Effect of sample volume and column length on transcortin determination by sephadex G-25 gel filtration

When Sephadex G-25 columns are used to separate transcortin-bound corticosterone from free corticosterone, there is a non-linear increase of total binding with increasing plasma volume filtered. However, the specific binding (total binding - unspecific binding) shows a linear relationship with the plasma volume filtered. Furthermore, the specific binding decreases with increasing Sephadex column length and absolute binding values are found by extrapolating to a zero length column.