Sample Loading in Column Chromatography

[NChaffin]

What's the appropriate amount of sample to load on in column chromatography? Is there a rule of thumb?

I have a column that is 4.5 cm x 27 cm

silica/THF

Thanks !

[tom jupille]

Is there a rule of thumb?

For "analytical-scale" separations (non-overloaded), the rule of thumb for a long time was that you could to up to about 100 micrograms on a 250 x 4.6 mm column. That would imply about 10 milligrams on your column (10x the diameter implies 100x the volume).

The catch is that prep separations are almost always run under "overload" conditions. How much you can overload on depends on the selectivity between your product and the closest interfering impurity.

I usually suggest a loading study on an analytical scale column (e.g., 250 x 4.6 mm), going up in geometric progression (e.g., 30, 100, 300, 1,000, . . .) to establish the maximum load for *your* sample. Then scale that up to the prep column in proportion to the column volume (in this case, 100x).