Chromatography Forum

I thought that temperature fluctuations in my lab were causing occasional waves in my UV detector baseline below 220 nm. I've since determined that the baseline actually varies almost in perfect sync with the relative humidity, not the lab temperature, especially on warm days where the RH is over 50%. I've calculated the approximate dew point and condensation inside the optics doesn't seem likely. Is there any other mechanism by which humidity could directly affect the baseline?

We are experiencing the same thing in our lab. We're baffled as well. What Make/Model instrumentation are you using? We have both Shimadzu LC 2010 and Shimadzu Prominence. We see the wave on both, although the amplitude is lower with the Prominence. Also, it happens independently of mobile phase/flow (i.e., with the detector flushed with MeOH or water and capped. Thoughts?

Jeff

I've experienced precisely this issue and correlated RH measurements with baseline preturbations perfectly. It's a bit of a PITA, honestly...Fix the building issue and your baseline will be fine. Anything short of that and you're stuck. One thing I noted was that the baseline waves occurred across all wavelengths, not just those below 220 nm. My new 2998 allegedly can have the flow cell "purged" with N2 and I may do that.

Are your assays being run at ambient temperatures?

Do you get the same effect if the column is run at temp above ambient ?

Column temperature appears not to matter. I run most separations at least 10 deg C above ambient, sometimes higher. There's not enough heat in the few micoliters of MP in the flow cell to matter, I think.

Not a problem I've come across before.

I'm running Waters (alliance and UPLC) and their site prep documentation states the environment is OK between 20 - 80%RH.

I'm having the baseline problem with a fairly new Waters Acquity-I do have a few Shimadzu modular systems but I haven't seen this with them.

Seconded what juddc said about column temperature. Also wavelength. We've seen this at multiple wavelengths from 210 through upwards of 254 nm. The magnitude of our "wave" also varies from about 0.1 mV (just a nuisance) to greater than 1 mV (affecting related compound analyses...).

The Shimadzu flow cell seems to be very well thermally insulated. We've monitored temp and found < a tenth of a degree fluctuation over a 24 hour period with our baseline wave (about 20 min x 1 mV) occurring the entire time.

Frustratedly,
Jeff

Is there any other mechanism by which humidity could directly affect the baseline?

Yes, by changing the refractive index of the air slightly. Absent a laser, it's impossible to totally collimate a light beam; there will always be some off-axis light, which will refract as it passes from air->quartz->mobile phase->quartz->air. A change in the RI of any of those media can cause the amount of transmitted light to change.

Remember that absorbance is a logarithmic function. Assuming 1V=1AU, a 0.1mV shift is equivalent to 0.02% change in light transmission.

I don't know whether any of your detectors have provision for nitrogen purging the monochromator, but I'd hazard a guess that would solve the problem.

I don't know whether any of your detectors have provision for nitrogen purging the monochromator, but I'd hazard a guess that would solve the problem.

Mine does and that's the plan when I get around to it...My (Waters) 2998 seems more sensitive to the problem than my 2996, but it's a more sensitive detector so it would be. I just have to run a "T" off of my N2 generator.