Up and down a pH gradient: Separating proteins by pH gradient IEC

Focusing on pH

Isoelectric focusing is a commonly used technique for separating proteins, usually forming the first separation dimension in 2D gel electrophoresis, and works by separating proteins according to their isoelectric point, the pH at which they have no electrical charge.

This is done by conducting electrophoresis in a gel with a pH gradient. At pHs above or below their isoelectric point, proteins have a negative or positive charge, respectively, and so will move through the gel under the influence of an electric field. Once they reach the region of the gel with a pH that matches their isoelectric point, however, they will lose their charge and stop moving. As different proteins have different isoelectric points, as determined by their specific amino acid sequences, the proteins will stop at various different points along the gel’s pH gradient.

There is also a chromatographic version of isoelectric focusing, known as pH gradient ion exchange chromatography (IEC), but it is far less popular. It works by using a mobile phase with a pH that changes over time to first load proteins onto an ion exchange stationary phase and then elute them by their isoelectric point.

If using an anion exchange stationary phase, the mobile phase initially has a high pH, giving all the proteins a negative charge and ensuring they bind with the stationary phase. The pH of the mobile phase is then gradually reduced, causing the proteins to lose their charge as the pH of the mobile phase falls to match their isoelectric point. As a consequence, they no longer bind to the stationary phase and so are eluted. The procedure is much the same with a cation exchange stationary phase, except that the mobile phase starts with a low pH that is then gradually increased.

 

Gradient over time

The reason pH gradient IEC is far less popular than isoelectric focusing is that it’s much more difficult to produce a pH gradient over time in a liquid mobile phase than a pH gradient over distance in a gel. In isoelectric focusing, the pH gradient is produced by applying a mixture of an acid and base at different ratios along the gel: the ratio of acid to base is high in the low pH region and low in the high pH region. In pH gradient IEC, the pH gradient is produced by titration, with increasing concentrations of an acid or base added to a liquid buffer.

Ideally, this liquid buffer would itself be an acid or a base; so if you wanted the mobile phase to go from a low pH to a high pH, you would simply add increasing concentrations of a base to an acid. Unfortunately, this doesn’t produce a smooth, linear pH gradient, which is essential for pH gradient IEC.

To produce a smooth, linear pH gradient, you need to utilize a liquid buffer that consists of a mixture of different strength acids and bases, to which you then steadily add a strong acid or base. Unfortunately, determining the most effective mixture of acids and bases for this liquid buffer has proved difficult, limiting pH gradient IEC to a fairly small range of pH values.

 

Computerized tool

Now, however, Frieder Kröner and Jürgen Hubbuch at Karlsruhe Institute of Technology in Germany have developed a computerised tool for calculating the optimum mixture of acids and bases for pH gradient IEC. Using this tool, they developed both an anion-exchange liquid buffer, consisting of six different strength acids and bases, and a cation-exchange buffer, consisting of eight different strength acids and bases.

Using these liquid buffers, the two researchers were able to produce mobile phases with a smooth, linear pH gradient for both anion and cation versions of pH gradient IEC, with the gradient spanning 7.5 pH units in each case. They then tested these two versions on a mixture of 22 different proteins.

Interestingly, although they were able to separate these proteins, not all of them eluted at the pHs that correspond to their isoelectric point. This is probably because the strength of a protein’s binding to a stationary phase isn’t just determined by its charge, but also by its three-dimensional structure. If this structure acts to weaken the binding strength, then the protein will be washed away before the pH of the mobile phase reaches its isoelectric point.