low sample energy for 0.5% tfa

[SandraAlbrecht]

Hi,

I'm a bit frustrated after trying the whole day to get our Waters HPLC system running. For the past 2-3 months I've been running samples with 0.5% TFA as mobile phase and got good results at 210 and 215 nm. Because of the nature of some of our samples we got some air into the system which accumulated in the flow cell (spikes in the chromatogram and very low ratio of sample to reference energy), but was successfully removed yesterday. The system was flushed with water and everything seemed happy (sample to reference energy more or less equal) when I left. This morning the UV detector had died and I exchanged it with the same model (W2487) from another of our systems. The reference energy of the UV lamp seemed rather low though (6-7 nA) but the ratio to the sample energy was okay as long as I used water as mobile phase. As soon as I try to switch to 0.5 TFA the sample energy goes down to 0.010-0.016 nA. I know that TFA absorbs at that wavelength, so I would expect the energy to be lower than using water. But the background noise is so high that sample analysis with low amounts is not possible. Does anyone have an idea what I could do? TFA is filtered and sonicated to get rid of dissolved air.

I appreciate every afford to help me solve the problem.
Sandra

[danko]

Hi Sandra,

Maybe a new lamp will do the trick.

Best Regards

[SandraAlbrecht]

Yep, that was my first idea. But it doesn't solve the problem. The new lamp shows 1/6 of the energy it's supposed to have when new.

[danko]

Calibrate the wavelength. Maybe the wavelength in reality is lower than the one you’ve chosen – f. x. 200 instead of 215nm (?)

Best Regards

[SandraAlbrecht]

Thanks, I'll try that.

[tom jupille]

If the energy on the reference side is low, I can think of three possibilities:
- lamp is misaligned
- your "new" lamp has been sitting on the shelf too long (D2 does diffuse out over time)
- your optics (mirrors, windows, etc) are dirty.

Since you had the same problem with the previous lamp, I would look at #3 as the most likely.

[tom jupille]

If the energy on the reference side is low, I can think of three possibilities:
- lamp is misaligned
- your "new" lamp has been sitting on the shelf too long (D2 does diffuse out over time)
- your optics (mirrors, windows, etc) are dirty.

Since you had the same problem with the previous lamp, I would look at #3 as the most likely.

[SandraAlbrecht]

The engineer was just here and the optics ARE dirty. He tried to clean it as much as he could, but we might have to face a replacement sooner rather than later.

Thanks for your help!