Affinity Chromatography

Affinity Chromatography

The goal of affinity chromatography is to separate all the molecules of a particular specificity from the whole gamut of molecules in a mixture such as a blood serum. For example, the antibodies in a serum sample specific for a particular antigenic determinant can be isolated by the use of affinity chromatography.

Step 1.

An immunoadsorbent is prepared. This consists of a solid matrix to which the antigen (shown in blue) has been coupled (usually covalently). Agarose, sephadex, derivatives of cellulose, or other polymers can be used as the matrix.

Step 2.

The serum is passed over the immunoadsorbent. As long as the capacity of the column is not exceeded, those antibodies in the mixture specific for the antigen (shown in red) will bind (noncovalently) and be retained. Antibodies of other specificities (green) and other serum proteins (yellow) will pass through unimpeded.

Step 3.

Elution. A reagent is passed into the column to release the antibodies from the immunoadsorbent. Buffers containing a high concentration of salts and/or low pH are often used to disrupt the noncovalent interactions between antibodies and antigen. A denaturing agent, such as 8 M urea, will also break the interaction by altering the configuration of the antigen-binding site of the antibody molecule.

Another, gentler, approach is to elute with a soluble form of the antigen. These compete with the immunoadsorbent for the antigen-binding sites of the antibodies and release the antibodies to the fluid phase.

Step 4.

Dialysis. The eluate is then dialyzed against, for example, buffered saline in order to remove the reagent used for elution.

Separations Based on Charge: Ion-Exchange Chromatography [IEC]For separations based on polarity, like is attracted to like and opposites may be repelled. In ion-exchange chromatography and other separations based upon electrical charge, the rule is reversed. Likes may repel, while opposites are attracted to each other. Stationary phases for ion-exchange separations are characterized by the nature and strength of the acidic or basic functions on their surfaces and the types of ions that they attract and retain. Cation exchange is used to retain and separate positively charged ions on a negative surface. Conversely, anion exchange is used to retain and separate negatively charged ions on a positive surface [see Figure T]. With each type of ion exchange, there are at least two general approaches for separation and elution.

Figure T: Ion-Exchange ChromatographyStrong ion exchangers bear functional groups [e.g., quaternary amines or sulfonic acids] that are always ionized. They are typically used to retain and separate weak ions. These weak ions may be eluted by displacement with a mobile phase containing ions that are more strongly attracted to the stationary phase sites. Alternately, weak ions may be retained on the column, then neutralized by in situ changing the pH of the mobile phase, causing them to lose their attraction and elute.

Weak ion exchangers [e.g., with secondary-amine or carboxylic-acid functions] may be neutralized above or below a certain pH value and lose their ability to retain ions by charge. When charged, they are used to retain and separate strong ions. If these ions cannot be eluted by displacement, then the stationary phase exchange sites may be neutralized, shutting off the ionic attraction, and permitting elution of the charged analytes.