Stationary phase size exclusion chromatography

Two classes of micron-sized stationary phases have been encountered in this section silica particles and cross-linked polymer resin beads. Both materials are porous, with pore sizes ranging from approximately 50 to 4000 A for silica particles and from 50 to 1,000,000 A for divinylbenzene cross-linked polystyrene resins. In size-exclusion chromatography, also called molecular-exclusion or gel-permeation chromatography, separation is based on the solute s ability to enter into the pores of the column packing. Smaller solutes spend proportionally more time within the pores and, consequently, take longer to elute from the column. 

The total stationary-phase volume required to process a given feed stream is proportional to the inlet concentration and volume of the feed. For example, for a typical inlet concentration of protein of 10 g/L, in a 100 L volume of feed, a column volume of at least 100 L is needed for size-exclusion chromatography. In comparison, an ion-exchange column having an adsorption capacity of 50 g/L would only require 20 L of column volume for the same feed. 

Figure 4-2. Size-exclusion chromatography. A A mixture of large molecules (diamonds) and small molecules (circles) are applied to the top of a gel filtration column. B Upon entering the column, the small molecules enter pores in the stationary phase matrix from which the large molecules are excluded. C As the mobile phase flows down the column, the large, excluded molecules flow with it while the small molecules, which are temporarily sheltered from the flow when inside the pores, lag farther and farther behind.

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. 

ISEC is a size-exclusion chromatography technique, in which the stationary phase is the CFP to be to characterized [16-18] and the eluates are geometrically well-defined steric probes. From the determined retention volumes in a given solvent and on the basis of suitable morphological models, ISEC analysis provides the... 

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. 

Many of the possible column combinations that are useful in 2DLC are listed in Chapter 5. Besides the actual types of column stationary phases, for example, anion-exchange chromatography (AEC), size exclusion chromatography (SEC), and RPLC, many other column variables must be determined for the successful operation of a 2DLC instrument. The attributes that comprise the basic 2DLC experiment are listed in Table 6.1. 

The difference in movement rates of various compounds through a column is attributed to differential migration in HPLC. This can be related to the equilibrium distribution of different compounds such as X, Y, and Z between the stationary phase and the flowing solvent(s), or mobile phase. The speed with which each compound moves through the column (ux) is determined by the number of molecules of that compound in the moving phase, at any moment, since sample molecules do not move through the column while they are in the stationary phase. The molecules of the solvent or mobile phase move at the fastest possible rate except in size exclusion chromatography, where molecular... 

Size exclusion chromatography. The separation occurs because the stationary phase particles are porous and the small molecules enter the pores and are slowed from passing through the column, while the large molecules pass through more quickly since they do not enter the pores. 

In actual practice, the inert gels of dextran (I)-a polyglucose or other types of polymers, for instance agarose and polyacrylamides, wherein the macromolecules invariably are cross-linked to afford a reasonably porous 3D-structure, served as the stationary phases in size-exclusion chromatography. 

Chromatographic approaches have been also used to separate nanoparticles from samples coupled to different detectors, such as ICP-MS, MS, DLS. The best known technique for size separation is size exclusion chromatography (SEC). A size exclusion column is packed with porous beads, as the stationary phase, which retain particles, depending on their size and shape. This method has been applied to the size characterization of quantum dots, single-walled carbon nanotubes, and polystyrene nanoparticles [168, 169]. Another approach is hydro-dynamic chromatography (HDC), which separates particles based on their hydro-dynamic radius. HDC has been connected to the most common UV-Vis detector for the size characterization of nanoparticles, colloidal suspensions, and biomolecules [170-172]. 

The present experimental approach is based on the chromatographic advantages provided by the diol or glycerol derivatives of porous silica stationary phases available for use in HPLC. These have recently become available for estimating the molecular size of polyelectrolytes using aqueous size exclusion chromatography. The conditions for reproducible polyelectrolyte size measurements, and their possible purturbations have been summarized by Barth (8). 

Note 3 Macroporous polymers are used, for example, as precursors for ion-exchange polymers, as adsorbents, as supports for catalysts or reagents, and as stationary phases in size-exclusion chromatography columns. 

Several variants of separation methods based on dialysis, ultrafiltration, and size exclusion chromatography have been developed that work under equilibrium conditions. Size exclusion chromatography especially has become the method of choice for binding measurements. The Hummel-Dreyer method, the vacancy peak method, and frontal analysis are variants that also apply to capillary electrophoresis. In comparison to chromatographic methods, capillary electrophoresis is faster, needs only minimal amounts of substances, and contains no stationary phase that may absorb parts of the equilibrium mixture or must be pre-equilibrated. 

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