gradient elution baseline problem

[ana]

Hello,
I wish you all a happy and succesful year...Which started with a problem for me ...
I have a strange decrease in the baseline during my gradient elution; that is, insted of line going straight and up and straight again, it goes straight then down...and straight again. I start with the mixture of acetate buffer, acetonitrile, methanol and THF, then move to the same without the buffer. The wavelenght is 248 nm. Help!! Thanks!!

[danko]

In your mobile phase, 2 components absorb some light at 248 nm – THF and acetate.
Your gradient goes from some acetate towards no acetate. This results in falling baseline.
So everything’s normal. I’d be troubled, if the baseline went upwards with the described mobile phase composition.

Best Regards

[ana]

Thank you!
So now I have a different kind of problem: what I'm trying to do is to validate a method developped in another lab; in their chromatograms the baseline goes upwards How's that possible?!

[danko]

I see. It’s not funny - to experience the opposite of what you’re expecting. So you have to find a plausible explanation.
I’ll suggest the following exercise: Confirm all the mobile phase components and remember to write down their concentrations in solvent A and B respectively.
Then I and I’m sure many others on this board might come up with more ideas of what is going on.

Best Regards

[ana]

Thank you, Danko. I hope I can find the solution for this.
I have solvent A: acetate buffer pH4 (as prescripted):acetonitrile:methanol:THF 50:42:7:1,
and solvent B: acetonitrile:methanol:THF 42:7:1.
The curve looks like this:
9min 100%A
next 6 min increasing to 50:50%
next 7min 50:50% steady
next 8min 100%A again
So I have a steady line until the 10th minute, then a rise followed by a steady line, and around the 13th minute it falls lower than at the beggining.
On the chromatograms from another lab, the line goes straight, up, a little bit down, straigt again, but higher than it was. But if what I'm getting is what s expected, how can they get a signal as this?
Thanks

[danko]

Hi ana,

It’s getting more and more mysterious. The time table and the description of the baseline are not consistent, so I’ll have to ask some more questions: What is the flow-rate? What are the column dimensions? What is the rest of the B-solvent (50 % milli-Q water)?
How is the described baseline generated (0 μL injection or do you actually inject something e.g. water)? How about the pressure – is it kind of stable through the run or is it fluctuating in a foreseeable or unforeseeable manner? Do you see the same baseline with the column removed?

Best regards

[scanter]

Hi Ana,

I have found batches and suppliers of THF can often differ in their properties even though they are all still 'HPLC grade' THF. During a money-saving change of suppliers in a previous position we found the gradient change in exactly the same manner although I will concede we were not using the same buffer as you are. Are you using denatured THF? Is it old? Are you using new freshly opened bottles each time?

Ah the joys of THF