flow cell in an HP 1100 detector.
I am at a loss, and any suggestions would be welcome and helpful!
Thank you.
Dear Jenn Rath,
In addition to comment above, check the detector settings again. Flush the system for enough period of time to remove the bubbles inside the cell. Then, try to run a standard responsible for wavelength rang around 240- 260 nm. Then will see the responses.
Piyal Aravinna
It does sound like the lamp may be failing (or the detection settings were not properly configured), but before we conclude that.. it is possible you have the two detectors set up improperly, have a leak or are using contaminated formic acid too (so besides the usual things we check, get a fresh bottle of formic and makes up new solutions).
Before testing the UV lamp, look at your UV/VIS settings. Please tell us exactly which wavelengths and bandwidth values you have selected (all of them) and also if you are using the software feature called "Reference Wavelength" (if so, please provide the settings used). If you can share you proposed method, that would be helpful too. This basic info is needed to insure that you have set up the detector correctly as incorrect settings can also cause the problems you have observed. *The incorrect use of the software feature, "Reference Wavelength" can result in all kinds of bizarre results.
To check the lamp: Start with some basic troubleshooting steps to verify correct output on the lamp. Fill the flow cell with Pure HPLC grade ACN (the same type you use in your methods), then run the lamp intensity tests and look carefully at the number of counts in the first two low wavelength ranges. Are they less than 2x the lower limit (spec)? If so, your lamp may be in need of replacement. If that looks ok, run a wavelength calibration test next and look at the results. Does it pass or need adjustment?
One additional item that you must verify is that your 1290 DAD (please, which model #) may have a broken and leaking flow cell (extremely common on this series when the 60 mm path length cell is used. Unlike the nearly indestructible 6 and 10 mm cells, the 60 mm cells are very easy to break.). You stated that you are running two UV/VIS detectors in series (Why is that? Two of the same types of detectors are not normally connected in-series. That is very unusual). Caution: When you place two detectors in-series you can easily break the flow cell on one or both detectors if one of the flow cells clogs or plugs up from forgetting to flush it regularly. This would be very likely to happen if the 60mm cell is last in line and plugs first. If the flow cell is leaking, then replace the flow cell before proceeding with other checks.
I would not disagree with any of William Letters suggestions. I do wonder why you are using Formic acid when you are monitoring 210 nm. Phosphoric acid may be a reasonable replacement.
- Badges
- Science method