Hello, I was radioiodinating insulin, which had previously been done 20 or so times in a lab by myself or others. The final step is HPLC purification. It uses a 60-minute time frame, and the retention period is typically between 35 and 45 minutes (varies between runs slightly). Anyway, this week, the separation of peaks remained the same although my retention time decreased from 35 to 15 minutes. It simply all eluted too soon.
I initially believed it to be poor column equilibration, but it was replicated twice more with the same RT (at 15 minutes). Any suggestions as to the potential cause? Is there any way the column has gone bad?