Chromatography Forum

In our laboratory, we develop and validate many analytical methods to assay various proteins by reversed phase HPLC. Often times, we have disagreement on how to perform the Accuracy part of the validation when the method requires purity determination by Area %. Do you have any suggestions/recommendations on how one could measure this? Is it really possible to check if everything eluted from the column? Anything you can help us will be greatly appreciated.

Some proteins are known to stick to the column and to not elute completely in the first run. This can commonly be seen, when a second blank run is performed (preferentially without injection).

If you are concerned about this, the blank run will reveal if you have a problem. If nothing shows up in the blank run, then you do not have a problem.

If you discover that a substantial amount of your protein(s) elutes in this second run, then more headscratching needs to be done.