Ion-exchange chromatography

Ion-exchange chromatography is a type of chromatography technique whose mechanism is based on the separation according to the electric charges of the molecules in the media. This makes the ion exchange chromatography principle very simple, relying on simple electrochemistry instead of more complex interactions like affinity for protein three-dimensional structure. This is used for the separation of proteins, as proteins are complex molecules with positive and negative surface areas. This can be a very precise technique, able to separate two proteins differing by very small electric charge properties, for example only one charged amino acid.

The same method is equally useful for separation of nucleotides or other biopolymers, which also can differ from each other by just a small electric charge difference. It can also be used for deionization of water, removing all anions and cations dissolved in the water. Similarly, it can be used to collect trace metal from seawater.

Ion-exchange chromatography resins exist in 2 types, negatively or positively charged, depending on the electrochemical characteristics of the target of the purification.

Another method for ion-exchange chromatography relies on membranes instead of resins. This is mostly relevant for low-volume buffer solutions, for quicker processing time, or for eliminating the need for column packing. Membranes are also more often used for the purpose of purifying viruses or virus-like particles.

The electric charge on the surface of a protein will vary depending on the pH. Every protein will have a point where it carries as much positive as negative electric charges, called its isoelectric point.

Above this isoelectric point, the protein will bind to a positively charged anion exchanger. Below this isoelectric point, the protein will bind to a negatively charged cation exchanger.

To perform the binding of the proteins onto the chromatography ion exchange column, several steps are followed:

1. Equilibration: once finished, this step loads all the stationary phase charged groups of the resin, which are bound with exchangeable counter-ion (so both negative and positive groups), for example, sodium or chloride.
2. Sample application and wash: The column is loaded with the proteins (or other molecules) media at low ionic strength, to let them bind strongly to the ion-exchange column’s resin.
3. Elution: Proteins (or other molecules) are released from the chromatography matrix thanks to an increase in ionic strength for example by applying a growing concentration of sodium chloride. A change in pH is another option for changing the electrochemical properties of the proteins and their bind to the column.
   1. The increase can be in discrete steps of continuous increase in concentration.
   2. Proteins are released (“desorbed”) successively in “batches” in function of the number of their charged groups on their surface.
4. Regeneration: this guarantees the separation from the resin of all molecules still bound to the resin, to ensure the full functionality of the ion exchange chromatography column for the next batch of purification.

Ion chromatogram displaying anion separation

A quality of resin in ion exchange chromatography is its high-throughput capacity, as this technique can relatively quickly separate large volumes of analytes. This is true for both organic and inorganic molecules. It can be used to separate multiple ions/molecules at once, making it often more economical or saving time. The technique also requires minimal preparation of the sample, reducing the risk of contamination and loss of analyte. Lastly, it is a relatively simple method, allowing for both experts and novices to use it. It also rely on simple and often non-toxic chemicals for its liquid phase, even if the acidity/basicity employed requires some precautions.

The main limitation of ion-exchange chromatography is its limited analytical capacity regarding structural information of the separated molecules. To define further the nature of the purifying product, additional analytical methods are required, like for example mass spectrometry or UV spectrophotometry.

Ion-exchange chromatography will also not work well for non-ionic molecules, for the obvious reasons that they will not interact with the chromatography column.

Optimizing ion exchange chromatography will rely on determining the right column material, as well as the right chromatographic conditions, like pH, salt concentration, or buffer system.

An important factor in the decision regarding ion-exchange chromatography is the knowledge about the isoelectric point of the protein.

If you know the isoelectric point, you should pick an anion exchanger + buffer above the isoelectric point or a cation exchanger + buffer below the isoelectric point. If you do not know the isoelectric point, it is usually best to use a strong ion exchanger, as they have a wider range of functional pH. A weaker ion exchanger system will be more selective, but you will likely need a correct “guess” of the possible isoelectric point of your target molecule.

Our experts can help you determine the best possible options according to the profile of your targeting molecule/protein, as well as depending on the goal of the purification (remove non-proteique impurities, multiple proteins purification, single protein purification, purification of molecules other than proteins, etc) and the composition of the initial media .