Resolution of proteins in linear gradient elution ion-exchange and hydrophobic interaction chromatography

The following equation was derived for the resolution, Rs, of proteins in linear gradient elution ion-exchange and hydrophobic interaction chromatography: Rs alpha [(column length.molecular diffusivity)/(slope of the gradient.column gel volume.linear mobile phase velocity.particle diameter2)]1/2. Experimental results obtained under a wide range of experimental conditions (column length, 1-30 cm; particle diameter, 37-92 micron; linear mobile phase velocity, 0.2-3.5 cm/min; temperature, 15-35 degrees C) have shown that this relationship is valid except for very short columns with a shallow slope of the gradient and for low flow-rates. The relationship is very useful for scaling-up the linear gradient elution of proteins from data obtained with small columns.