Steric exclusion separation

Unlike the pellicular packings used for ion exchange, the packings used in ion exclusion are derived from totally sulphonated polymeric materials. Separation is dependent upon three different mechanisms Donnan exclusion, steric exclusion and adsorption/partitioning. 

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 ... 

Therefore, for a separation based solely on steric exclusion, Kp is unity. 

Criteria that may be used in judging whether or not the separation is based strictly on steric exclusion include measuring elution as a function of sample concentration, temperature, and flow rate. Also, in the absence of secondary separation mechanisms, the universal calibration should hold. 

Compared to hydrocarbonaceous silica RPC sorbents, not as much commitment has been made to the development of bonded, polar-phase sorbents suitable for the high-performance chromatographic separation of peptides. Due to polar, notably hydrogen bonding, interactions between the peptide and the hydrophilic surface of the sorbent useful selectivity effects can, however, be achieved. In fact, at least two types of separation mechanisms can be identified with bonded polar-phase sorbents. In the first mode, the peptides do not interact per se with the bonded polar-phase sorbent but, rather, are separated on the basis of their ability to permeate into the pores and elute in order of their hydrodynamic volume. In this mode, peptides are separated by steric exclusion effects, with the retention (in terms of elution volume, Ve) of a partial retained peptide, Pb described by the following relationships ... 

In the wide field of possible applications for MTS, the use of their properties of adsorption and steric selectivity is still to be explored. However, such applications require well-defined particles, especially spherical particles in the micrometric range. The synthesis of MSU-X silica that exhibits these shapes allowed us to test their properties in adsorption HPLC. Non polar solvent such as hexane are suitable to allow a significant separation. Further analyses and testing for size exclusion separation processes are under progress. 

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. 

Pozo, C. 1993. Application of steric exclusion chromatography for the separation of degradation products of the solvent used for the reprocessing of the nuclear fuels. Rapport CEA-R-5647. 

In steric exclusion (gel permeation) separations, the retention of partially retained solutes is described by... 

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. 

In the gel network there are zones, where the polymers interact, and large segments, where the macromolecules are randomly extended. The lattice is responsible for the elasticity and the textural strength of the product. In multicomponent gels all constituents may form separate or coupled networks, or else one component, not involved in network formation, may indirectly affect the gelling by steric exclusion of the active molecules. Such exclusion increases the concentration of the active component in the volume of the solution where the gel is formed. In gels made from the mince of squid meat at 1.5% NaCl, the added carrageenan and egg whites form separate networks that support the structure made of squid proteins, while added... 

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. 

Ultraflltration involves the pressure-activated separation of chemical species which have different permeability through a membrane. Solute retention is achieved on the basis of steric exclusion, that is, a sieving-type of mechanism and solvent passes through by pore flow. As an initially homogeneous solution is pressurized over a selective menijrane, solvent permeates through while rejected solute accumulates in the vicinity of the membrane. The net result is a layer of solution adjacent to the membrane surface of substantially greater solute concentration than that of the bulk solution. This phenomenon of concentration polarization always operates to reduce the solvent permeation rate which may become pressure independent in some cases. 

A sequence (using P-388 guided bioassay) of steric exclusion and partition-type chromatographic separations of the active butanolic fraction by reversed-phase semipreparative HPLC led to the isolation of cephalo-statin 12 (22), CaHyeNzOjz, [a]D 157° (CH3OH), (M + H)+ m/z 945.5444, and cephalostatin 13 (23), [a]D 108° (CH3OH), (M + H)+... 

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 ... 

It is assumed that the steric exclusion from the gel pores is the decisive separation mechanism in the ideal gel chromatography, and both the latter mechanisms contribute positively (but within a limited extent only) to the overall separation effect. 

The procedures where the basic separation mechanism (steric exclusion) remains unchanged (recycling, continuous GPC, differential, vacancy and flat bed techniques). 

An extra group of special working procedures in gel chromatography are those separations where the steric exclusion mechanism is intentionally combined with an auxiliary additional separation mechanism to increase the selectivity. Such combinations can be realized either in one step, i.e., in one system gel-eluent, or in several steps, e.g., by the subsequent elution of the sample with two different mobile phases, or from two different column-filling materials. 

The one-step procedures can be utilized for the separations of the sample components with similar dimensions but different adsorption characteristics in the given system gel-eluent. The non-adsorbed part of the sample is separated according to the steric exclusion mechanism, while the elution of the adsorbed part of the sample is retarded [26]. Another combined procedure utilizes the thermodynamic partition of the sample as the auxiliary separation mechanism the gel is either swollen by a solvent immiscible with eluent [27] or a mixed eluent is used, one component of which is preferentially sorbed within the gel matrix [28]. In both cases, the composition of the stagnant phase within the gel differs significantly from the composition of the mobile phase and thermodynamic partition takes place. The extent of the partition is controlled by the porous structure of the gel so that steric exclusion remains the principal separation mechanism. 

Large potentiality of the combination of different separation mechanisms within the same gel bed offer bidimensional columns [30], in which the bed is formed by a layer (as in TL GPC) in a sandwich arrangement. In one direction, the sample can be eluted in the steric exclusion mode and, using another eluent in the perpendicular direction, the elution is completed (as in the mode of sorption liquid chromatogra-phy). 

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. 

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