PDA

[MassSpecM]

I thought I knew something about HPLC -PDA, but this one has got me.

Can someone please guide me in the general direction of this problem.

I have a HPLC attached to a PDA detector.
When I run a isocratic run with 65%MetOH I get perfect baseline, everthing is perfect.

As soon as i start running a gradient of low organic to high organic (10% MetOH -> 90%Metoh) I get huge baseline drift into -12000 mAU as soon as I introduce the High organic solvent. I know organic can do that because of absorbance but this much???

I've tried zero'ing the detector with 100%MetOH as instructed in the manual. But now it does the complete opposite. Huge baseline drift up to + 12000mAU.

the column I use was bought second hand but tested in our lab (Isocratic 65%MetOH) when we bought it and it seemed fine. Can this be the problem? bleed??
I Equilibrated the column before I tried running the gradient.

thanks in advance for your help.

[Ron]

Is the baseline rise due to the solvent effects at short wavelengths? If so, try collected data over a narrower wavelength range, maybe starting at 230 nm for example.

[grzesiek]

I really don't remember any problems using PDA attached to HPLC, maybe this is the problem, try attaching them this way

but seriously i remember sth simmilar a long time ago, but don't remember what was wrong - something simple

1. check if gradient is ok - A and B lines mistakenly changed with eachother?
2. check pda settings
3. change your solvents - purity issue?
4. i don't think the column has anything to do with it but if you like then run a gradient without the column
5. call your local support and cry

that should do it

[tom jupille]

Check the basline at various wavelengths.

If the drift is linear and worse at short wavelength, it's most likely a solvent purity problem

If the drift is non-linear (and especially if it comes down a bit at the end to look like a "hump") and is worse at longer wavelength, then it's most likely a flow cell alignment problem.

This kind of gradient drift problem is actually common enough that we've put together a "mini-seminar":
http://www.lcresources.com/more_resourc ... hp?f=3&t=5

You will have to register (free) to view the seminar.

[MassSpecM]

thanks for the replies.

need to do other analyses for now but will play with it and keep you posted

help is much appreciated!

[MassSpecM]

This kind of gradient drift problem is actually common enough that we've put together a "mini-seminar":
http://www.lcresources.com/more_resourc ... hp?f=3&t=5

Tom, you have a great resouce centre here, really helped a lot! altough i tend to loose track of time, working through them. kept me up till 1 am the other night!

[tom jupille]

Flattery will get you *everywhere*

Glad they were useful!