|Related||High performance liquid chromatography
Aqueous Normal Phase Chromatography
Size exclusion chromatography
Micellar liquid chromatography
Ion-exchange chromatography (or ion chromatography) is a process that allows the separation of ions and polar molecules based on their affinity to the ion exchanger. It can be used for almost any kind of charged molecule including large proteins, small nucleotides and amino acids. The solution to be injected is usually called a sample, and the individually separated components are called analytes. It is often used in protein purification, water analysis, and quality control.
Ion-exchange chromatography retains analyte molecules on the column based on coulombic (ionic) interactions. The stationary phase surface displays ionic functional groups (R-X) that interact with analyte ions of opposite charge. 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:
Anion exchange chromatography retains anions using positively charged functional group:
Note that the ion strength of either C+ or A- in the mobile phase can be adjusted to shift the equilibrium position and thus retention time.
The ion chromatogram shows a typical chromatogram obtained with an anion exchange column.
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. 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 will displace the analyte ions from the stationary phase. For example, in cation exchange chromatography, the positively charged analyte could be displaced by the addition of positively charged sodium ions. The analytes of interest must then be detected by some means, typically by conductivity or UV/Visible light absorbance.
Proteins have numerous functional groups that can have both positive and negative charges. Ion exchange chromatography separates proteins according to their net charge, which is dependent 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, then it will bind to a column of negatively charged beads, whereas a negatively charged protein would not. By changing the pH so that the net charge on the protein is negative, it too will be eluted.
Elution by increasing the ionic strength of the mobile phase is a more subtle effect - it works as ions from the mobile phase will interact with the immobilized ions in preference over those on the stationary phase. This "shields" the stationary phase from the protein, (and vice versa) and allows the protein to 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 cation-exchange resin such as DEAE-Sepharose or 6 glutamates for binding to anion-exchange resin).
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.
Allows for quantitative testing of electrolyte and proprietary additives of electroplating baths. It is an advancement of qualitative hull cell testing or less accurate UV testing. Ions, catalysts, brighteners and accelerators can be measured.
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- Robert E. Smith (31 December 1987). Ion Chromatography Applications. CRC Press. ISBN 978-0-8493-4967-6.
||This article includes a list of references, but its sources remain unclear because it has insufficient inline citations. (March 2010)|
- 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.
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