Baseline movement

[padtwo]

We've recently changed our HPLC method from TFA/Acetonitrile buffers on a C4 column to Formic Acid/Acetonitrile buffers on a C18 column and are now experiencing a shift in the baseline during analysis.

After a set time the baseline steps up about 70mAu and then levels off and stays at that level. I have tried using the TFA buffers again on the C18, and although not completely successful there is a difference so I am assuming it is something to do with the column. Has anybody else experienced anything similar.

Any help would be much appreciated!

[zokitano]

Is your method gradient or isocratic?

At which wavelength is set your detector?

Give us more information about your method in order to be able to help you.

Regards

[danko]

Hi Padtwo,

In addition to previous questions:
If gradient, how does the gradient program look like?
Do you perform an actual injection?
If so, what do you inject and how much?

Best Regards

[padtwo]

It is a gradient program, set to run over 22 minutes, with the gradient starting after 5 minutes. Rise in baseline is seen consistently at approx 11 minutes. Column has been well washed with elution buffer on a number of occasions. I'm looking at peptides dissolved in water at 0.5 mg/ml and am injecting 10ul. Detector is set at 214nm

Sorry first post, wasn't sure exactly how much information was required.

[zokitano]

Can you post the gradient program also? It is easier for us to give any comment if we know the composition of your mobile phase during the gradient program? (% buffer solvent vs % organic AcN during time).

Are you adding formic acid to your (A) buffer solution as well as to your (B) AcN prior analysis?

At which concentrations is present the formic acid in your mobile phase?

Formic acid and TFA absorb UV light at 214nm.

Formic acid
http://www.atmosphere.mpg.de/spektrum_i ... OH_lin.JPG

TFA
http://books.google.com/books?id=RYOHFn ... &ct=result

Regards

[mohan_2008]

Padtwo:

1. Your detection wavelength is very close to the cut-off wavelengths for both TFA & HCOOH.

What it means is: Theoretically even if your operating wavelength is 4 nm above their cut-offs, still it is not sufficient. Due to presence of impurities - they can still absorb over the range.

Due to this selective absorbance by either TFA or Formic acid, and specifically if they are present only in mobile phase component - can create this drifting slope.

2. Do this. Replace your TFA/HCOOH with phosphoric acid. Phosphoric has a lower cut-off and will not absorb at your operating wavelength.

Caution: Phosphate can precipitate out if excess 50 mM in 100%ACN. Hence, deduce an appropriate buffer strength.

I would revert to an isocratic if using phosphate just to avoid precipitation problems during gradient.

[HW Mueller]

mohan_2008, may I suggest that you take a look at the links given by zokitano? You will see that impurities do not have to be invoked, it will also give you an idea of the meaning of "cutoff".
Others have already hinted at possible control of the drift without resorting to a different buffer.

[padtwo]

Thank you for the replies so far!

Buffers are 0.1% Formic in water for buffer A, and 0.1% Formic in ACN for buffer B

Column prep would be washing the column with 100% B for 5-10 minutes, then equilibrating it with 90% A and 10% B for 10 minutes or until the baseline looks ok.

Program then runs for 5 minutes at 90%A after loading then the conc of B increases from 10% to 100% over 15 minutes, then continues at 100% for a further 2 minutes to make sure everything is off. A total run of 22 minutes.

I know the 214 is close to the limits, elsewhere we use 280, but we've been ok in the past and this is a relatively new problem. Machine has a DAD which I thought was one of the better detectors, hence getting away with reading near the limits, but I'm quite new to this and would welcome any advice!

[zokitano]

Dear Padtwo,

I don't mean to offend you with this question, but is it possible that your AcN/Formic acid solution is put in opened reservoir (or is prone to evaporation, with other words) or maybe you're using offline degassing with helium, which could facilitate evaporation? If evaporation has occured, then the concentration of the formic acid will rise and the absorptivity of your solvent B (AcN/0.1%FA) will rise too. Hence you'll see rising baseline

Just a thought

Something in this context, that I thougth it would be useful to post it:
http://www.msdshazcom.com/WEB_DOCS/EMD/ ... d01cc2.pdf

Best regards

[danko]

Hi Padtwo,

There is nothing wrong with the column (in context of your fist post) or the detector (last post).
Your baseline is - metaphorically speaking - a “drawingâ€