For size exclusion, there are two considerations - sample volume and sample concentration.
As a general rule, load no more than 5 % of the column volume. Theoretically, a 300
x 4.6 mm column, with a column volume of ~ 5 mL, would limit injection volume to
200 μL. In practice, volumes of 10-30 μL are common.
Another important consideration is sample concentration; the higher the concentration
of protein, the higher the viscosity of the sample. This difference in viscosity
can lead to peak shape distortion. The distortion can occur either through exclusion
effects or a solvent front referred to as “viscous fingering.” A good starting concentration
is 1 mg/mL, though optimal concentrations must be determined experimentally.