Modern chromatographic techniques for the separation of biomolecules arguably stem from the work of British scientists Archer J. P. Martin and Richard L. M. Synge, who developed liquid-liquid partition chromatography in the 1940s to separate amino acids. Though Russian botanist Michael Tswett was the first to describe the concept of chromatography at the turn of the 20th century, Martin and Synge were the first to employ a solid support for a stationary liquid phase, over which a mobile solvent containing the mixture to be separated could be passed. Martin and Synge separated a mixture of amino acids by applying it to a column filled with water and a silica gel support and running chloroform through the column; they later used paper as the solid support, with similar results. In subsequent experiments, the researchers successfully used the same technique to resolve mixtures of small peptides.
In a speech presenting the 1952 Nobel Prize in Chemistry to Martin and Synge, Nobel laureate Arne Tiselius cited the invention of partition chromatography as key to the development of advanced chromatographic methods for biology, chemistry, and medicine. These advancements sparked a chromatography explosion in the latter half of the century. With the introduction of HPLC in the mid-1960s, liquid chromatography techniques, such as ion exchange separation, became more widely used for biomolecular applications.
Offering scalability, high specificity, and a wide choice of column materials, ion exchange chromatography (IEX or IEC) has become one of the best-known methods for protein and peptide purification. IEX separation relies on reversible charge interactions between a charged biomolecule (such as a protein or nucleic acid) and an oppositely charged resin-based matrix. IEX chromatography requires minimal equipment—pump, column, injector, fraction collector, and detector—and takes place in five steps. First, the column is equilibrated with a starting buffer at a pH that allows the protein of interest to bind selectively to the matrix. The sample—a cell lysate, for instance—is then applied to the column, and proteins of opposite charge bind to the matrix, while other sample components elute from the column. Bound proteins are displaced by applying a pH or salt concentration gradient to the column; as the ionic strength increases, more tightly bound proteins are displaced. Finally, the column is washed with high ionic strength buffer to remove all bound proteins, and re-equilibrated with starting buffer for subsequent purifications.
IEX resins—most commonly composed of agarose, dextran, or cellulose—are covalently bound to a charged group, which is classified according to charge type (cationic or anionic) and strength (strong or weak). A strong ion exchange functional group shows no loss or gain of charge with varying pH, while a weak functional group’s ion exchange capacity can vary with pH. Weak ion exchangers are more flexible in terms of selectivity, but strong ion exchangers are more frequently used for initial development and optimization of protocols because their binding capacity does not change with pH. Commonly used weak ion exchangers include diethylaminoethyl (DEAE), a negatively charged group, and carboxymethyl (CM), a positively charged group; strong ion exchangers include quarternary ammonium (Q), which is negatively charged, and methyl sulfonate (S), which is positively charged.
In addition to charge type and strength, matrix materials can be classified according to size and porosity. For example, larger particles are frequently used in initial protein capture steps that require fast elution rates and high capacity but low to intermediate resolution. Smaller particles are ideal for final purification steps requiring high resolution. Commercially available resins range in size from 10 to 400 microns and have binding capacities ranging from less than 2 mg/ml to more than 150 mg/ml.
Another important consideration for IEX protocols is buffer choice. The buffering ion should be the same charge as that of the ion exchanger. For example, Tris buffers are often used with DEAE; phosphate and acetate buffers are frequently used with CM. Buffer pH should permit the protein of interest to remain stable throughout the purification process while allowing it to bind reversibly to the matrix. It should also be close enough to the pH at which the protein begins to dissociate from the column to prevent the need to adjust the pH or ionic strength to levels that would destabilize the protein. Most protocols recommend that the buffer pH be within 1 pH unit of the protein’s isoelectric point. For most proteins, the ideal pH range is between 4 and 8.
A variety of suppliers sell ion exchange resins for applications ranging from process-scale separations to isolation of milligram quantities of protein. Some vendors sell pre-packed IEX columns for use with standard liquid chromatography setups or HPLC systems; ion exchange matrices can also be purchased in microplate for high-throughput applications. Some examples of IEX-related products are listed below.