Select an appropriate filtration medium depending on the nature of your sample. Choose media with a fractionation range that spans the molecular weights of components present in your sample. If many media have similar fractionation ranges, select the one with the steepest selectivity curve for achieving the best resolution.
Use multiple columns coupled in a series to increase the effective bed height. This ensures more efficient separation, provided the columns all contain the same separation medium.
Determine the exact sample volume to be used. Generally, this should not be greater than 2 percent of the total column volume. Begin with a volume of 0.5 percent and carry out test runs at increasing sample volumes to find the value that yields best resolution. Avoid using sample volumes less than 0.5 ml because low volumes impair resolution.
Concentrate samples before applying them to the gel filtration column. This improves the degree of separation; however, ensure you use concentrations less than 70 mg/ml with protein samples to prevent interference due to the effects of viscosity.
Degas buffers you need to use in the separation by filtering them under a vacuum. If air bubbles enter the filtration column, they affect column performance.
Use the lowest possible flow rate to avoid peak broadening. Broad peaks yield results that are not as accurate as narrow ones.