Displacement chromatography versus pH zone refining CCC
The biggest drawback of “elution mode” chromatography, which I am using to refer to methods that rely solely on the differential attraction of analytes to the mobile phase versus the stationary phase for separation, is that they often require low loading. On an analytical scale, these problems are not significant. When your working on a preparative scale this is when high loading capacity is critical.
Displacement chromatography and pH zone refining offer two different approaches that make high loading, and high purity possible on a preparative scale. In both cases, modifying agents are used to dramatically change the dynamics of separation within the column.
In displacement chromatography, which is a type of column chromatography, analytes are separated based on competitive binding of the analytes to biomolecules in the chromatography matrix. This process works by using a displacer molecule, a molecule that has a very high affinity for the binding sites within the column, to compete with and displace the analytes in the column. The analytes that have the weakest interactions are displaced faster than analytes that have stronger interaction with the column. The analytes come off of the column as rectangular peeks, with minimal overlap and high purity.
pH zone refining, which can be preformed on any countercurrent chromatography column, uses a series of “retainer,” acids are used to modify the hydrophobicity of ionizable analytes within the column sequentially, based on their pI. That was a mouthful-in other words-the pH within the column is constantly changing during the process of separation. Modifying agents referred to as retainer acids (or conversely as eluter acids when running in reverse phase), buffer the pH of within the CCC column allowing stepwise elution of analytes based on pI. This creates very different results when compared to using a pH gradient alone. pH gradients produce chromatograms similar to those created by displacement chromatography. This image I pulled from Yoichiro Ito’s pH-zone-refining patent shows a chromatogram produces by a pH gradient.
separation of DNP amino acids using a pH gradient on CCC
When buffers are used to control the pH change within the CCC column, complete separation of the DNP-amino acids occur. The buffers allow the sequential elution of analytes to occur in discrete steps, rather.
separation of the same DNP amino acids with the addition of retainer acids (pH zone refining)
Although pH zone refining and displacement chromatography are similar in that they can both allow for high loading capacity and produce nice robust rectangular peeks, with minimal overlap in the case of displacement chromatography and no overlap in the case of pH zone refining. It is important to remember that the basis of separation is completely different because this allows you to quickly deduces which technology is more suitable for your application. For example if you know you in advance that the species you are trying to separate are not ionizable, or have pI’s that have a difference of less than (0.3), displacement chromatography would be the best fit for your preparative needs. However, if you need to completely eliminate peek overlap pH zone refining is for you. Or if you know or suspect that the compounds you are working with have quite different pI’s look into pH zone refining. Luckily, if you have a CCC column the only additional material required for pH zone refining are the retainer acids. However, with displacement chromatography you’re going to need to purchase a special displacement column and a specialized displacer agent.
