Peaks in established HPLC assay now not resolving

[Merrin]

Hi all,

So I have an established assay for separation of paracetamol, paracetamol sulphate (PS) and paracetamol glucuronide (PG) using UV-HPLC (IS=3-acetamidophenol). Mobile phase is 0.1M KH2PO4: 1.5% isopropanol, 0.1% THF - pH 3.7. Column is a 60mm 3um rocket column, flow rate 2mL/min, oven 35 C.

Until resently para had an RT of ~3.1 min and PS was 2.7 min however now they are coleuting at 2.9. I have no idea what the problem is. I have remade the standards, altered the flow rate, made up several batches of MP etc so it's none of the obvious (to me anyway). The only thing I can think of is that the pH meter might be off - is this likely to make that big a difference?

Any suggestions would be fantastic!

Thanks.

Merrin

[tom jupille]

Since 3.7 is outside the buffering range of phosphate, yes, pH could well be a problem. The other (and more likely) possibility is that your column has died and needs to be replaced.

[qfiviera]

I agree with Tom. It will probably be the column the problem. Try to change for a new one, and if it´s not the problem, check the pH of your MP after running.
Another but really really really strange thing that could be happening os that you have a void volume before the cell that is getting them together. But the bigger chance is the column. Could you reproduce the conditions from the certificate and check the initial parameters to behave well?

[Merrin]

I have put an older column on now and am going to see how things go. Thanks for your suggestions.

M.

[Consumer Products Guy]

One should ALWAYS have a brand-new column as backup, sitting in the drawer, for any regulated-product assays. Substituting that in is a good first check to troubleshoot.

No $$$ for back-up new column?? Try telling that to the FDA or EPA. Then get out of the pharmaceutical business before FDA moves that decision along for you.

[Merrin]

In a perfect world we would always have a backup but paritcularly in academia this is not always possible - we are lucky to have enough money for one let alone two. Not everyone doing chromatogrpahy is with a pharma company. But thanks for the lecture.

Tom and qfviera - it was the column - resolution is fine with the column I used previously however the run time is significantly longer without the rocket column. Thanks for your help and advice though.