Anion-exchange chromatography is a process that separates substances based on their charges using an
ion-exchange resin
An ion-exchange resin or ion-exchange polymer is a resin or polymer that acts as a medium for ion exchange. It is an insoluble matrix (or support structure) normally in the form of small (0.25–1.43 mm radius) microbeads, usually white or ye ...
containing positively charged groups, such as diethyl-aminoethyl groups (DEAE).
In solution, the resin is coated with positively charged counter-ions (
cations
An ion () is an atom or molecule with a net electrical charge.
The charge of an electron is considered to be negative by convention and this charge is equal and opposite to the charge of a proton, which is considered to be positive by convent ...
). Anion exchange resins will bind to negatively charged molecules, displacing the counter-ion. Anion exchange chromatography is commonly used to purify
proteins
Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, respo ...
,
amino acids
Amino acids are organic compounds that contain both amino and carboxylic acid functional groups. Although hundreds of amino acids exist in nature, by far the most important are the alpha-amino acids, which comprise proteins. Only 22 alpha am ...
,
sugars
Sugar is the generic name for sweet-tasting, soluble carbohydrates, many of which are used in food. Simple sugars, also called monosaccharides, include glucose, fructose, and galactose. Compound sugars, also called disaccharides or double s ...
/
carbohydrates
In organic chemistry, a carbohydrate () is a biomolecule consisting of carbon (C), hydrogen (H) and oxygen (O) atoms, usually with a hydrogen–oxygen atom ratio of 2:1 (as in water) and thus with the empirical formula (where ''m'' may or may ...
and other acidic substances with a negative charge at higher
pH 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.
Unbound proteins are collected in the flow-through and/or in subsequent buffer washes. Proteins that bind to the positively charged resin are retained and can be
eluted
In analytical and organic chemistry, elution is the process of extracting one material from another by washing with a solvent; as in washing of loaded ion-exchange resins to remove captured ions.
In a liquid chromatography experiment, for exa ...
in one of two ways. First, the salt concentration in the elution buffer is gradually increased. The negative ions in the salt solution (e.g. Cl
−) compete with protein in binding to the resin. Second, the
pH of the solution can be gradually decreased which results in a more positive charge on the protein, releasing it from the resin. Both of these techniques can displace the negatively charged protein which is then eluted into test tubes fractions with the buffer.
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.
References
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Chromatography