Column Selection and Optimal Flow Rate

The gel-filtration medium is normally packed into a stainless steel column characterized by an i.d. between 4 and 25 mm and a length between 300 and 600 mm. Four important factors in characterizing SEC packings are the average particle size, the particle size distribution, the average pore diameter, and the pore size distribution. The particle size and the particle size distribution have a significant influence on the column efficiency, which is most decisive in SEC, because the biopolymers are eluted isocraticaUy in a small elution window with 

To extend the separation range, SEC columns with two different pore sizes, e.g. 

5-30 nm and 100 nm, are combined. The columns should be coupled in order of decreasing molecular weight exclusion limit 

By coupling more columns or using very long columns, it is possible to improve the resolution and the fractionation window. However, the back-pressure and the analysis time will also increase. A reasonable compromise should therefore be sought 

Mobile phase 0.1 m sodium phosphate buffer/0.1 m NaCI at pH 6.8, X 206 nm. (K. K. Unger, High-Performance Size-Exclusion Chromatography, Enzyme Purification and Related Techniques, ed. by W. B. Jakoby, Academia Press, Inc.). 

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