6.2. Ion exchange chromatography

Ion exchange chromatography is one of the most efficient methods for the separation of charged particles.

Ion exchange chromatography is most often performed in the form of column chromatography. However, there are also thin-layer chromatographic methods that work basically based on the principle of ion exchange. In the following, we will exclusively deal with column chromatographic applications.

Column materials used for ion exchange chromatography contain charged groups covalently linked to the surface of an insoluble matrix. When suspended in an aqueous solution, the charged groups of the matrix will be surrounded by ions of the opposite charge. In this “ion cloud”, ions can be reversibly exchanged without changing the nature and the properties of the matrix.

The charged groups of the matrix can be positively or negatively charged. A positively charged matrix will bind negatively charged ions from the solution. Therefore, it is called an anion exchanger. Cation exchanger matrices have negative charges.

Based on the structure of the ion exchange matrix, we distinguish ion exchangers with hydrophobic and hydrophilic matrices. Ion exchangers with a hydrophobic matrix are most often highly substituted polystyrene resins. These are suitable for the binding of inorganic ions, e.g. in water softening applications. However, they tend to denature proteins due to the high hydrophobicity of their matrix and their high surface charge density.

Ion exchangers with hydrophilic matrices were first produced from modified cellulose. However, cellulose has disadvantageous mechanical properties: cellulose fibres are prone to break, making it difficult to create a well-utilisable column. This disadvantage has been partially remedied in Sephadex (dextran-based) ion exchange matrices.

In recent years, regular spherical and monodisperse matrices have been produced from synthetic hydrophilic polymers. The best known of such resins is the MonoBead-based ion exchange matrix.

Table 6.I summarises the charged groups linked to ion exchange matrices.

Anion exchangers

Functional group

diethyl-aminoethyl (DEAE)


quaternary aminoethyl (QAE)


quaternary ammonium (Q)


Cation exchangers

Functional group

carboxymethyl (CM)


sulfopropyl (SP)


methylsulfonate (S)


Table 6.I. Functional groups of ion exchangers

Ion exchangers containing sulfonyl and quaternary ammonium groups are called strong ion exchangers. These are practically completely charged between pH 3.0 and 11.0. The degree of dissociation of DEAE and CM groups—and thus their ion exchange capacity—depends on the pH of the medium.

The theory of ion exchange

Most ion exchange experiments comprise five different phases (Figure 6.7).

Phases of ion exchange chromatography (salt gradient elution)

Figure 6.7. Phases of ion exchange chromatography (salt gradient elution). In the figure, a positively charged anion exchanger particle is shown, with counterions on its surface in the starting state (first phase). In the second phase, the binding of the ions to be separated takes place. At the start of the elution (third phase), weaker-binding ions are desorbed. At the end of the desorption, the stronger-binding ions are also desorbed (fourth phase). During regeneration (fifth phase), the starting state can be reconstituted via washing the column with the starting buffer.

The first phase is the equilibration of the ion exchange column with a so-called starting buffer, setting the conditions of the experiment (pH and ionic strength). In this phase, the charged groups of the ion exchanger will bind easily replaceable simple ions (e.g. chloride or sodium).

The second phase is the loading of the sample and its reversible binding to the column. If some of the contaminating materials do not bind to the column, these can be removed via washing the column with the starting buffer.

The third and fourth phases comprise the elution, i.e. the desorption of the bound molecules, which can be achieved via changing the composition of the elution buffer. The simplest form of elution is achieved via an increase in ionic strength, i.e. in the concentration of the counterions present. Another means of desorption is the change of the pH of the medium. The most effective method is the continuous change of the ionic strength or the pH, i.e. the application of a gradient elution. During gradient elution, molecules with smaller net charges (i.e. the weaker-binding ones) will be the first to leave the column.

An important property of an ion exchange column medium is its ion exchange capacity. This parameter reflects the amount of counterions that can be bound to the column. Three types of capacity can be distinguished:

Total capacity: The number of charged groups per gram dry weight of the ion exchanger or per millilitre of swollen gel. This can be determined by titration with a strong acid or base.

Free capacity: Due to steric reasons, only a part of the full capacity is accessible for macromolecules (proteins, nucleic acids). This is the free capacity.

Dynamic capacity: The so-called dynamic capacity is determined when the binding of the given macromolecule to the column is measured during buffer flow.

The free and the dynamic capacity values are dependent on the properties of the material to be separated, the properties of the ion exchanger, and the applied experimental conditions.

With regard to separation, the important properties of the material to be separated are the size of the molecules and the pH dependence of their charge. This implies that the capacity of ion exchangers will de different for different proteins.

Frequently used Sephadex-based ion exchangers

Via the modification of the Sephadex G-25 and G-50 gel filtration matrices with four different groups, eight different ion exchangers were created (Table 6.II). Matrices derived from G-25 (A-25 and C-25) contain more crosslinks. Therefore, they are more rigid and swell to a smaller extent than the ones derived from G-50.

Ion exchanger

Total capacity

Total capacity

Functional group




DEAE Sephadex A-25

3.5 ± 0.5




DEAE Sephadex A-50




QAE Sephadex A-25

3.0 ± 0.4




QAE Sephadex A-50




CM Sephadex C-25

4.5 ± 0.5




CM Sephadex C-50




SP Sephadex C-25

2.3 ± 0.3




SP Sephadex C-50




Table 6.II. Properties of Sephadex-based ion exchangers

With the use of specially-treated polysaccharide and synthetic polymer matrices that are more pressure-resistant than Sephadex, it was possible to develop fine-grained regular spherical polymer beads for ion exchangers with significantly increased efficiency. Such matrices include monodisperse MonoBeads with a bead size of 10 μm, or MiniBeads with a 3-µm bead size. Such ion exchangers are used in FPLC techniques (see below).

Important parameters to consider during the planning of ion exchange-based separation

  1. The charge of components present in the sample to be separated

In the case of proteins, this will depend on the isoelectric point. Below the isoelectric point, proteins are cations; above the isoelectric point, they are anions. The applied chromatographic buffer should be chosen in a way that the proteins to be separated should bind to the given anion or cation exchange column, from which they can be eluted after the washout of the non-binding components. More rarely, it can occur that the unwanted contaminants are bound to the column, whereas the component to be isolated will freely flow through.

  1. The amount of the sample

The size of the column should be chosen so that the dynamic capacity of the medium should somewhat exceed the amount of the sample. If the column is too small, the sample will saturate it, and part of the sample will not bind to the column. If the column is too large, a significant loss can occur during elution.

  1. The molecular mass of the protein to be isolated

The pore size of the ion exchange matrix should be chosen in a way that gel filtration effects—i.e. size-based separation—do not occur during ion exchange.