Ion exchange chromatography is a type of liquid chromatography where the molecules in the sample are separated according to their charge.

It is commonly used to separate charged biological molecules such as proteins, peptides, amino acids, or nucleotides. For instance, the amino acids in the proteins contain both positively and negatively charged chemical groups. Proteins may carry a net positive or negative charge or even no charge depending on the pH of their environment.

The pH where the protein has no charge is called an isoelectric point. Therefore, the choice of buffer pH determines the net charge of the protein of interest. If the buffer pH is greater than the isoelectric point of the protein of interest, the protein will carry a net negative charge. On the other hand, a pH lower than the isoelectric point will make the protein carry a net positive charge.

Depending on the net charge of the molecules to be separated, the stationary phase should be selected accordingly. Also, since samples are mixtures of different compounds with different charges, a buffer gradient is normally used to elute each different compound separately (Figure 1).

Principle of separation molecules in ion exchange chromatography. First, a mix of proteins, shown as colorful spheres with plus or minus symbols on them, flows through the column, from the top to the bottom. On the sides of the column are immobilized cations, depicted as orange spheres with plus signs. As the time passes, first, negatively charged molecules bind to cations, and positively charged molecules are eluted. After addition of the elution buffer, slightly negatively charged molecules are eluted. Lastly, increased concentration of the elution buffer separates highly negatively charged molecules from the cations and eluted them. The elution curve presented below the workflow presents two peak, one at the time of elution of slightly negatively charged molecules, and second one at the time of elution of highly negatively charged ones.

Figure 1. Example of ion exchange chromatography.