Using Waters PDA detector with ACN

[jong]

When I use ACN/water as MP, the baseline shots up even near 1 AU when detected under 220 nm. However, it is not the case when I used UV detector at 220 nm.

I contacted Waters, but their answer is intriguing. Any suggestions would be highly appreciated!

-------------- Waters' answer

it's quite possible that the PDA model type is the issue here. There is a large enough difference between how the 2487 UV detector works and your model (the 2487 has the ability to do some filtering since it uses a reference) but in your case, what your detector sees is what you get. The bandwidth of the cell and the function of the diodes all play a role with what you're seeing and it can't be altered to achieve a similar result as in the 2487. In addition, the UV cut off for ACN is below 200 but close enough to the regions of your interest that the noise generated can't be filtered out on this detector type.

However, there's one thing you might be able to try that could help the situation. If you're using 100% ACN and 100% H2O, you could try to use a premix of say 95% ACN with 5% H2O and 0.1% TFA for solvent A and for solvent B you could use 95% H2O with 5% ACN and 0.1% TFA. This composition seems to work better than the straight 100% composition in decreasing the amount of out-gassing to the cell. If this is something you think you could use in your analysis, I'd give it a try. Otherwise if you need to use ACN, you might have to use a different detector type.
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[danko]

Hi Jong,

....even near 1 AU when detected under 220 nm.

I don’t think the explanation offered to you, can elucidate fluctuations in the order of 1 AU. Neither am I convinced that the offered solution/suggestion – although sensible enough – would solve the described problem.
Unfortunately, I’m missing a lot of details, such as detector setting and more importantly illustrative graphical data and that makes trouble-shooting quite difficult – if not impossible.

Best Regards

[DR]

^ agreed.

There are three different approaches, both may be helpful...
Waters is onto something in that premixing your mobile phases as suggested can make your baseline a bit quieter. Also, you may need to clean up your water as 1AU is higher than would be expected for a RI related gradient ramp. Try using a C18 guard column or Empore Extraction Disk to pretreat your A-phase water.

Failing the above, add something to the B-phase so that the RI is a closer match to that of the A-phase (I'd be more specific, but I can't remember what most people use for this...).

[Hollow]

Some suggestions:

What about the PDA-cell?
Is it clean?
Did you thoroughly rinse it with 100% organic or even with HNO3 6 mol/L (Caution!!! Don't let the nitric acid get in contact with your organic solvents (Waste or solvent lines)!!!!)

What about the optics? Are the optic windows still ok?

What about the lamp? How many hours have it been on? Is it still ok?
Did you caried out the PDA-Diagnostics if you're using Empower/Millenium?
Do you have enough energy below 220 nm?

How does the baseline for a 0-100% Water:ACN gradient looks like?
Is it smooth and straight up or does it exhibit some absorbance maximum in the mid-range?

PS: Waters mentioned something about TFA in your mobile phases, but you didn't say that to us. Are you just using ACN/Water or also with TFA in it?

[Kwet]

What's your acetonitrile quality? Some can pretty abosrb as far as 240 nm. Have you try a different quality?
Please also describe your gradient.

[jong]

Thanks for all your reply and suggestions and I truly appreciate. BTW, this the a new Waters system we just started to test and run it.

The water quality is the issue here. I switch my Nano-Water (from the NANopure DIamond Life Science ultrapure water system) to HPLC grade water from Fisher and the baseline is fine now. There is a little increase (~ 5-10 mAu) when ACN content is above 60%.When I switched back to the Nano-water, the baseline starts jump up and down again, although to a less extent ( ~ 150mAu). Therefore, I am pretty much convinced that the "Nano-Water" cannot be used for HPLC.

The problem was not caught easily because the baseline is fine when MeOH/Nano-water was used. Again, the Nano-Water purification system was also recently installed ( less than 2 months).

However, I am still a little uncomfortable to say that the Nano-water is bad ( for HPLC), because we use it for preparing many biological buffers. And I use similar water purification system in my former lab and use the water for HPLC as well. I checked the spectrum of the Nano-water using Nano-Drop, it looks similar to the HPLC grade water from Fisher. Any comments here?

Thank you.

[Chromatographer2010]

You also want to make sure you water system has a carbon filter and a uv detector to filter out organic contaminants and to give a measurement of TOC. The Nanopure comes with and without this option.

Also the bandwidth of the UV detector (not changable on a Waters PDA) and the spectral resolution affect what wavelength range you are actually looking at on any UV detector. Make sure you have your spectral resolution set to the lowest value to minimize how far away from your set point you are actually looking at.

[grzesiek]

"The water quality is the issue here. I switch my Nano-Water (from the NANopure DIamond Life Science ultrapure water system) to HPLC grade water from Fisher and the baseline is fine now. " - We only had some problem with our nano pure when it was not working fine, apart from this we hadn,t seen any problems

Did you change ACN? Try another vendor