Theory of Gel Chromatography

The theory of gel chromatography has been comprehensively reviewed by Determann25 and Altgelt.26 In the present article, emphasis is laid upon the fundamental aspects most likely to arise in carbohydrate chemistry. 

In a column packed with a swollen gel, two solvent phases may be distinguished, one within the gel (the stationary phase), and the other outside (the mobile phase). The volume of solvent in the gel is known as the internal volume, designated V4, and the volume of solvent outside the gel particles is the void volume of the column, designated V0. A solute will distribute itself between the two phases to an extent measured by the distribution coefficient Kd, a constant determined by the nature of the solute, the solvent, and the gel, but independent of column geometry.27 The volume of solvent required for eluting the solute from the column in maximum concentration is called the elution volume and is designated Ve it is equal to the sum of the void volume and the volume of the stationary phase available to the solute, given by Kd V. Hence, 

If the solute molecules are so large that they are completely excluded from the gel, Kd = 0. In this case, the solute passes through the column entirely in the mobile phase, and its elution volume equals the void volume V0. Small solute molecules that can freely penetrate the gel pores will have a Kd value of 1 here, Ve = V0 + Vf. Between these two extremes lie all solutes that can enter the gel phase to various, limited extents these will have Kd values lying between 0 and 1, and elution volumes between V0 and V0 + Vt, with both Kd and Ve increasing as the molecular size of the solute decreases. 

Fractionation Ranges of Some Poly(acrylamide) Gels 

In some cases, but not in others, the concentration of the sample has been found to have a small effect on the elution volume, and, consequently, on Kd. Winzor and Nichol40 noted a slight increase in the elution volumes of certain proteins on Sephadex G-100 as the sample concentration was increased from 1 to 12 mg per ml, but no such effect was observed with a dextran of Mw (weight-average molecular weight) 500,000. The effect observed with the proteins was ascribed to dependence of their migration rates on the concentration. 

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