UPLC

[gooshi81]

Does anybody know what kind of method revalidation do i have to do when converting HPLC method to UPLC by downscaling (changing flow and column dimention only) ?

[Uwe Neue]

Your UPLC system comes with software that helps you in the down scaling of all column parameters. In principle, you change injection volume, gradient volume, flow rate first by the ratios of the old column volume to the new column volume. Then you increase the flow rate by the ratio of old particle size to new particle size. This applies 100% when the column length changes in proportion to the particle size, but you can get away with the same first guess if the ratio of column length to particle size changes.

[danko]

Gooshi81,

Regarding re–validation, I would focus my efforts especially on the precision, because of the injection volume reduction which I assume you are forced to implement.
Another parameter you might need to consider is precision of the retention time - if your analytical procedure prescribes a certain time window for your analyte/s to elute in.
Finally – especially if the analysis is based on gradient elution – you should consider the most important parameter in chromatography: Resolution.
I’m sure there will be several more ideas if you wait a couple of days

Best Regards

[Uwe Neue]

Sorry, another post that I read too quickly. I gave you a response on the downscaling, not on method revalidation.

Some of the revalidation is associated with the change in the instrument, and is similar to what you would do when you take a method from one instrument to another: linearity, LOD, LOQ, etc...

I assume that with the change in column dimensions comes a change in the particle size. I pointed out the rules for this procedure above. If you are sticking with the same column chemistry, the revalidation is the same as you would do with a classical method changing the particle size.

[gooshi81]

thank you all!