Size-exclusion chromatography molecular weight separation range

The range of the (molecular) size of the analytes usually exceeds that which can be determined by classical laboratory analytical methods such as size exclusion chromatography, etc. [351]. Reports on investigated substances are widespread and cover applications such as the separation and characterization of proteins [450] and enzymes [240, 241], of viruses [132], the separation of human and animal cells [50, 51], the isolation of plasmid DNA [367], and the molecular weight and particle size distribution of polymers [216,217]. The approach is relatively new in biotechnology therefore, practical experiences are not yet abundant. Langwost et al. [229] have provided a comprehensive survey of various applications in bio-monitoring. 

Differentiation and separation only occurs over a certain range of molecular sizes, typically between molecular weights of 2 kDa and 200 kDa, although this can be increased up to 1,000 kDa by the use of more specialised gels. This size range is dependent on the sizes of the pores and pore size distribution in the gel matrix. Retention volumes Vr are often used in size exclusion chromatography instead of retention times tR. The total volume V, of the separation column is the sum of the volume of the gel particles Vg, the volume of the solvent inside the pores, also called the intrinsical volume V and the volume of the free solvent outside the pores, the inter particle volume Vq ... 

Despite the fact that the plate count of a mixed bank is inferior to the performance of a bank of columns of the same type, the gain in selectivity obtainable with a mixed column bank may offset this disadvantage. Many examples are found in size-exclusion chromatography, where often only a coml ation of columns with different pore sizes covers the molecular-weight range that needs to be separated. One of the rare examples of such a separation problem in retention chromatography is shown in Figure 14.1, where the selectivity of a CN column is combined with the selectivity of a Cg column. 

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