Theories of retention in SEC

Since the inception of size exclusion chromatography, many models have been considered in an attempt to describe the retention of macromolecules. Most of these models treated the pores as having various well-defined shapes, and examined the effects of purely physical exclusion mechanisms. In practice, these models gave good agreement with observed retention behaviour. 

More recently the tendency has been to examine the retention in thermodynamic terms. Under normal chromatograph conditions, solute molecules distribute themselves between the mobile and stationary phases and are in thermodynamic equilibrium. This has been demonstrated experimentally in a number of ways. It has been shown that retention volume is independent of flow rate [19]. A further confirmation was provided by static mixing experiments. These involved measuring the equilibrium polymer concentration in a mixture of a polymer solution and a porous packing material. The results supported the equilibrium theory [20]. 

Several other theories have been put forward to explain retention in SEC. 

One of these proposed a non-equilibrium process in which the separation was controlled by differing rates of diffusion for different molecular masses [23]. Other workers have proposed a separation by flow mechanism [24, 25] in which the larger molecules are excluded from the surface of the gel particles and remain in the centre of the solvent channels and are thus eluted first. The original theory did not invoke a porous structure for the gel, but this was modified later. The mechanism bears resemblance to that proposed for hydrodynamic chromatography (see Chapter 10). A further model suggested that the pore size distribution of the gel was directly responsible for its ability to separate molecules by size, and that there is a one-to-one correspondence with size of pores and size of molecules [26]. All these theories have been critically reviewed in the book by Yau et al [6]. 

In conclusion, the results suggest that the size exclusion process is an equilibrium, entropy-controlled process and that the separation depends on the differential degree of permeation of molecules rather than the differential rate. This is equivalent to stating that the retention mechanism is a simple physical exclusion mechanism, and it is normally sufficient to consider the mechanism in these terms in order to understand what is happening in a size exclusion column. 

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