RP-HPLC baseline drift?

[holmerz]

Hi
Recently I have observed relatively high baseline drifting on our Agilent 1100 system. I'm running a linear gradient of 5-90% Acetonitrile in water (0.1% TFA). Initially the tubing was 0.10mm but I changed it to 0.17mm, which somewhat helped on issue. As I only observe the drifting at 215nm and not 280, I suspected it could be related to the ACN, however exchanging it with a fresh bottle didn't solve it, neither does it seem to be the column, as I have tried with 3 different columns. The inserted pictures is of a run where the sample is a bit too concentrated, but the baseline at 215nm is drifting up to approx. 350 mAu.


280nm


Any help appreciated
Erik

[tom jupille]

Baseline drift with TFA / ACN systems is almost unavoidable because of the interactions between TFA and ACN and the effects on the absorbance spectrum of TFA:
http://www.chromatographyonline.com/lcg ... rticle.pdf

[danko]

Some people use 0.1% TFA in eluent A and 0.05% TFA in eluent B. This approach tends to reduce - if not eliminate - the upwards drift but it might infuence the separation due to the pH rise during the gradient. But it might not be an issue here judging from the separation task simlicity in this particular case.

Best Regards

[holmerz]

Thanks Tom , quite comprehensive paper! I had a closer look at the degasser module, and it might here the issue arises, since one of the vacuum tubes had tenderised. I ordered a replacement and hopefully that'll help.

Danko I'll try your advice and only use 0.05% TFA in the ACN. But what do you mean by "pH rise" during gradient? Will the pH not decrease with an increasing eluent B percentage if you only have half the concentration of TFA in eluent B?

Thanks again
Erik

[danko]

Hi Erik,

Will the pH not decrease with an increasing eluent B percentage if you only have half the concentration of TFA in eluent B?

Less acid = higher pH

Not very significant in this case though. So you don't need to worry. just try and see

best Regards

[holmerz]

yes yes yes of course, it's me not thinking.

[holmerz]

Hi again
So now I have exchanged the damaged vacuum tubing, but the baseline drifting still persist - no difference at all. I'll try the 0.05% TFA in eluent B and see if that helps, although the issue must have occurred due to some change in the system, it wouldn't just occur out of the blue!

\Erik

[danko]

If it doesn't help I'll buy you a beer - or two

Best Regards

[holmerz]

I did help as stated, thanks again.

\Erik