Chromatography Forum

We've been performing a gradient method that uses 10% of LC grade unstabilized THF and varying levels of buffer and acetonitrile. The method is an impurities test and the THF is needed to resolve the active ingredient in our pharmaceutical product from an impurity that is very similar to it.

The analytical wavelength is 226 nm (unfortunately!).

The sensitivity of the method was acceptable when we started out but has deteriorated since then and the baseline noise can be terrible at times. We suspect that there's interference from the THF peroxide so we use fresh THF each time. We have tried using small bottles of THF so that we use a fresh unopened bottle each time but the results are not reproducible.

I've been reading the threads on THF and we will try blanketing it in nitrogen. I'd also like to test the solvent to determine peroxide levels. Someone mentioned peroxide test strips. I'd like to have more information on these, especially their sensitivity.

Any other tips regarding the use of THF would be great as well.

Ahalya

I can't remember which salt I added (anh CuSO4 or something), but I routinely distilled THF via round bottom/mantle heater/water jacketed condenser just before use to eliminate peroxides in one very sensitive assay.

N2 blanketing is essential. If you know you're going to need several runs worth of 10% THF MP, make it in advance and store He or N2 sparged portions in tightly capped bottles as THF actually degrades more slowly when diluted in water.

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Thank you very much DR. That's a big help. I hope we never have to resort to distillation!

Ahalya

Another way to purify THF is to pass it through a column of activated basic alumina. Of course this is an expensive option, and distillation is cheaper in the long run.

At times we have used THF. Sometime difficult to get a good baseline for low level impurities, but the THF was needed for specificity. In these cases, left the THF concentration constant, and varied the amount of water/acetonitrile to get the desired gradient.

Thank you very much DR. That's a big help. I hope we never have to resort to distillation!

It's not so bad. Once the rig is setup, you can knock out a liter in ~20 min. It can take a lot longer than that to integrate around garbage peaks...

I had a similar situation using 20% THF at 228nm for a neutral compound, isocratic. The baseline noise was really terrible. What I did was turning off the degasser (agilent 1100 series) and voila, problem solved. I hope this helps in your case.

The baseline noise was really terrible. What I did was turning off the degasser (agilent 1100 series) and voila, problem solved.

How could the degasser be the source of noise problem? Any explanation?

Maybe the degasser was yanking a little of the THF out of the mobile phase. It's a vacuum/membrane unit that runs periodically, so it there's any preferential evaporation, you would see it in the baseline as RI artifacts, if nothing else.

How could the degasser be the source of noise problem? Any explanation?

For the same reason we degas...

If your degasser is, instead of degassing, doing nothing or adding bubbles, you can face a big problem!

I have seen cross-talk between the degasser and the detector baseline also, not just with THF. I really can't explain it. There are new degasser designs that use a variable speed motor that runs continuously; the cycling effect is eliminated, and I have not seen cross-talk with this design.

Gentlemen,
It was my bad. The baseline I experienced was really wavy. I didn't have any problem running the same method on the old Agilent 1100 series (DOM:1997), but I do have problem with newer one (DOM:2003+). This is due to the new design of the degasser which allows small volume for the solvent to pass through compared to the old one, which causes the cyclic effect as Mark puts it.
Sorry for the confusion,