Chromatography Forum

We have had a persistent problem with this assay following the BP procedure which is a gradient method up to 30% MeOH.
The Allopurinol peak, which greatly overloads the column varies from injection to injection. The peak can then swamp the small related substance peaks.
I thought I had solved the problem by introducing a wash step going up to 70% MeOH. This did improve things but still there is an occasional broad allopurinol peak.

What strength solvent is your sample dissolved in. Plus what concentration / injection volume are you putting on the column? Could be an overload problem by the sounds of it.... however the fact that the problem is intermittant suggests not. Are you allwoing enough time for re-equilibration to your starting conditions?

PS. Any chance of posting an actual chromatogram?

The sample is dissolved in 10ml 0.1M NaOH then made up to 200ml with the starting mobile phase. 20ul containing 10ug of allopurinol is injected.

We have just tried a new column and that appears to have solved the problem for now. It would still be satisfying to find out why the problem occurs.

I can't easily post chromatograms as our system generates WMF - I realise it is possible to convert them to GIF files but it doesn't seem to be very straightforward