I am a new analyst in a stability group in a large biotech company. I just started working with a new gradient method for a peptide map that has a 2 min. isocratic hold before the gradient starts. Nothing elutes during the isocratic hold and I was wondering what the purpose of it was. Thank you for any insights.

Dear Anon,

Most likely this is to help separate out some of the early peaks even though they don't elute during this time. It could also be to help with a solvent concentration effect, where the analytes are focused on the head of the column when the sample is injected in a much weaker solvent the the initial LC solvent. Without more info and particulars it is harder to give more of an answer.

Regards,
Mark

The isocratic hold is most likely to compensate for the difference in dwell volume of the LC units.

If the method is developed using a higher dwell volume unit and is performed using a lower one, the isocratic hold is necessary in order to have reproducible results.

Just a couple of quick thoughts. Without any method details it is difficult to provide much help. Assuming a 4.6X150mm column, 5um, and a 1ml/min flow rate I would expect t0 to be about 1.5 min thus 2 min doesn't seem sufficient to perform an isocratic seperation of early eluting impurities.

I would check the forced degradation study section of the method validation report, or better yet the method development report if your company produces these.

I suspect the isocratic hold was used to compensate for different dwell volumes in differect systems. There could be an earling eluting pair of peaks and their resolution couldn't be maintained when run on systems with different dwell volumes. In this case the pair could have been pushed into the isocratic hold and their resolution would be more robust to dwell volume changes.

Similarly, the isocratic hold could be used to load the sample at low organic concentration and provide you enough time to switch a high dwell volume autosampler to bypass and thus reduce the dwell volume before the gradient starts.

Another possibility is that there is no reason, the person who developed this method just followed what the previous person did. I have seen lots of methods like that, with artifacts that probably made sense in their original context but make no sense in their current context. Good luck.

My guess is that it is there to allow the system to stabilise at the isocratic concentration before the gradient starts ... a conditioning phase.
Check the baseline stability of the gradient without actually making an injection and see if time period is required.
good luck

I am a fresh person in the pharma area of research and would like to have good messages and suggestions on the HPLC techniques.