Separation of serum proteins by high-performance gel-permeation column systems

Combined TSK-Gel G4000SW-G3000SW and Zorbax GF450-GF250 columns and a Superose 6 column were evaluated for the analytical gel permeation chromatographic separation of serum proteins. Serum elution profiles showed four distinct peaks, which were attributed to immunoglobulin M (IgM), alpha 2-macroglobulin, IgG and albumin. A reproducible shoulder on the IgG peak could be attributed to IgA. These purified serum proteins and other commercially obtained proteins with relative molecular masses between 1000 kDa and 10 kDa were chromatographed on the combined systems and the separate columns. The frictional coefficient-based hydrodynamic radii of these proteins showed a linear relationship with the inverse error function complement of their partition coefficients. Using this relationship, theoretical summation plots of the data obtained from the separate columns of a system correspond to plots that were calculated by treating the combined systems as one column. The best resolution was obtained with the TSK colums, but all three column systems were suitable for the separation of clinically important immunoglobulins in serum. The retention of positively charged proteins by the TSK columns becomes noticeable after relatively short usage and is a first sign of progressive loss of resolution.