Chromatography is a technique in use since the early 1900s. Mainly used in laboratories, chromatography helps to separate mixtures into their components. This technique was first used to study the composition of plants and to understand organic chemical compounds.
Studying the constituents of these mixtures helped scientists further understand the workings of the world. While this traditional chromatography method is seldom used today, more advanced techniques like High-Performance Liquid Chromatography, Ion Exchange Chromatography (IEC), Aqueous Normal Phase Chromatography, Micellar Liquid Chromatography, etc., are in use.
These advanced techniques help separate more complex mixtures; IEC (as the name suggests) helps separate mixtures containing charged particles. Ions are atoms that are electrically charged.
Using IEC, a scientist or a laboratory technician can easily separate the constituents of a mixture containing ions or polar molecules (molecules with opposite charges on each pole), for example, amino acids.
Before elaborating on IEC, it is essential first to understand the basics of chromatography. In any chromatography, the fundamental process is the same; a mixture is dissolved into a mobile phase or solvent, which has the capability of containing the mixture without altering its properties.
This mobile phase moves over the stationary phase. The mobile phase is usually a liquid or semi-solid, or even gaseous, capable of mobility over the stationary phase. During this movement, the mixture’s properties and the mobile phase interact with the stationary phase, causing a reaction.
This reaction results in the separation of the various constituents of the compound at different intervals. This difference is because each component reacts differently with the stationary phase.
This process is the underlying principle of every chromatography technique. Depending on the particles, the procedure becomes more complex.
UNDERSTANDING ION EXCHANGE CHROMATOGRAPHY
The mobile phase in IEC is an eluent, and the stationary phase an ion exchanger resin. The principles of IEC rely on the exchange of ions between the mixture and the resin. The stationary phase contains ligands, which form an immobile matrix within the resin.
When the mixture moves through these ligands, the matrix causes an electrostatic interaction between the components and the resin, resulting in ion exchange. These ligands contain opposite charges to that of the eluent and the mixture, which helps neutralize the latter.
This neutralization occurs because the element consists of exchangeable counterions. The counterions and the ionizable particles in the mixture’s components compete to bind with the stationary phase’s matrix charges.
The particles which bind weakly or do not bind sufficiently wash away first. Therefore, just like basic chromatography, the compound separates into its various components along the resin column at different intervals.
The eluent helps the compound travel over the immobile matrix and provides the perfect environment for the components to separate. While this principle explains the basics of IEC, the technique is versatile enough for various other uses.
Laboratory technicians and scientists also use it to separate a particular component from a complex compound. But to understand how that works, one has to be familiar with the concept of an isoelectric point.
An isoelectric point (also referred to as pH) refers to a state or point wherein an atom is no longer carrying an electrical charge. The pH of an atom changes or becomes neutral when introduced to an environment which increases its proton or electron count.
When introduced to such a carefully calibrated environment, this increase results in the neutralization of the ion. This neutralization process is what occurs during the IEC process. Scientists have to calibrate the eluent such that its pH is one unit more/less than that of the compound.
Usually, technicians and scientists choose the most suitable eluent for the particular compound based on its pH. But they can also calibrate the eluent’s pH such that only a particular component gets released/separated from the mixture.
Thus, by making adjustments in the eluent, they have complete control over the chromatography process.
Categorization of Ion Exchange Chromatography
Scientists and technicians can make adjustments to the eluent or change the type of immobile phase to suit their experiment. Categorizing the different IEC techniques depends on the adjustments or changes they make during or before the process.
One method is gradient elution, which calls for the gradual change in the concentration of counterions in the eluent. This process elutes the components at different intervals or can also elute a single component. Another method would be to change the number or concentration of counterions in each step of the process, known as step elution.
Conversely, changing the resin or the stationary phase is also possible in two ways. These different procedures are subdivisions of the step elution IEC process.
One method is cation exchange chromatography which uses resin-containing cations that make positive ions in the molecules bind with the resin. The alternative is anion exchange chromatography, negatively charged ions bind with the resin containing anions.
