Size exclusion chromatography pore volume

Because the separation in size exclusion chromatography takes place only in the range of the pore volume, this parameter has a great impact on the resolution. A large pore volume will result in that peaks are separated further apart. In... 

Most size exclusion chromatography (SEC) practitioners select their columns primarily to cover the molar mass area of interest and to ensure compatibility with the mobile phase(s) applied. A further parameter to judge is the column efficiency expressed, e.g., by the theoretical plate count or related values, which are measured by appropriate low molar mass probes. It follows the apparent linearity of the calibration dependence and the attainable selectivity of separation the latter parameter is in turn connected with the width of the molar mass range covered by the column and depends on both the pore size distribution and the pore volume of the packing material. Other important column parameters are the column production repeatability, availability, and price. Unfortunately, the interactive properties of SEC columns are often overlooked. 

In 1971, Hiatt et al. found that polyethylene oxide (PEO) of molecular weight about 100000 prevented the adsorption of rabies virus to porous glass with an average pore diameter of 1250 A. The support was modified by passage of one void volume of 0.4% solution of the polymer in water, followed by 5 or more volumes of distilled water or buffered salt solution. The virus was effectively purified from the admixtures of brain tissue fluid by means of size-exclusion chromatography on the modified glass column [28]. 

Size exclusion chromatography (SBC) is a separation process by which molecules are fractionated by size on the basis of differential penetration into porous particulate matrices. Blution volume (Vq) of any given molecular species relative to another of different size is dependent on the pore diameter of the matrix, pore-size distribution, pore volume (Vp, interstitial volume (Vq) and column dimensions. Use of SBC to estimate molecular size is achieved by plotting the log of the molecular weight of a series of calibrants against their elution volume. Since Vg is a function of Vg and Vj, its magnitude will be dependent on the geometry of a column. 

The specific pore volume is important in size-exclusion chromatography (SEC) [10] because the separation takes place there. In retentive chromatography, it is necessary to provide the surface area... 

Before considering how the excluded volume affects the second virial coefficient, let us first review what we mean by excluded volume. We alluded to this concept in our model for size-exclusion chromatography in Section 1.6b.2b. The development of Equation (1.27) is based on the idea that the center of a spherical particle cannot approach the walls of a pore any closer than a distance equal to its radius. A zone of this thickness adjacent to the pore walls is a volume from which the particles —described in terms of their centers —are denied entry because of their own spatial extension. The volume of this zone is what we call the excluded volume for such a model. The van der Waals constant b in Equation (28) measures the excluded volume of gas molecules for spherical molecules it equals four times the actual volume of the sphere, as discussed in Section 10.4b, Equation (10.38). 

Exclusion Volume—In size-exclusion chromatography, Vo, the volume of solvent necessary to washout unretarded compounds too large to penetrate the pores of a size-separation column. The inclusion volume, 2Vo, is the elution volume needed to elute all compounds small enough to fully penetrate the pores. 

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),... 

The separation range in size-exclusion chromatography for a particular column is relatively narrow, and it lies between the total volume of the liquid phase in the column (void volume) and the exclusion volume, Ve. The difference between these two volumes is the total pore volume of the packing material in the column. Indeed, if some molecules of studied polymers are small enough to penetrate inside all pores of the packing material, they will elute with the column void volume. On the other hand, polymers with significant molecular size that cannot penetrate inside the particles will all travel together around the particles and elute early with exclusion volume. 

Separation in size-exclusion chromatography requires careful matching of the pore size of the stationary phase material with the size of the molecules to be separated. Small molecules in a sample will be able to penetrate all the pores of the stationary phase and will elute with an elution volume, Fe, which is equal to the void volume of the column, Fm- Very large molecules will be excluded from all the pores of the stationary phase and will elute with an elution volume, which is equal to the interstitial volume of the Uquid between the particles, Fj. Molecules of intermediate size will be able to penetrate some but not all of the pores and will elute with an elution volume that is between the interstitial volume and the void volume of the column. The void volume is the total volume of the liquid in the column and is related to the interstitial volume and the pore volume, Fp by equation (3.38) ... 

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