Ion chromatography

Ion exchange chromatography

Acronym	IC, IEC
Classification	Chromatography
Other techniques
Related	High performance liquid chromatography Aqueous Normal Phase Chromatography Size exclusion chromatography Micellar liquid chromatography

Ion-exchange chromatography (or ion chromatography) is a chromatography process that separates ions and polar molecules based on their affinity to the ion exchanger. It works on almost any kind of charged molecule—including large proteins, small nucleotides, and amino acids. It is often used in protein purification, water analysis, and quality control. The water-soluble and charged molecules such as proteins, amino acids, and peptides bind to moieties which is oppositely charged by forming covalent bonds to the insoluble stationary phase.^[1] The equilibrated stationary phase consists of an ionizable functional group where the targeted molecules of a mixture to be separated and quantified can bind while passing through the column. This method applies the idea of the interaction between molecules and the stationary phase which are charged oppositely to each other. Cation exchange chromatography is used when the desired molecules to separate are cations, and an anion exchange chromatography is to separate anions meaning that the beads in the column contain positively charged functional groups to attract the anions.^[2] The bound molecules then can be eluted and collected using an eluant which contains anions and cations by running higher concentration of ions through the column or changing pH of the column.

History

Ion exchange chromatography

The boom of Ion exchange chromatography primarily began between 1935-1950 and it was through the "Manhattan project" that applications and IC were significantly extended. It was in the fifties and sixties that theoretical models were developed for IC for further understanding and it was not until the seventies that continuous detectors were utilized, paving the path for the development from low-pressure to high-performance chromatography. Not until 1975 was "ion chromatography" established as a name in reference to the techniques, and was thereafter used as a name for marketing purposes. Today IC is important for investigating aqueous systems, such as drinking water. It is a popular method for analyzing anionic elements or complexes that help solve environmentally relevant problems. Likewise, it also has great uses in the semiconductor industry.

Because of the abundant separating columns, elution systems, and detectors available, chromatography has developed into the main method for ion analysis.^[3]

Principle

Ion Chromatography

Ion-exchange chromatography separates molecules based on their respective charged groups. Ion-exchange chromatography retains analyte molecules on the column based on coulombic (ionic) interactions. Essentially, molecules undergo electrostatic interactions with opposite charges on the stationary phase matrix. The stationary phase consists of ionizable functional groups combined to a matrix material that is immobile.^[4] The stationary phase surface displays ionic functional groups (R-X) that interact with analyte ions of opposite charge. To achieve electroneutrality, these inert charges couple with exchangeable counterions in the solution. Ionizable molecules that are to be purified compete with these exchangeable counterions for binding to the immobilized charges on the stationary phase. These ionizable molecules are retained or eluted based on their charge. Initially, molecules that do not bind or bind weakly to the stationary phase are first to wash away. Altered conditions are needed for the elution of the molecules that bind to the stationary phase. The concentration of the exchangeable counterions, which competes with the molecules for binding, can be increased or the ph can be changed. A change in ph changes the charge on the particular molecules and, therefore, alters binding. The molecules then start eluting out based on the changes in their charges from the adjustments. Further such adjustments can be used to release the protein of interest. Additionally, concentration of counterions can be gradually varied to separate ionized molecules. This type of elution is called gradient elution. On the other hand, step elution can be used in which the concentration of counterions are varied in one step.^[5] This type of chromatography is further subdivided into cation exchange chromatography and anion-exchange chromatography. The ionic compound consisting of the cationic species M+ and the anionic species B- can be retained by the stationary phase.

Cation exchange chromatography retains positively charged cations because the stationary phase displays a negatively charged functional group:

$\text{R-X}^-\text{C}^+\,+\, \text{M}^+ \, \text{B}^- \rightleftarrows \,\text{R-X}^-\text{M}^+ \,+\, \text{C}^+ \,+\, \text{B}^-$

Anion exchange chromatography retains anions using positively charged functional group:

$\text{R-X}^+\text{A}^-\,+\, \text{M}^+ \, \text{B}^- \rightleftarrows \,\text{R-X}^+\text{B}^- \,+\, \text{M}^+ \,+\, \text{A}^-$

Note that the ion strength of either C+ or A- in the mobile phase can be adjusted to shift the equilibrium position, thus retention time.

