Vydac® Case Study – Scaling Gradient Volume To Column Volume

In order to maintain the same elution pattern when scaling a separation between columns of the same phase but different inner diameters, gradient volume must be scaled in proportion to column volume.

In separations on a reversed-phase column, smaller molecules (like heme) tend to partition between the stationary phase and the mobile phase in a ratio that changes gradually with mobile phase composition. Larger biopolymers (like the α and β polypeptides) tend to be retained in an all-or-nothing manner, adsorbing firmly to the stationary phase below a critical mobile phase solvent concentration and releasing rapidly once the critical concentration is attained.

When the gradient develops in too small a volume, there is not sufficient mobile-phase flow to sweep the partitioning heme molecules from the column before the critical concentration is reached for releasing the β polypeptide. Hence the elution order changes.

In more complex reversed-phase separations of peptides of various sizes, changes in elution patterns may be more subtle but nonetheless problematic.