Ion-Exchange HPLC (IEX-HPLC) separates analytes based on differences in their surface charge. The basic principle governing IEX-HPLC separations is that analytes with an electrical charge are attracted to ligands on the stationary-phase particles which bear the opposite charge. The mobile phase typically consists of a pair of aqueous buffers, with the A phase containing a low concentration of salt and the B phase containing much more salt.
Two types of ion-exchange chromatography exist – anion exchange separates negatively-charged particles, and cation exchange separates positively-charged particles. In ion-exchange HPLC the stationary phase is composed of small stationary-phase particles to which charged ligands are attached on the surface. These particles are packed into a dense three-dimensional matrix within a glass or steel column.
When using anion exchange chromatography, all negatively charged particles within a sample will have some degree of interaction with the stationary phase beads which have a positive charge. The opposite is true for cation exchange chromatography. Upon entering the column the sample interacts with the stationary phase electrostatically, causing the sample to be retained on the stationary phase. The sample remains bound to the stationary phase until displaced by negatively-charged ions. This concentration of negative ions is reached by progressively increasing the salt concentration in the mobile phase. Once this critical salt concentration is reached, the interaction between the negative ions of the mobile phase and the stationary phase is stronger than that of the sample particles and the stationary phase, and the sample particles are displaced from the stationary phase.
This disruption can be described as an exchange between the sample particle and the ions of the mobile phase, hence the name ion exchange chromatography. The concentration of charged ions in the mobile phase necessary to cause exchange is dependant upon the degree of charge on the sample particle. Particles with a high degree of charge will be strongly attracted to the stationary phase and a significant amount of salt ions will be necessary to cause exchange. However, for particles that are slightly charged only a small amount of salt will be necessary for exchange. Therefore separation is the result of different sample particles requiring different concentrations of salt ions to displace them from stationary phase beads, and these concentrations of salts being reached at different times.
Since the investigator determines the length and slope of the mobile phases salt gradient they can manipulate the range in which maximum separation occurs. Additionally, a variety of stationary phases are available with varying degrees of charge which also allow the investigator to manipulate the separation range. At Analytical Ventura we possess a wide variety of stationary phase columns and will perform a variety of tests to ensure that conditions are located which maximize the type of separation your experiments require.
Figure 1. Separation of particles by AEX chromatography A. Initially the mobile phase has a low salt concentration and the negatively charged ions of the mobile phase are shown binding to the stationary phase beads. B. The sample is loaded onto the column in a low concentration of salt, the charged particles of the sample are therefore able to displace the mobile phase ions and bind to the stationary phase beads. C. As the salt concentration in the mobile phase increases the salt ions are able to exchange with the weakly charged sample particles. D. Weakly charged sample particles are no longer bound to the stationary phase and are eluted from the column. E. The salt concentration in the mobile phase increases further, displacing the highly charged sample particles from the stationary phase. F. Highly charged sample particles are no longer bound to the stationary phase and are eluted from the column.