Size-exclusion chromatography stationary phase interactions

The stationary phase in gel permeation (also called size exclusion) chromatography contains cavities of a defined size distribution, called pores. Analytes larger than the pores are excluded from the pores and pass through the column more rapidly than smaller analytes. There may be secondary effects due to hydrophobic adsorption, ionic interaction, or other interactions between the stationary phase and analyte. Gel permeation and non-ideal interactions in gel permeation are described more fully in Chapter 6. 

In size exclusion chromatography K < or = 1, excepting cases where interactions develop between the solute and stationary phase since this creates a partition phenomenon which superimposes itself upon the diffusion in the pores. 

In the case of adsorption chromatography in contrast to exclusion chromatography, the macromolecules interact with the stationary phase, which makes the method sensitive to both the size of the macromolecule and its local structure, since the interaction of monomer units with the surface is restricted to the distance of several Angstroms. It is one of the ways of solving the above-mentioned problems. This review presents the principal ideas underlying the method of chromatographic... 

Figure 13.2. Illustration of molecular separation according to their size in a size-exclusion chromatography (SEC) column. The underlying principle of SEC is that particles of different size will elute through a stationary phase at different rates Larger molecules will take less time (or elution volume) to reach outlet of the column as compared to the smaller ones.The prerequisite of a direct correlation between elution time and molecular size is an absence of interactions between the stationary phase and an analyte. Otherwise, nonexclusion effects such as electrostatic repulsion or sorption must be considered.

Such precise control of porous properties is expected to be very useful in the design of specialized CEC columns for separation in modes other than reversed-phase. For example, size exclusion chromatography (SEC) is an isocratic separation method that relies on differences in the hydrodynamic volumes of the analytes. Because all solute-stationary phase interactions must be avoided in SEC, solvents such as pure tetrahydrofuran are often used as the mobile phase for the analysis of synthetic polymers, since they dissolve a wide range of structures and minimize interactions with the chromatographic medium. Despite the reported use of entirely non-aqueous eluents in both electrophoresis and CEC [65], no appreciable flow through the methacrylate-based monoliths was observed using pure tetrahydrofuran as the mobile phase. However, a mixture of 2% water and tetrahydrofuran was found to substan-... 

Konas M, Moy TM, Rogers ME, Shultz AR Ward TC, McGrath JE (1995) Molecular weight characterization of soluble high performance polyimides. 1. polymer-solvent-stationary phase interactions in size exclusion chromatography, J. Polym Sci, Part A Polym Phys 33 1429... 

The effect of limited penetration of the pores by the largest molecules may also be applied beneficially for the separation of very large molecules. Depending on the size of the molecules (in solution), they will be more ore less excluded from the pores, and hence the retention times will be affected. This effect is used in size exclusion chromatography (SEC) or gel permeation chromatography (GPC). In this technique, any interactions between the solute molecules and the stationary phase are purposefully avoided. The solute molecules remain exclusively in the mobile phase, but the accessible mobile phase volume, and hence the retention volume, may vary between the total volume of the mobile phase and the so-called exclusion volume, which is the total volume of mobile phase outside the pores. The latter elution volume applies to very large solute molecules (excluded solutes),... 

Size exclusion chromatography (SEC, also known as gel permeation chromatography) is a method of separating compounds of different molecular masses and sizes. Because steric interactions between analytes and the stationary phase are relatively weak, unstable forms of metals can be separated from more stable complexes and from adducts stabilized by ionic interactions. Unfortunately, the process of sorption and ionic interactions between the investigated substances and the stationary phase can decrease metal recovery by as much as 50 % these interactions are also responsible for the instability of retention times [146]. The separation can be performed both in the aqueous environment and in the presence of organic solvents. Because the technique is not selective, it is utilized primarily as the first stage of multidimensional chromatography [147]. 

Specific and essentially stand-alone mode of liquid chromatography is associated with the absence or suppression of any analyte interactions with the stationary phase, which is called size-exclusion chromatography (SEC). In SEC the eluent is selected in such a manner that it will suppress any possible analyte interactions with the surface, and the separation of the analyte molecules in this mode is primarily based on their physical dimensions (size). The larger the analyte molecules, the lower the possibility for them to penetrate into the porous space of the column packing material, and consequently the faster they will move through the column. The schematic of this classification is shown in Figure 1-1. 

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