Cation exchange chromatography is a type of ion exchange chromatography. Now what is ion-exchange chromatography? Well, this article is the right place to know everything you need to know about ion exchange chromatography and its sub-types.
As the name suggests, ion exchange chromatography relates to the exchange of ions where ions are charged species. These ions could be positively charged called cations and/or negatively charged ions called anions. Thus, ion exchange chromatography is particularly valuable for analytical separations concerning charged particles.
Depending upon the type of ions targeted for separation, different stationary phase packings are involved. Therefore, ion exchange chromatography has two main types: cation exchange chromatography and anion exchange chromatography. This article presents cation exchange chromatography to you in detail while a subsequent one will deal with all the information regarding anion exchange chromatography.
What is cation exchange chromatography
Cation exchange chromatography is a type of ion exchange chromatography in which the stationary phase packing in the chromatographic column consists of a cation exchange resin. Salt solutions or buffers of varying concentrations are used as mobile phases. Positively charged species from the analyte mixture gets retained onto the stationary phase, by replacing the labile positive charges already present on it. A denser positive charge from the mobile phase then replace these analyte components from the stationary phase, eluting them out of the column.
Stationary phase in cation exchange chromatography
The stationary phase packings in cation exchange columns usually consist of resins; the amorphous, non-crystalline substances that act as a support surface in the column. These surfaces are consequently coated with covalently anchored charged species. In case of a cation exchange resin, these species are negatively charged. Opposite charges attract thus labile, positively charged ions are held close to these negative charges by electrostatic forces of attraction. Cations having a higher positive charge from the analyte mixture can easily replace these exchangeable ions by developing a stronger force of attraction with the covalently bonded functional groups present on the resin. Thus, this resin acts as a cation exchanger.
One of the most reliable natural materials for these resin syntheses is cellulose. Cellulose is a polysaccharide; a polymer consisting of a large number of β-D-glucose monomers, condensed together via β-1,4-glycosidic linkages. Every second β-glucose monomer flips 180° in order to facilitate the polymer synthesis. Glucose is a 6-carbon compound that exists as a pyranose ring where the 6th carbon is exposed outside the ring, in a hanging position. This position can be efficiently exploited to covalently attach charged functional groups.
A sulfonate (-SO3–) group covalently attached to the cellulose resin forms a strong cation exchanger while a carboxyl (-COO–) functional group attached forms a weak cation exchanger. Strong cation exchangers can bear strongly acidic conditions for e.g., the -SO3– group stays ionized over a high pH range (2-14). Meanwhile, weak cation exchangers cannot tolerate extremely acidic pH conditions. A -COO– group gets protonated below pH 4, losing its cation exchange property. Knowing this, we should be able to understand how important a role pH plays in ion exchange chromatography.
You may find useful a general read on how to calculate pH here.
Strictly monitoring the pH conditions is important in order to keep the charges present on the resins intact i.e., maintaining the integrity of the stationary phase packing in an ion exchange column which in turn controls the overall chromatographic separation. Other than resins, porous gels are also sometimes used for performing ion exchange chromatography such as a dextrose, agarose or a polyacrylamide backbone-based gel.
How to perform cation exchange chromatography
Step I: Column packing
While packing the column with a cation exchange stationary phase, the following factors must be considered:
- Choice of exchanger
- Analyte stability
The column is packed similarly as for other column chromatographic methods. The type of packing material is as discussed in the above section. pH should be maintained within the stability range for both the exchanger as well as the analyte of interest.
Step II: Equilibration
The mobile phase buffer is passed through the column. This step ensures good column packing. Any excess ions stuck on the stationary phase get washed out of the column. A single, sharp peak must be observed at the recorder as a result of this step.
Step III: Sample loading and analyte separation
The analyte mixture is then loaded onto the stationary phase packed column. Cations from the analyte mixture displace the positively charged ions present on the cation exchanger by repulsion. The incoming cations from the sample mixture are in turn retained onto the cation exchanger by electrostatic forces of attraction. We must be aware of the fact that only readily exchangeable cations can get replaced while the negatively charged functional moieties stay intact via covalent bonding. Here the principle of ‘opposite attracts’ applies.
Step IV: Washing
The cation exchange column is once again washed with the buffer we talked about in step I. This ensures removal of any unwanted ions such as all the negatively charged ions/anions present in the analyte mixture as well as the displaced cations from the stationary phase all get eluted out with this buffer.
Step V: Targeted Elution
The buffer pH is then changed for the final elution process. The pH of the buffer is gradually increased for cationic elution in cation exchange chromatography while its pH is decreased for anionic elution in anion exchange chromatography. Both an isocratic as well as a gradient mode of elution can be applied.
Conversely a salt solution can also be used as an eluent in ion exchange chromatography. For instance, a high concentration of sodium (Na+)ions from NaCl solution can displace the analytical cations strongly retained onto the stationary phase by overcoming their forces of attraction with the cation exchanger. Na+ ions get attached onto the exchanger, while targeted cations repel each other and elute out of the column as shown in the figure below.
The eluate reaches the detector which sends a signal to the recorder and a chromatogram is plotted same as that for high-performance liquid chromatography (HPLC).
In other words, HPLC can be performed in both an ion exchange as well as in a size exclusion mode depending upon the stationary phase preparation and the purpose of analysis.
Cation exchange chromatography aids in both analytical as well as preparative separations. It is particularly useful for protein analysis and purification. It is also important for pre-concentration of trace compounds in an analytical mixture.
For more information on the uses of different types of chromatography, consult: what are the uses of chromatography?.
- 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. Kilislioglu, A. (2016). Ion Exchange Technologies.