Total permeation limit

The selective separation range of P-6/S-200 was determined with Blue Dextran (Vexdi exclusion limit) and fructose (V,o total permeation limit). Molecular weight (degree of polymerization) calibration (Fig. 16.22) was established with dextran standards and low dp pullulans (dp 3, 6, 9, 12, 15, 18) formed by the controlled hydrolysis of high dp pullulan. 

Hi) The curve, thus obtained, normally approximates to a straight line within the exclusion and total permeation limits,... 

Ion exchange of sample (bands elute aher total permeation limit) Hydrophobic lelentioii (bands dute after total permeation limit)... 

Vo is the exclusion limit of the column and equal to the interstitial volume of the packed SEC column (volume of eluent between the particles of stationary phase). Fi is the pore volume of the packing (the internal solvent volume in the pores). Ft is the total permeation volume. The volume of solvent at which a solute elutes from the column or the volume of liquid corresponding to the retention of a solute on a column is known as the retention volume (Fr). Fr can be related to the physical parameters of the column as follows ... 

One of the most serious disadvantages of SEC is limited peak capacity that is, only a few separated bands can be accommodated within the total chromatogram, as all peaks must elute between the total exclusion volume and the total permeation volume. 

Molecules of various size are drawn on the photo and show that the large molecules cannot fit at all and would be washed out of the column. The smallest m. w. molecule that does not enter the gel is called the exclusion limit, and the volume required to elute the large molecules is called the void volume, V. The smaller molecules can permeate the gel the smaller the molecule, the farther the penetration and the greater the retardation. This results in a separation because it requires more solvent to wash the smaller molecules out of the gel. This has a limit. If the molecules become too small, then their molecular size compared to the pores is so small that they behave the same and require essentially the same amount of solvent to elute them. This volume is called the total permeation volume, Figure 17-2, p. 172... 

Fig. 2.13. Molecules are selectively retarded if their molecular weight is between the exclusion limit and the limit of total permeation.

Even though the total permeation rate is still acceptable, data suggests that suitable precautions have to be adopted to limit as much as possible stress and film deterioration. 

We are concerned with two major limits of this selectivity the case where the total permeate pressure is close to the equihbrium vapor pressure and the case where the total permeate pressure is near zero. We consider these two cases sequentially. 

In exclusion chromatography, the total volume of mobile phase in the column is the sum of the volume external to the stationary phase particles (the void volume, V0) and the volume within the pores of the particles (the interstitial volume, Vj). Large molecules that are excluded from the pores must have a retention volume VQ, small molecules that can completely permeate the porous network will have a retention volume of (Vo + Fj). Molecules of intermediate size that can enter some, but not all of the pore space will have a retention volume between VQ and (V0 + Fj). Provided that exclusion is the only separation mechanism (ie no adsorption, partition or ion-exchange), the entire sample must elute between these two volume limits. 

Efficiencies of 24,000 plates at permeation and 23,000 plates at total exclusion were measured at flow rates of 1.0 ml/minute for this column. Separation speed is quite good also, but does not represent the limit which can be attained. 

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