HPLC separation difficulties

[Paxton00]

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?

[Multidimensional]

The two most likely reasons for the retention time to be too early are:
(1) Your mobile phase composition has changed (IOW: It is not what you think it is due to programming, hardware defect, evaporation etc) OR (2) your flow rate has changed (always measure it using a timer and graduated cylinder. Do not rely on the instrument's displayed values).

[Multidimensional]

Columns can and do go "bad", but this is one of the reasons why we dedicate a specific standard (and method) for each column so we can always go back to verify the column's performance (Btw: NEVER use one of the manufacture's "QC" check standards as your column performance std (use a real sample with strong K prime which is related to your actual analysis). These Qc check samples are often worthless for evaluating a column's true performance. They are chosen to show high plate counts and quick QC results during column packing.

[Vlad Orlovsky]

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?

You might have a partial collapse of your stationary phase (assuming it is C18)