Designing a Cation Exchange Separation

This web site is designed to help you study the three steps in ion exchange chromatography: sample application, washing the column to remove weakly bound proteins, and elution of the protein of choice with a properly designed gradient.

It is necessary to select a resin and buffer so that the protein of interest will bind to the resin. In a cation exchange separation, the protein of interest needs to be positively charged to bind to the stationary phase in the column.

Table 1. Charge on Proteins for Cation Exchange.

The column heading indicates the pH for the separation and the stationary phase. For example, pH4.8 cm is a test mixture with a pH 4.8 mobile phase and a carboxymethyl-Sephadex stationary phase.

Protein pI pH4.8 cm pH7.2 cm pH8 cm
Carbonic Anhydrase 7.0 +16.5 -0.4 -2.7
Carboxypeptidase B 6.2 +12.0 -3.3 -6.3
Chymotrypsin 8.0 +9.0 +2.7 0.0
Lysozyme 9.8 +14.1 +7.9 +6.9

For the protein mixture in Table 1, all the proteins will bind to the cation exchanger at pH 4.8, since all are positively charged. The more positively charged the protein, the stronger it will bind. Therefore, if you elute the four proteins in this mix with a 0 to 0.2 M NaCl gradient, the elution order will be chymotrypsin, carboxypeptidase B, lysozyme, and carbonic anhydrase. Try it.

If you use pH7.2 cm, then carbonic anhydrase and carboxypeptidase B will elute in the wash (before the gradient is initiated), followed by chymotrypsin, then lysozyme during the salt gradient.

In the mixture pH8 cm, only the lysozyme will bind to the stationary phase, the other three proteins will elute in the wash.

It is also possible to refine the salt concentration in solvents A and B so that the more weakly bound proteins will wash off the column in the wash, even if they are positively charged. For example, if you modify pH4.8 cm to have 0.1 M NaCl in solvent A and 0.2 M NaCl in solvent B, you find that the most weakly bound protein (chymotrypsin) elutes in the wash, while the other three elute in the gradient.

A similar set of test mixtures may be used for anion exchange chromatography. The same four proteins and buffers may be used as are shown in Table 1, but the stationary phase is DEAE-cellulose. Experiment with these text mixtures to see their binding and elution behaviors.