Anion exchange chromatography is a type of ion exchange chromatography. This article is the second part of our articles on ion exchange chromatography. By now, we know that ion exchange chromatography is a chromatographic separation and purification tool for charged species.
Previously, we learnt cation exchange chromatography which allows targeted separation of positively charged species called cations from a complex mixture by packing the chromatographic column with a cation exchanger. So, you may have guessed by its name that anion exchange chromatography contrarily allows chromatographic analysis of negatively charged ions called anions.
What is anion exchange chromatography
Anion exchange chromatography is performed by packing the column with an anion exchange resin as the stationary phase. Negatively charged species from the analyte mixture get retained onto this stationary phase, replacing the labile negative charges initially held to it by electrostatic forces of attraction. Buffers of varying pH and concentration and/or salt solutions are then used as a mobile phase to elute the targeted anions out of the column.
Stationary phase in anion exchange chromatography
Similar to what was discussed for cation exchange chromatography, covalently anchored support surfaces such as a cellulose based resin is used for column packing in anion exchange chromatography. But in this case, positively charged species are covalently bonded to this resin while the oppositely charged anions are held close to these positive charges by electrostatic forces of attraction.
In accordance with this, the extended cellulose carbons, we talked about in cation exchange chromatography, are treated with functional groups such as a quaternary ammonium group (triethyl amino ethyl) and/or a tertiary amine (diethylamino ethyl). A quaternary ammonium group (-CH2NR3+) is strongly basic in nature so it forms a strong anion exchanger which can bear highly alkaline conditions upto pH 13-14. Meanwhile, a tertiary ammonium group (-CH2NHR2+) gets deprotonated above pH 10 that means the stationary phase will lose its permanent charge above this pH, in turn losing its ion exchange property. Therefore, tertiary ammonium groups form weak anion exchange resins.
In this way, we are once again reminded of the integral role of pH monitoring while performing ion exchange chromatography. As a general rule of thumb, the pH of the buffer solutions is increased for elution purposes in cation exchange chromatography while their pH is gradually decreased for analyte elution from an anion exchange column. A cation exchanger is acidic in nature thus it liberates the sample ions on increasing the pH upto a basic level. In contrast, an anion exchanger is basic in nature henceforth it liberates the retained sample ions on decreasing the pH inside the column.
How to perform anion exchange chromatography
Anion exchange chromatography is performed using the following set of steps:
- Column Packing: The column is packed with an anion exchange resin as discussed above.
- Equilibration: The mobile phase buffer is passed through the column to remove any trace impurities and/or unwanted ions from the stationary phase packing.
- Sample loading: A small quantity of the sample containing the charged species is loaded from the top of the column. Anions from the sample replace the exchangeable ions present onto the column and get attached to the resin by opposite charge attraction.
- Washing: Displaced anions, sample cations and any other ions stuck onto the stationary phase are removed by a subsequent washing step with the same buffer as used while equilibration.
- Elution: The targeted anions are eluted out of the column by decreasing the pH of the mobile phase and/or by using a salt solution containing anions that can displace and replace the targeted anions. Both an isocratic as well as a gradient mode of elution can be used.
All the other details on how to perform anion exchange chromatography are exactly as we learnt for cation exchange chromatography.
For the concept of chromatographic elution and other such details, we recommend our article: high-performance liquid chromatography (HPLC).
Where do we need anion exchange chromatography
Anion exchange chromatography is particularly useful for:
- Separation of complex mixtures: Complex mixtures such as sugar nucleotides, monosaccharides and proteins can be separated by anion exchange chromatography. Nucleotides are negatively charged species therefore they get retained onto an anion exchanger.
The hydroxyl groups present in carbohydrates can also form anionic complexes. Similarly, proteins contain negatively charged amino acids such as glutamic acid and aspartic acid exhibit negative charges in their ionized forms. Once retained onto the anion exchanger, the pH inside the column can be decreased to re-protonate them and liberate the targeted compounds from the column.
- Water purification: Ionized water containing hydroxyl (OH–) functional groups can be deionized on passing it through an anion exchanger.
Conclusion:
Both types of ion exchange chromatography are specifically valuable for the chromatographic analysis of macromolecules. One restriction associated with this very important chromatographic type however is that it can only analyze charged species.
Watch a video tutorial on ion exchange chromatography here.
Do you know ?
Ion exchange chromatography is an ”adsorption” type of chromatography. We talked about different types of chromatographies in: what is chromatography.
Q.1) What is the other main adsorption type of chromatography?
Ans: Well, it is thin-layer chromatography (TLC).
Q.2) What is the difference between adsorption in ion exchange and that in TLC?
Ans: Ion exchange chromatography allows ionic adsorption on stationary phase by electrostatic forces of attraction. While for TLC? Read it for yourself here: Thin-layer chromatography.
References
1. Jungbauer, A. and R. Hahn (2009). Chapter 22 Ion-Exchange Chromatography. Methods in Enzymology. R. R. Burgess and M. P. Deutscher, Academic Press. 463: 349-371.
2. S.Hage, D. (2018). Chromatography Clinical chemistry and molecular diagnostics 266-294.
