Real gel chromatography

In the preceding section, we briefly discussed the separation mechanisms that are exclusively and directly connected with the dimensions of the separated species. Let us designate these mechanisms primary. In the real gel chromatographic systems, however, several other processes are operative, affecting both the retention volumes and the widths of chromatographic zones. These secondary processes can be classified into  

The secondary processes often exhibit dynamic character, i.e., their extent may vary in the course of elution (e.g., as a consequence of progressive dilution of the separated substances within the column). 

In some cases, several secondary processes may manifest themselves simultaneously. A typical combined secondary process is the so-called concentration effect in gel chromatography, i.e., the often observed rise of the retention volumes of macromolecular substances with the increased sample concentration. The concentration effect is caused by a decrease in the macromolecular coils dimensions in solution with growing concentration, further by viscosity and osmotic effects as well as by secondary exclusion — and in the case of soft gels also by altering the pore geometry due to deswelling of gel particles in the zone of the sample. 

The nature of particular secondary processes and their influence on the results of gel chromatographic separation are discussed in detail, e.g., in Refs. 6,10,11. 

The retention volume in real gel chromatography can be expressed thus 

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