Amino Acids, Peptides and Proteins - Dévényi T., Gergely J. 1976

Ion-exchange chromatography on a fixed bed of ion exchanger
Equilibration of "Fixion 50x8" plates

Ion-exchange Chromatography employs a resin in the appropriate ionic form. In Column chromatography, the resin is first converted from one form to another (e.g., from the H⁺ to the Na⁺ form), after which the column is equilibrated with the starting buffer solution. When using Fixion 50 x 8 plates, The First stage—conversion from one form to another—is not required, as the layer is prepared from ultra-pure resin already in the Na⁺ form. However, resin equilibration is necessary in some cases, especially when developing new Methods. This is easily achieved by descending chromatography with a continuous supply of buffer solution. The Fixion 50 x 8 plate is placed back-to-back on a Glass plate of the same size. Four sheets of filter paper (20 x 15 cm) are folded with a 1 cm wide strip bent at one end, and applied to the upper edge of the plate so that the bent one-centimeter paper strip covers the ion-exchanger layer; it is secured to the plate with a suitably sized rubber band or two 21 cm glass rods tied tightly at the ends (Fig. 48). The free portion of the paper sheets is folded back onto the reverse side of the glass. The paper sheets must be wider than the plate; otherwise, they may wrinkle during fastening, resulting in loose contact that disrupts the uniform flow of the buffer solution and impairs the equilibration process.

For equilibration, a very dilute buffer solution should be used, with a molarity an order of magnitude lower than that of the chromatography buffer solution. It is not recommended to use a buffer solution with a pH below 3, because during prolonged chromatography (16–24 h) under such conditions, the ion-exchanger layer may adsorb heavy Metal Ions, and equilibration will then do more harm than good.

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Fig. 48. Equilibrating Fixion plates.

Equilibration typically employs a citrate buffer solution with an Na⁺ concentration of 0.02 M and a pH of 3, used at a 10-fold dilution as the first buffer solution (A) in an amino acid analyzer. During plate equilibration, the lower part of the layer (immersed in the buffer solution) may delaminate, slide off the backing, or detach completely. Therefore, during equilibration, the end of the plate should be immersed by no more than 1 cm, and after drying, it is advisable to trim off this strip of the ion-exchanger layer.

Equilibrated plates can be stored indefinitely at room Temperature. It is only necessary to ensure they do not adsorb ammonia vapors. To rule out this possibility, smoking, in particular, should be prohibited in the laboratory.