Bioequivalence and Bioavailability Forum

Dear Geokad!

❝ Does anyone test or evaluate the impact of different injection volumes?

Yes.
The smaller the volume, the less dispersion of the solution in the column head - and less peak broadening. If you have to inject larger volumes for any reason, try this trick: reconstitute the residue not in mobile phase, but in a mixture of lower elution power (more buffer, less organic modifier). The analyte will concentrate on the column head during injection and will be flushed with mobile phase resulting in sharper/higher peaks. You have to find a compromise between this effect and limited solubility of the analyte in solutes with very reduced percentages of organic modifier. In most cases 10%-20% less organic will be sufficient.

❝ If yes, is it done during method validation?

No - during method development. Once you finished your experiments, your method should be laid down in a working instruction or SOP. Then prepare a validation protocol - and start working.