The ion chromatogram shows a typical chromatogram obtained with an anion exchange column.

Weak and strong ion exchangers

A "strong" ion exchanger will not lose the charge on its matrix once the column is equilibrated and so a wide range of pH buffers can be used. "Weak" ion exchangers have a range of pH values in which they will maintain their charge. If the pH of the buffer used for a weak ion exchange column goes out of the capacity range of the matrix, the column will lose its charge distribution and the molecule of interest may be lost.^[6] Despite the smaller pH range of weak ion exchangers, they are often used over strong ion exchangers due to their having greater specificity. In some experiments, the retention times of weak ion exchangers are just long enough to obtain desired data at a high specificity.^[7]

Resins of ion exchange columns may include functional groups such as weak/strong acids and weak/strong bases. There are also special columns that have resins with amphoteric functional groups that can exchange both cations and anions.^[8] Examples of functional groups of strong ion exchange resins are quaternary ammonium (Q), which is an anion exchanger, and sulfonic acid (S), which is a cation exchanger. These types of exchangers can maintain their charge density over a pH range of 0-14. Examples of functional groups of Weak ion exchange resins include diethylaminoethyl (DEAE), which is an anion exchanger, and carboxymethyl (CM), which is a cation exchanger. These two types of exchangers can maintain the charge density of their columns over a pH range of 5-9.^[9][10]

Typical technique

Metrohm 850 Ion chromatography system

A sample is introduced, either manually or with an autosampler, into a sample loop of known volume. A buffered aqueous solution known as the mobile phase carries the sample from the loop onto a column that contains some form of stationary phase material. This is typically a resin or gel matrix consisting of agarose or cellulose beads with covalently bonded charged functional groups. Equilibration of the stationary phase is needed in order to obtain the desired charge of the column. If the column is not properly equilibrated the desired molecule may not bind strongly to the column. The target analytes (anions or cations) are retained on the stationary phase but can be eluted by increasing the concentration of a similarly charged species that displaces the analyte ions from the stationary phase. For example, in cation exchange chromatography, the positively charged analyte can be displaced by adding positively charged sodium ions. The analytes of interest must then be detected by some means, typically by conductivity or UV/visible light absorbance.

Control an IC system usually requires a chromatography data system (CDS). In addition to IC systems, some of these CDSs can also control gas chromatography (GC) and HPLC.

Separating proteins

Preparative-scale ion exchange column used for protein purification.

Proteins have numerous functional groups that can have both positive and negative charges. Ion exchange chromatography separates proteins by their net charge, which depends on the composition of the mobile phase. By adjusting the pH or the ionic concentration of the mobile phase, various protein molecules can be separated. For example, if a protein has a net positive charge at pH 7, it binds to a column of negatively charged beads—whereas a negatively charged protein does not. By changing the pH so that the net charge on the protein is negative, it too is eluted.

Elution by increasing ionic strength of the mobile phase is more subtle. It works because ions from the mobile phase interact with the immobilized ions on the stationary phase, thus "shielding" the stationary phase from the protein, and letting the protein elute.

Separation can be achieved based on the natural isoelectric point of the protein. Alternatively a peptide tag can be genetically added to the protein to give the protein an isoelectric point away from most natural proteins (e.g., 6 arginines for binding to a cation-exchange resin or 6 glutamates for binding to an anion-exchange resin such as DEAE-Sepharose).

Elution from ion-exchange columns can be sensitive to changes of a single charge- chromatofocusing. Ion-exchange chromatography is also useful in the isolation of specific multimeric protein assemblies, allowing purification of specific complexes according to both the number and the position of charged peptide tags.^[11][12]

Uses

Clinical utility

Detection limits as low as 1 μM can be obtained for alkali metal ions.^[13] It may be used for measurement of HbA1c, porphyrin and with water purification. Ion Exchange Resins(IER) have been widely used especially in medicines due to its high capacity and the uncomplicated system of the separation process. One of the synthetic uses is to use Ion Exchange Resins for kidney dialysis. This method is used to separate the blood elements by using the cellulose membraned artificial kidney.^[14]

