Ion Exchange Chromatography (IEX) Technique

Ion exchange chromatography (IEX) is a commonly used technique for biomolecules purification based on their affinity to the ion exchanger. This technique exploits the ionic (or electrostatic) interactions between charged molecules in analytes and oppositely charged moieties in the stationary phase of chromatography matrix. IEX is to some extent superior than other techniques since the charges carried by the analytes can be easily manipulated by the pH of buffer used.

How Does It Work?

Fig. 1 Schematic illustration of Ion exchange chromatography (IEX).

IEX uses a resin, bearing either positively or negatively charged chemical groups, to separate charged biomolecules such as proteins, peptides, amino acids, or nucleotides according to their surface charges. Resins with positive charges can attract negatively charged solutes and are referred to as anion exchange resins, while resins with negative charges are cation exchangers, which are shown as follow. Generally, by increasing the salt concentration (often is a linear salt gradient) the molecules with the weakest ionic interactions begin to elute first, whereas the molecules with a stronger ionic interaction will require a higher salt concentration and elute later in the gradient.

As regards to proteins, the amino acids that make up proteins are zwitterionic which contain both positive and negative charges. Depending on the pH of their environment, proteins may carry a net positive charge, a net negative charge, or no charge. The pH at which a molecule has no net charge is called its isoelectric point, or pI. The buffer pH can determine the net charge of the protein of interest. For example, if the buffer pH is greater than the pI of the protein, the protein will carry a net negative charge; therefore, an anion exchange resin will be chosen. By contrast, if buffer pH is lower than the pI, the protein will carry a positive net charge and a cation exchange resin should be chosen.

Similarly, proteins of different degrees of PEGylation can also be separated by IEX since for each PEG molecule attached to an amino group, for example, a PEGylated protein has one less positive charge and this chromatographic technique separates proteins based on net surface charge. By choosing the optimal ion exchanger and separation conditions, high resolution can be obtained.

General Considerations

• Separated ions & ion exchangers

Generally speaking, the higher the valence of the ion, the greater the atomic number, and the smaller the radius of water and ions, the greater the selectivity ability of the ion.

The selectivity order of strong acid cation exchanger for cations is:

Fe³⁺>Al²⁺>Ba²⁺≥Pb²⁺>Sr²⁺>Ca²⁺>Ni²⁺>Cd²⁺≥ Cu²⁺ ≥ Co²⁺ ≥ Mg²⁺ ≥ Zn²⁺ ≥ Mn²⁺>Ag⁺>Cs>Rb⁺>K⁺ ≥ NH₄⁺>Na⁺>H⁺>Li⁺

The selectivity order of strong base anion exchanger for anions is:

Citrate>PO₄^3->SO₄^2->I^->NO^3->SCN^->NO^2->Cl^->HCO^3->CH₃COO^->OH^->F^-

• Composition of the mobile phase

The mobile phase composed of ions with strong exchange capacity and high selectivity coefficient has strong elution capacity. The ionic strength of the mobile phase increases, its elution capacity increases, and the retention value of the components decreases.

• pH of the mobile phase

The adjustment of pH value mainly reflects its control of the dissociation of weak electrolytes: the dissociation of solutes is inhibited, and its retention time becomes shorter. Therefore, the change of pH value has a greater impact on the exchange capacity of weak ion exchange resin.

What Can IEX Be Used For?

• In recent years, IEX has been the most used downstream technique for the separation of proteins, peptides, nucleic acids, and other charged biomolecules, offering high resolution and group separations with high loading capacity.
• At the same time, IEX is also able to separate mono-, di-, or multi-PEGylated proteins from their native form and positional isomers based on protein net charge alteration, showing weak retention of by-products and un-conjugated PEG species.

Strengths & Weaknesses of IEX

References

1. Shen, C. H. Quantification and Analysis of Proteins. Diagnostic Molecular Biology 2019: 187-214.
2. Ramos-de-la-Peña A M, et al. Progress and challenges in PEGylated proteins downstream processing: a review of the last 8 years. International Journal of Peptide Research and Therapeutics, 2020, 26(1): 333-348.