Size separation, steric exclusion

Theory. The most widely accepted mechanism of size separation is based on steric exclusion (1). In terms of thermodynamic properties, the distribution coefficient consists of enthalpic and entropic contributions ... 

Separation is dependent on three different mechanims Donnan exclusion, steric exclusion, and adsorption/partitioning. Donnan exclusion causes strong acids to elute in the void volume of the column. Weak acids that are partially ionized in the eluent are not subject to the Donnan exclusion and can penetrate into the pores of the packing. Separation is achieved through differences in acid strength, size, and hydrophobicity. The major advantage of ion exclusion is the ability to handle samples that contain both strong and weak acids. 

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]. 

Size-exclusion chromatography, also known as gel-filtration, gel-permeation, steric-exclusion, molecular-exclusion, or molecular-sieve chromatography, separates solutes on the basis of their molecular sizes (Figure 6-5 see Fig, 6-3). Molecular shape and hydration are also factors in the process. 

Di- and tricarboxylic acids such as oxalic and citric acid elute between the excluded and the total permeated volume. Apart from Donnan exclusion, the predominating separation mechanism is, in this case, mainly steric exclusion. The retention is determined by the size of the sample molecule. Since the pore volume of the resin is established by its degree of crosslinking, the resolution can only be improved by applying another or by coupling with another separator column, respectively. 

The above simple model of a steric exclusion mechanism was considered by several authors attempting to describe quantitatively the gel chromatographic separation process. Distribution coefficients were expressed on the basis of the model considerations of the dimensions of both the separated molecules and the pores of gel, as well as of the stochastic model approaches (for reviews see e.g.. Refs. 1, 3-6), and also of the thermodynamic reasoning on the changes of conformational entropy of macromolecules due to their transfer from the interstitial volume into the pores in the course of separation [7]. However, besides the steric exclusion from the pores, at least two other size-based mechanisms are operative in the ideal gel chromatography ... 

Because in gel chromatography steric exclusion is the dominating mechanism, the most common application of this type is the purification of a mixture of substances differing from one another by particle size. We think of purification in a broad sense depending on the degree which can be reached by the separation process itself or which is required. Various modes of GPC application will be discussed from this point of view. 

Size exclusion, steric exclusion chromatography, SEC components in a sample are separated according to their molecular size by selective retention on a stationary phase having pores of varying sizes see gel filtration. 

To illustrate the results obtainable in steric exclusion chromatography. Figure 21.24 depicts an HPLC separation of polystyrene standards in a column packed with cross-linked polystyrene particles (average pore size 260 A) of 10-/im d, using tetra-hydrofuran as a mobile phase. Unlike mobile phases in other forms of chromatog-... 

The size ratio of the fractionated macromolecules or particles to the channel thickness must be taken into account. A decrease in channel thickness can lead to an important contribution of steric-exclusion to the mechanism of separation whenever the distance of the center of gravity of the concentration distribution of the retained species from the accumulation wall, I, becomes commensurable with the size of the retained species. Whereas the elution order in normal (polarization mode) TFFF is from the small to the large species, it is inverted in purely steric-exclusion mode. Consequently, the fractionation deteriorates in the vicinity of the inversion point The dependence of the retention ratio R on the particle radius r can be... 

Although the chromatographic principles apply equally to polymers and low molar-mass analytes, liquid chromatography of polymers has its peculiarities, which need to be understood. The most obvious difference is the much larger size of polymers as compared to low molar-mass molecules. While HPLC-separations of low molar-mass analytes usually are determined by differences in the enthalpic interactions between the analytes and the stationary phase, the majority of polymer separations are entropically driven, based on steric exclusion of... 

One final criteria for the choice of SEC column is that thrae should be no interaction between the solute and the surface of the stationary phase so that there is nothing eluting after the smallest molecule, that is separation occurs exclusively by size-exclusion. The problem of excessive interaction of the protein with the stationary phase, especially common with silica based materials, is solved by bonding the surface with a modifier. However, bonding on silica is often incomplete for steric reasons, so these materials... 

The two techniques differ in that HDC employs a nonporous stationary phase. Separation is affected as a result of particles of different size sampling different velocities in the interstitial spaces. Size exclusion chromatography is accomplished by superimposing a steric selection mechanism which results from the use of a porous bed. The pore sizes may vary over a wide range and the separation occurs as a result of essentially the same processes present in the gel permeation chromatography of macromolecules. 

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