Recycling gel partition method

The method tenned recycling gel partition by Ford and Winzor (ref. 35) is similar to the batch method, but it has the advantage of permitting convenient binding determinations for an entire series of different ligand concentrations in a way related to the steady-state dialysis method of Colowick and Womack (ref. 36). The partition equilibriun 

The volumes and Vq are measured first by L and by M or by suitable inert marker compounds. The partition of L between and Vq is preferably measured in several successive concentrations. This enables one to detect deviations from the ideal gel chromatographic partition of L, (i.e. possible adsorption onto the gel). The sane is repeated with M, and eventually with various concentrations of L and a fixed concentration of M. This series of experiments yields curves such as shown in Fig. 5, which will allow calculations of the successive binding constants. 

If a normal chromatographic column, as shown in Fig. 4,is employed a uniform distribution of samples throughout the voids will take an unpractically long time. Two different solutions for this problem have been offered. The first of them employs a construction similar to the stirred batch enzyme reactors used in the analysis of immobilized enzymes (ref. 35,37). In that mode, an open chromatographic column containing appropriate gel slurry is stirred continuously (ref. 35). The apparent advantages of this method are its simple construction, short equilibration and lack of need of special sanpling port. Equilibration times of about 10 minutes are sufficient with the apparatus (ref. 

However, a drawback is that the void volume cannot be minimized because an excess of solvent must be used for mixing of the gel slurry. The ratio of Vp/Vi of about 1.5 is appropriate for the system (ref. 35). 

Wong and Porter solved the equilibration problem with a special double conical dispersion column in which the band spreading of the sample was much more facilitated than in the cylindrical column (ref. 38). In their system the total mixing was achieved in about 30 minutes or less while the ratio Vq/ could be kept as low as 0.76. As the sensitivity of the recycling gel partition method is inversely proportional to l+Vp/ V, the dispersion column system is advantageous whenever a longer time is allowed for the measurement (ref. 38). 

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Figure 5. Typical curves obtained by the "recycling gel partition" method (see Fig. 4). I he lowest curve refers to a situation when the gel adsoti>s ligand while the dashed line refers to an ideal case where only partition mechanism is working. In the presence of M an extra ligand is drawn into the mobile phase as a result of binding between M and L.

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