Anion-exchange chromatography
Acronym | HPAE Size exclusion chromatography Micellar liquid chromatography |
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Anion-exchange chromatography is a process that separates substances based on their charges using an
levels. The tightness of the binding between the substance and the resin is based on the strength of the negative charge of the substance.General technique for protein purification
A slurry of resin, such as DEAE-Sephadex is poured into the column. The matrix that is used is insoluble with charged groups that are covalently attached. These charged groups are referred to as exchangers like cation and anion exchangers. After it settles, the column is pre-equilibrated in buffer before the protein mixture is applied. DEAE-Sephadex is a positively-charged slurry that will have electrostatic interactions with the negatively charged atoms, making them elute later than the positively-charged molecules in the interested sample. This is a separation technique used widely to discover specific proteins, or enzymes in the body.
The separation of proteins will depend on the differences in total charge. Composition of ionizable side chain groups will determine the total charge of the protein at a particular pH. At the isoelectric point (pI), the total charge on the protein is 0 and it will not bind to the matrix. If the pH is above the pI, the protein will have a negative charge and bind to the matrix in an anion exchange column. The stability of the protein at values above or below the pI, will determine if an anion exchange column or cation exchange column should be used. If it is stable at pH values below the pI, the cation exchange column be used. If it is stable at pH values above the pI then the anion exchange column can be used.[7]
References
- PMID 2281856.
- ^ PMID 7300338.)
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: CS1 maint: multiple names: authors list (link - .
- PMID 24674065.
- ^ "FPLC Ion Exchange and Chromatofocusing: Principles and Methods". Pharmacia Biotech. Pharmacia Biotech AB. 1985.
- ^ Guide to Ion-Exchange Chromatography. Harvard Apparatus. p. 2.
- ^ Guide to Ion-Exchange Chromatography. Harvard Apparatus. p. 3.