IonIon exchange chromatography, alias ion chromatography, is an analytical chromatography technique used frequently to separate and identify ionic compounds.
It separates ionic and polar compounds based on their interaction with another ionic substance, typically known as an ion exchanger.
Not only for ionic solutions, but it can also be used to separate any charged molecule, e.g., amino acids, nucleic acids, and proteins.
There are two types of ion-exchange chromatography: anion- and cation-exchange chromatography. As can be implied from their names, cation exchange chromatography is used when the molecule of interest to be separated is a cation or positively charged molecule.
Quite logically, if the molecule to be separated contains a positive charge, then the ion exchanger should be of negative charge. Similarly, anion exchange chromatography is used for anions or negative ions, and the rest of the conditions are vice versa.
Although there are many pros associated with this technique, it has many applications; some cons are also relevant to ion-exchange chromatography. Let’s take a look at them one by one.
No Direct Information on Actual Events
Although being extensively used in various applications, the exact mechanism of this technique is still elusive. This leads to an important disadvantage: no direct information is obtained about what events are occurring during ion exchange with the resin or substrate used to separate substances.
This process basically depends on the equilibrium between the ions so that ions exchange can occur. This equilibrium is always determined by either the interaction of the solute with the resin (exchange phase) or the interaction of eluent (elusion phase).
Therefore, getting the exact information becomes impossible under such circumstances.
Microporous Beads Prevent Proteins’ Penetration
Different types of beads are used in ion-exchange chromatography, broadly classified as micro, macro, and non-porous. One disadvantage of using microporous beads is that they can hinder or slow down the penetration of molecules when used to separate proteins from other substances like carbohydrates.
The major reason is the binding of protein molecules and their adherence to the surface of the beads, which functions to prevent their further movement and penetration in the pores of the beads.
Such problems can be efficiently overcome by using macroporous beads of the size the molecule of interest can penetrate easily.
Ions at Low Concentrations Pose Preconcentration Problems
When ions have to be determined present in meager amounts or concentrations, then concentrating the samples before running ion-exchange chromatography becomes a prerequisite.
This is generally known as preconcentration or trace enrichment.
By this, samples are concentrated, resulting in volumes coming in the range of lower detection limits. However, this requires some additional efforts such as a concentrator column, additional valves for switching samples in and out of the columns, and most importantly, extra time is required to carry out all the processes of preconcentration.
Inconsistency in Columns
The ion-exchange chromatography technique is constantly evolving, leading to new columns in the market and industry day by day.
This leads to a major disadvantage associated with the technique that the new columns differ from the previous ones and produce quite an amount of inconsistency in the results.
As the new columns are developed containing new ionic groups bound to the stationary phase, ion-exchange columns are not always well established, leading to considerable irreproducibility of the results.
Another downside of ion-exchange chromatography is the price associated with its columns. These columns are typically much more expensive than the reverse phase chromatographic columns.
Moreover, reverse phase columns also present an additional advantage in that they are quite reliable and established because of their use for so many years now.
Keeping in view all the shortcomings associated with this technique, one should be cautiously selective for ion-exchange chromatography when adding this technique in their experimental system based on the molecule being separated and its final purpose for which the separation is being done.
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