Size exclusion

In size exclusion chromatography the solid support is a porous polymer with a controlled pore size, and the solute molecules are separated according to their size in solution. The larger molecules are excluded most and thus they have the shortest retention times. The size exclusion may be performed in aqueous systems (gel filtration), where water soluble macromolecules can be separated, or in non-aqueous systems (gel permeation). By proper calibration the method can also be used for determination of molecular weight or molecular weight distribution. 

In the GPC experiment, polymer molecules are separated by size or their hydro-dynamic volume because of their ability to penetrate part of the pores volume of the gel particles, i.e., the stationary phase. As the sample moves along the column with the mobile phase, the largest molecules are almost entirely excluded from the pores of the stationary phase, whereas the smallest find almost all the stationary phase accessible. The smaller the molecule, the more of the stationary phase volume is accessible to it and the longer it stays in that phase. Consequently, small molecules are eluted from the column later (Fig. 18.1). 

The separation of a solute of a given size in solution is determined by a distribution coefficient, Ksec, which governs the fraction of internal pore volume of the gel, Vu that is accessible to this solute. The value of the retention volume, Vr, for this solute is given by 

Vr on solute is shown in Fig. 18.2 for solutes with a range of sizes, e.g., for calibration standards. 

Benoit and co-workers [18] proposed that the hydrodynamic volume, Vr which is proportional to the product of [17] and M, where [17] is the intrinsic viscosity of the polymer in the SEC eluent, may be used as the universal calibration parameter (Fig. 18.3). For linear polymers, interpretation in terms of molecular weight is straightforward. If the Mark-Houwink-Sakurada constants K and a are known, log [t7]M can be written log M1+ + log K, and VT can be directly related to M. The size-average molecular weight, Mz, is defined by this process  

For branched polymers, molecular size is crucial because the material eluting at any value of Vr consists of a mixture of species having different molecular weights and degrees of branching but constant hydrodynamic volume. 

Although the oldest type of columns, these are presently the most popular nonpartition columns because they can separate large biological molecules such as proteins and nucleic acids. By means of controlled pore sizes, they separate compounds by their molecular size and shape. The resolution achieved by a size resolution column is not nearly as great as that shown by ion exchange or by partition. You will need a 100% difference in molecular weights to achieve a clean separation. Partition can separate on the basis of a proton up or down out of 13 protons on a compound. 

Although we often describe these as molecular weight columns, the separating parameter actually is their Stokes radius, the major axis of the molecule in its current configuration. The shape and folding of a protein molecule under differing solvent conditions affect their maximum radius and, therefore, their retention times. Only when extreme conditions are used to force all molecules into the same shape are we able to obtain a direct molecular weight relationship. 

Both polymeric and silica-based columns are in common use.The polymeric columns are heavily used in the analysis of synthetic polymers and plastics where organic solvents are required. Silica-based columns with hydrophilic bonded phases are used to separate aqueous solutions of macromolecules. Finally, polymeric size-separation columns with hydrophilic phases are available for separation of polysaccharides, peptides, and very small proteins. 

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Wei G T, Liu F K and Wang C R C 1999 Shape separation of nanometre gold particles by size-exclusion chromatography Anal. Chem. in press... 

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. 

In size-exclusion chromatography, the smallest solute that can be separated from other solutes all smaller solutes elute together. 

Examples of the application of size-exclusion chromatography to the analysis of proteins. The separation in (a) uses a single column that in (b) uses three columns, providing a wider range of size selectivity. (Chromatograms courtesy of Alltech Associates, Inc. Deerfield, IL). 

Calibration curve for the determination of formula weight by size-exclusion chromatography. 

Size-exclusion chromatography can be carried out using conventional HPLC instrumentation, replacing the HPLC column with an appropriate size-exclusion column. A UV/Vis detector is the most common means for obtaining the chromatogram. 

A series of polyvinylpyridine standards of different molecular weight were analyzed by size-exclusion chromatography, yielding the following results. 

SI units stands for Systeme International d Unites. These are the internationally agreed on units for measurements, (p. 12) size-exclusion chromatography a separation method in which a mixture passes through a bed of porous particles, with smaller particles taking longer to pass through the bed due to their ability to move into the porous structure, (p. 206)... 

Figure 9.15 Schematic illustration of size exclusion in a cylindrical pore (a) for spherical particles of radius R and (b) for a flexible chain, showing allowed (solid) and forbidden (broken) conformations of polymer.

Use the model for the size exclusion of a spherical solute molecule in a cylindrical capillary to calculate for a selection of R/a values which... 

Another example of vims clearance is for IgM human antibodies derived from human B lymphocyte cell lines where the steps are precipitation, size exclusion using nucleases, and anion-exchange chromatography (24). A second sequence consists of cation-exchange, hydroxylapatite, and immunoaffinity chromatographies. Each three-step sequence utilizes steps based on different properties. The first sequence employs solubiUty, size, and anion selectivity the second sequence is based on cation selectivity, adsorption, and selective recognition based on an anti-u chain IgG (24). 

Fig. 3. Overview of puriftcation sequence for the nonrecombinant tissue plasminogen activator (t-PA) which also contains urokinase plasminogen activator (u-PA). Serum-free culture conditional media is from normal human ceU line. The temperature for aU. steps, except for size-exclusion chromatography...

An example of a size-exclusion chromatogram is given in Figure 7 for both a bench-scale (23.5 mL column) separation and a large-scale (86,000 mL column) mn. The stationary phase is Sepharose CL-6B, a cross-linked agarose with a nominal molecular weight range of 5000-2 x 10 (see Fig. 6) (31). 

Fig. 7. Chromatograms of size-exclusion separation of IgM (mol wt = 800,000) from albumin (69,000) where A—D correspond to IgM aggregates, IgM, monomer units, and albumin, respectively, using (a) FPLC Superose 6 in a 1 x 30 — cm long column, and (b) Sepharose CL-6B in a 37-cm column.

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