Industrial applications

Allows for quantitative testing of electrolyte and proprietary additives of electroplating baths.^[15] It is an advancement of qualitative hull cell testing or less accurate UV testing. Ions, catalysts, brighteners and accelerators can be measured.^[15]

See also

• Isoelectric point
• High performance liquid chromatography
• Chromatofocusing
• Anion-exchange chromatography

References

1. Luqman, Mohammad, and Inamuddin. Ion Exchange Technology II. N.p.: Springer Netherlands, 2012. Print. p.1
2. Fritz, James S. “Ion Chromatography.” Analytical Chemistry Anal. Chem. 59.4 (1987): n. pag. Web.
3. Eith, Claudia, Kolb Maximilian, and Seubert Andreas. "Introduction." Practical Ion Chromatography An Introduction. Ed. Viehweger Kai. Herisau: Metrohm, 2002. 160.
4. , Acikara, Özlem Bahadir. "Chapter 2." Ion-Exchange Chromatography and Its Applications. 10 Apr. 2013. Web. 02 May 2016
5. , Ninfa, Alexander J., David P.Ballou, and Marilee Benore. Fundamental Laboratory Approaches for Biochemistry and Biotechnology. Hoboken, NJ: John Wiley, 2010. Print
6. Appling, Dean; Anthony-Cahill, Spencer; Mathews, Christopher (2016). Biochemistry: Concepts and Connections. New Jersey: Pearson. p. 134. ISBN 9780321839923.
7. Alpert, Andrew J.; Hudecz, Otto; Mechtler, Karl (2015-05-05). "Anion-Exchange Chromatography of Phosphopeptides: Weak Anion Exchange versus Strong Anion Exchange and Anion-Exchange Chromatography versus Electrostatic Repulsion–Hydrophilic Interaction Chromatography". Analytical Chemistry 87 (9): 4704–4711. doi:10.1021/ac504420c. ISSN 0003-2700. PMC 4423237. PMID 25827581.
8. Dragan, E. S.; Avram, E.; Dinu, M. V. (2006-07-01). "Organic ion exchangers as beads. Synthesis, characterization and applications". Polymers for Advanced Technologies 17 (7-8): 571–578. doi:10.1002/pat.755. ISSN 1099-1581.
9. "Ion Exchange Chromatography Selection Guide". www.pall.com.
10. "Ion Exchange Chromatography". www.bio-rad.com.
11. Sakash, J.B.; Kantrowitz, E.R. (2000). "The contribution of individual interchain interactions to the stabilization of the T and R states of Escherichia coli aspartate transcarbamoylase.". J Biol Chem 275 (37): 28701–7. doi:10.1074/jbc.M005079200. PMID 10875936.
12. Fairhead, M. (2013). "Plug-and-Play Pairing via Defined Divalent Streptavidins.". J Mol Biol 426 (1): 199–214. doi:10.1016/j.jmb.2013.09.016. PMID 24056174.
13. Hauser, Peter C. (2016). "Chapter 2. Determination of Alkali Ions in Biological and Environmental Samples". In Astrid, Sigel; Helmut, Sigel; Roland K.O., Sigel. The Alkali Metal Ions: Their Role in Life. Metal Ions in Life Sciences 16. Springer. pp. 11–25. doi:10.1007/978-3-319-21756-7_2.
14. Luqman, Mohammad, and Inamuddin. Ion Exchange Technology II. N.p.: Springer Netherlands, 2012. Print. p.169
15. Robert E. Smith (31 December 1987). Ion Chromatography Applications. CRC Press. ISBN 978-0-8493-4967-6.

Bibliography

• Small, Hamish (1989). Ion chromatography. New York: Plenum Press. ISBN 0-306-43290-0.
• Tatjana Weiss; Weiss, Joachim (2005). Handbook of Ion Chromatography. Weinheim: Wiley-VCH. ISBN 3-527-28701-9.
• Gjerde, Douglas T.; Fritz, James S. (2000). Ion Chromatography. Weinheim: Wiley-VCH. ISBN 3-527-29914-9.
• Jackson, Peter; Haddad, Paul R. (1990). Ion chromatography: principles and applications. Amsterdam: Elsevier. ISBN 0-444-88232-4.

External links

• Ion Exchange Chromatography Handbook
• LC Instruments at DMOZ