Column parameters and separations

A number of theoretical treatments of gel chromatography have been published, including applications of the theoretical plate concept similar to those outlined in Chapter 2. It is appropriate here to mention only a few simple parameters of most value in practical work. 

As in other forms of partition chromatography the elution volume can be related to the coliunn dead volume (F ) and the volume of stationary phase 

Ka is a constant for a given solute and gel under constant operating condition of eluant composition and temperature, hence the determination of characterises the retention behaviour of the solute independently of the dimensions of the chromatographic bed. 

In practice, not all these volumes are readily determined. V can be measured as the volume required to elute a completely excluded solute, and Ve for a particular solute is equally readily measured. On the other hand, the methods which can be used for determining Vi (requiring, for example, observation of the elution of tritiated water) give only approximate results, since exchange reactions on the Sephadex gel cause the peak to emerge after the bed volume [34]. is often, therefore, replaced by an alternative distribution coefficient, which is defined [35] as 

If Ki or is plotted against the logarithm of the molecular weight for a series of solutes similar in molecular shape and density, an S-shaped curve is obtained. Over a small range of the fractionation range, the curve approximates to a straight line 

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