Hi,chomatographers,
I'm developping a gradient elution method :
Mobile phase A :buffer pH2 + heptane sulfonic acid + MeOH +THF
Mobile phase B :buffer pH2 + hept.sulf.acid + CH3CN+THF
The tested gradient is : 1' to 15' : 100% A to 100% B
15' to 16' : return to 100% A
I observe a strong increase of the baseline during the first 15' of mobile phase changement.
The wavelength is fixed at 205nm and can't be changed.
1) Is it possible to minimize this slope's problem? Especially if impurities elute during this changement of solvant.
2) After the positive slope , there is a changement in 1' : it generates a dramatical decrease of the baseline and a 2' wide hill just one minute after which would hide impurities.What can be the cause of this wide "peak" ?

Thanks very much for advices.

At 205 nm you will see the changing organic content of your mobile phase throughout the gradient run. There is not really a lot you can do about this. You can try baseline subtraction techniques but I find that they don't really work too well. A 205 nm you are going to have to live with it. Ultimatly, your may need to investigate non-UV detection technology to solve you problem.

Thank you for your answer.I wonder if an amount changement of organic solvent could minimize the slope?
You don't have any solutions about question 2?

Thanks.

Have you run the gradient without actually injecting anything (not even water - use a 0uL injection volume or just get your system to start the gradient and data acquisition)? This will show if this extra "hill" you are observing is from you mobile phase or the solutions you are injecting.

It's a way I didn't investigate because I didn't know it was possible to run methode without sample ! I'll try it next time.(coming back holidays) I've read that it could come from air sample too;HPLC has got a online degasser but I don't know if it's enough?
Thank you for your help .

When detecting at this low wavelenght, first you should check the absorbance of the mobile phase, especially when using THF (same to some buffers or ion pair, but I'm focusing right now only on THF).
To do this without having a long equlibration time for your column, disconnect the column and run water or water/ACN. That is your zero baseline. Now switch to your mobile phase. When using THF baseline absorbance can easily go out of the lienar range of your detector - depending on the quality of the THF. If that happens, use only fresh(!!!) THF, and keep it under nitrogen and cool!

I made some tests and got for fresh THF a max. absorbance of 0.4 AU, for older THF up to 3 AU (bottle was opened some weaks or month ago). Stabilized THF didn't make any real difference when it is old enough.

From my experience as long as you use THF and an UV detector at low wavelenght you have to live with storng baseline shifts when running gradients.

Andreas

Thanks a lot Anon and Andreas ,
I'll investigate all these strategies.