Chromatography Forum

Hi,
i would apreciate your opinion about a problem of ours.
we are facing increasing retention times ,all the times, in a method that uses a Phenomenex Prodigy ODS2 and MP is 30%ACN/70% H2O + 2ml orthophosphoric acid. My opinion is that we destroy the column with too low pH. Does this make sense? Do you have another option or experience?

Hello

First of all please provide more details:
-paste chromatograms (pressure profile and signal)
-what is the type of pump - binary or quat
-are you using 2 lines (online mixing) or isocratic mix?
-method parameters

Retention time shift in 90% cases is linked with pump problems (flow or mixing) so more details you give, better answer you'll have

Regards

Tomasz Kubowicz

Agree with Tomasz, more information would be helpful. Unlikely that your pH is too low, however. Assuming a 1 L prep, you have 0.2% phosphoric acid, putting your apparent pH in the low 2s, which is within range of modern C18 columns (typically 2-8).

My direct question would be are you premixing your mobile phase or letting the pump mix the 30% ACN and 70% water?

We use a quat pump(Agilent) but this happens in every instrument we have and all the time. It is not instrument related. We premix the MP so there is a possibility that ACN is evaporated, but the PH is below 2.I am not sure what pH 2-8 means. We measure the pH in the aqueus phase but we may have 10% or 90% aqueus so the final pH will change

Most modern reverse phase columns have an operating range of pH from 2 to 8. Your mobile phase is likely around pH 2 which means it should be just fine. I have spent the last few years at a peptide CMO and the typical mobile phase for HPLC purity methods on C18 columns is 0.1% TFA in water and 0.1% TFA in acetonitrile. This pH is around 2 and the columns have held up just fine.

My point was that I don't believe your shifting retention times are a function of your pH being so low that it's destroying your column.

Is your method isocratic or gradient? You originally stated that you were 70% water 30% acetonitrile but then you said you might be 10% water or 90% water. Please clarify.

It's isocratic 70-30% as i mentioned. The other was a general question about the pH of M.phases. we measure the pH in the aqueus phase but we may have 10% organic or maybe 90% organic so the pH will change. So i wondered what exactly the manufactures mean with pH 2-8 since we do not know the actual pH value

Hello

Could you explain sentence below:

we may have 10% organic or maybe 90% organic so the pH will change.

If you have isocratic mobile phase pH should be the same all the time.

Regards

Tomasz Kubowicz

If you are stripping the phase off the column with strong acid than I would have thought that retentions would decrease, not increase. Exceptions would be analytes (which you do not reveal) that would be retained by bare particles.

Without details everyone is just guessing. How much extra retention do you see, over how many runs. What are you analysing ? The simplest explanation for extra retention on an isocratic system is slower flows. Have you measured the flow ?

The more you don't tell us, the more we can't help you.

Peter

.

Hello

Could you explain sentence below:

we may have 10% organic or maybe 90% organic so the pH will change.

this is a general question, it doesn't concern this specific analysis. one measure the pH in the aqueus phase and the pH is 1.8. Does he know the final pH of the m.phase when he adds 90% ACN or 10% ACN?


It has nothing to do with the instrument. We have tried at least 5 different Agilents.
I don't really care about retention time shift but for areas changing so the %RSD may be too high (3-10%)

Hello

In first reply I asked you to paste chromatograms and give method parameters. You didn't send it so I'm sorry I can't help you...I don't like guessing.

Best regards

Tomasz Kubowicz

we used a Gemini (pH 1-8) and the problem solved. Βut i really would like to know how could i know the pH of a M.phase since i measure the aqueus phase and then i add 10%, 50% or 90% organic

You can't. You can take a measurement and document it, but by definition it's not a true pH value. I've seen this referred to as an apparent pH in some documents at different companies.

Hello

Just think...
If you measure pH in your buffer it is fine but if you measure pH in mobile phase (Buffer:organic) you will have false result because your pH meter is (probably) calibrated with buffers that contains water not organic solvent.
So even if you measure pH for your mobile phase , result is meaningless.

Regards

Tomasz Kubowicz

Re pH issues, check these links:
http://lcresources.com/tsbible/hs2150.htm
http://lcresources.com/tsbible/hs2160.htm
http://lcresources.com/tsbible/hs2170.htm

thank you very much fot your replies