Displacement Chromatography on Synthetic Ion - exchange Resins

In Part 1 of this series of papers (Partridge & Westall, 1949) it was shown that, whilst the baseexchange resin Zeo Karb 215 may be used with success for the separation of the neutral and acidic amino-acids, certain difficulties arise when the separation of basic substances is attempted. These difficulties were traced to the phenolic hydroxyl groups of the resin. Resins of the sulphonated phenol-formaldehyde type are multifunctional when in equilibrium with solutions of high pH. Titration curves (Partridge & Westall, 1949, Fig. 4) show that over the rangepH 1-7 the sulphonic acid groups only are dissociated, but when the pH rises from 7 to 12 the phenolic hydroxyl groups also dissociate and thus over this range the resin retains cations on both types of group. Measurement of the boundary widths of fronts due to the retention of sodium ions from a range of buffered solutions of different pH showed that sharp boundaries were obtained over the range pH 1-7, but that under more alkaline conditions the boundaries became progressively wider (Partridge & Westall, 1949, fig. 7). This suggested that the rate of exchange between sodium and hydrogen ions was rapid under acid conditions when the sulphonic acid groups only were involved, but was much slower when phenolic hydroxyl groups also played a part. Other experiments showed that the effect was also important when one base displaced another from the resin, and successful separations were obtained only with components of lower basic strength than ammnonia; in the experiments on the fractionation of a protein hydrolysate, described in Part 3 of this series (Partridge, 1949a), ammonia was used as the displacement developer, in spite of the fact that this reagent was insufficiently basic to displace arginine. In practice it was found that the affinity ofammonia for the resin was not much greater than that of lysine and thus this amino-acid also was not readily displaced and could not be recovered quantitatively. The difficulty with which arginine and lysine are recovered from a column of Zeo Karb 215 either by elution with hydrochloric acid or by displacement with sodium hydroxide has been stressed by Hems, Page & Waller (1948), who were unable to obtain satisfactory recoveries of these two amino-acids in experiments with protein hydrolysates. In order to overcome this difficulty experiments have now been carried out with samples of another type of resin which contained no phenolic hydroxyl groups. This was a resin of the sulphonated crosslinked polystyrene type. As will appear below, attempts to use a commercial sample of this resin resulted in failure, and for this reason variously modified samples of sulphonated crosslinked polystyrene resin were prepared in the laboratory. It soon became apparent that the rate of exchange of large organic bases varied considerably with the degree of crosslinking of the resin, and sharp boundaries were only obtained on resin samples in which the structure was relatively open. However, resins of open structure suffered from the corresponding disadvantage of excessive shrinkage with increases in the ionic strength of the solution phase, and it was necessary to take special steps to minimize disturbance of the boundaries due to this cause.