Size-exclusion chromatography diameters

Generally, size exclusion chromatography is carried out using columns with an internal diameter of 7.8 mm. However, some SEC applications require the use of expensive solvents. For this purpose, size exclusion columns with a smaller internal diameter (4.6 mm) have been developed. Of course one should use proportionally lower flow rates with narrow-bore columns. If the standard column size uses a flow rate of 1 ml/min, then the smaller 4.6-mm columns should be used at a flow rate of 0.35 ml/min. This provides the same linear velocity as 1 ml/min on 7.8-mm columns. The decreased flow rate reduces solvent consumption and solvent disposal cost. The performance of the smaller diameter columns is not compromised if properly optimized instrumentation is used. 

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]. 

The ideal packing should be relatively large in diameter, 80-100 nm, and be available with a pore size range from 500 to 10,000 A. The ores should be uniform in size distribution and shallow to reduce diffusion times. Improvements of this type will lead to the use of size exclusion chromatography for particle size determinations in the routine manner in which it is now employed for molecular weight determinations. 

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. 

Size exclusion chromatography (SEC), also called gel filtration when the mobile phase is aqueous, and gel permeation (GPC) when the mobile phase is organic, is based on the differing degrees of penetration of the sample molecules into the pores of the stationary phase (Fig. 7.1). These pores must have a diameter similar to the size of the species to be separated when these species are in solution in the mobile phase (Fig. 7.5). 

A more modern method to determine the MMD is GPC, gel permeation chromatography, also named size-exclusion chromatography, SEC. A polymer solution is passed over a column with a porous structure. The residence time of the chains on the column depends on the diameter of the coiled chain smaller chains can migrate through more pores (they can also enter into the smaller ones), and it takes a longer time for them to pass along the column. The bigger ones cannot enter into any of the side-pores and pass in the shortest time. 

Figure 7. Size Exclusion Chromatography peak average diameters for three batch runs with different emulsifier levels runs BIO — Bll are replications.

As the pore diameter increases in size (s decreases) relative to molecular or colloidal dimensions, less restrictions are imposed on the motions of contained species. Thus the exclusion effect gradually subsides as the pore size increases and consequently K-+1. For the separation of two molecules of different size, it is important to pick a pore diameter that will substantially exclude one species but not another. Pore size selection is thus of utmost importance in membrane science and in choosing a support for size exclusion chromatography (SEC). Aspects of pore size optimization in SEC based on the above partitioning theory have been developed [28]. 

These dendrimers have branch cell segments which are approximately one-half the dimensions of the PAMAM series (5 A versus 10 A) and possess chemical linkages which have much greater chemical and physical stability. A comparison of theoretical molar masses, number of terminal groups, CPK predicted diameters, and measured diameters (size exclusion chromatography, SEC) as a function of generation is shown in Table 3. 

In Fl-FFF, the channel is created by placing a mylar spacer with the channel cut out between two porous frits. A membrane hlter of a specihc molecular weight cutoff is placed on one of the frits and acts as the accumulation wall to permit flow, without loss of particles. The applied force is then a perpendicular flow of the carrier solution across the porous frits. Fl-FFF is a versatile technique capable of separating macromolecules as small as roughly 1000 Da, in which case it is comparable to gel permeation (size exclusion) chromatography. However, Fl-FFF can also be applied to the separation of colloidal particles. In this case the hydrodynamic diameter of the colloidal particle is related to the retention volume, V by the equation... 

Conventional Size Exclusion Chromatography, SEC, was performed on a Spectra Physics apparatus with two PL gel columns (5 pm particle size, 300 mm length, one with 50 A and one with 100 A pore size) and a Styragel HR2 column (7.8mm internal diameter x 300mm length). The detection was achieved with a SP8430 differential refractometer. The tolnene was elnted at a flow rate of 0.8mL.min . 

Commercially prepared silica is also characterised by the size of the pores. For the separation of low molecular weight compounds it is recommended that the pore width should be at least 5 nm in diameter. For the separation of macromolecules, it is more common to use a material with a pore width of at least 30 nm. If the silica has a pore width smaller than these values then the analytes will be wholly or partially excluded from the pores and a form of size exclusion chromatography will result. 

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