Gel Matrices for Size Exclusion Chromatography

The separation temperature also affects resolution, in that diffusion coefficients increase with temperature, so that partitioning equilibria are faster at higher T. This equilibration time is the factor that usually limits resolution. 

The length of the column is important, because the number of theoretical plates (.N) increases with the square root of column length however, practical limitations are imposed on column length. Components eluted from the column are diluted with respect to their initial concentrations, and reasonable separations yield dilution factors of 2, for standard column lengths (20 cm). 

Finally, the viscosity of the samples may be important if macromolecule concentrations are high enough to contribute to band broadening as a result of unstable flow. 

The trade names of the stationary phase media represent particular polymers formed under well-controlled conditions. Sephadex consists of dextran, cross-linked with epichlorohydrin in alkaline solution. Sephacryl is an allyl dextran, cross-linked with iV.iV -methylenebislacrylamide). Sepharose is a beaded form of agarose gel, where aggregates of the polysaccharide chains are formed. 

Bio-Gel P is a beaded polyacrylamide gel, and has separation properties similar to Sephadex . Bio-Gel A is a beaded agarose gel, like Sepharose it has the largest pore sizes known, and is even capable of separating small virus particles. 

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