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).
Figure 1. Example of ion exchange chromatography.