Scaling Up a Separation

When looking to increase the sample loading on a column, there are a number of factors to consider:

1. Always try to improve the separation first
2. Use software to help calculate the loadings
3. Longer columns give better resolution
4. Wider columns have higher sample loading

The diagrams below show the effect of mass on sample resolutions. This illustrates how increased sample loading results in poorer separations.

1mg Sample RfA = 0.40 RfB = 0.20	10mg Sample RfA = 0.50 RfB = 0.35

It is possible to predict sample loading based upon the difference in CV values. The table on the right shows an example of such a prediction when using a 40x150mm silica cartridge.

By carefully choosing the solvent, you can maximise the number of column volumes the sample is eluted in and so maximise the sample loading.