Affinity Chromatography

What is Affinity Chromatography?

Affinity chromatography is a chromatographic method of separating biochemical mixtures, based on a highly specific biologic interaction such as that between antigen and antibody, enzyme and substrate, or receptor and ligand.

Principle Of Affinity Chromatography

Affinity chromatography involves the covalent attachment of an immobilized biochemical (called an affinity ligand) to a solid support. When the sample is passed through the column, only the solute that selectively binds to the complementary ligand is retained.The other sample components elute with out retention. The separation is well expressed by lock and key binding. The retained solute(s) can be eluted from the column by changing the mobile phase conditions. The major advantage of this technique is its tremendous specificity, which permit rapid isolation with a good yield in a single step.

Principle:-

Components Of Affinity Chromatography

Ligand:It uses a specific ligand, that has been immobilized chemically to an insoluble matrix to adsorb reversibly a single molecule species (affine components) from a mixture of solutes. It exploits the unique property of extremely specific reversible biological interaction to achieve separation an purification. The separation exploit Lock n Key binding that prevalent in biological system.

Ligands
It is of two types: Monospecific Group specific Monospecific:- binds only to one specific antibody/ antigen Disadvantages: It has a high effectivity so special ligand matrix combination is require for every substance to be purified. Bind more strongly and require harsh elutents than group specific. Eg. Steroid hormone, vitamins, certain enzyme inhibitor

Group Specific
It binds to specific group on target species. Imp. For the elucidation of the molecule. It is possible to immobilized the ligand via different chemical groups of the ligand.one can identify those structures that are essential for binding by means of their adsorption behaviour.

Choice Of Ligand or properties Of Ligand

Specifically form a reversible complex with the substance to be purified. A chemically modified group through which the covalent linkage to matrix can occur should be present. Form stable complex on binding to substance to be purified. Shd be able to withstand harsh condition used during process. Easy to dissociate the complex by simple change in medium without by adversely affecting the component. Consistently available and economical.

Examples of ligands

Spacer
For a ligand with low molecular wt. it is usually necessary for stearic reasons that it should not be bound directly to the matrix but via a side chain called a Spacer. Problem rarely occur with high mol.wt compounds.

How Spacer is introduce?

Two possibilities: One end of spacer is 1st bound to matrix and then the biological ligand attached to other end of spacer using various methods for matrix fixation. The spacer can 1st bound to ligand as a substitutes and then other end of spacer can fixed to the matrix.

Spacer
Spacer arms are aliphatic, linear hydrocarbon chain with two functional groups one at each end of the chain, one group is attached to matrix and other to ligand. It is used when active site located deep within the sample molecule. If too long it can interact with sample species on its own. If too short it cant reach active site on sample molecule. Eg. Hexamethelene diamine, 6-amino hexanoic acid,

Support or Matrix

Properties of matrix:It shd be of adequate size and shape i.e increase in particle size reduces the flow resistance and separation power & decrease in size leads to increase flow resistance and clogged. Irregular shape leads to unequal path lenghths for substance and band brodening.So spherical shape is best suited. Mechanically and chemically stable and resistance against microorganism. It shd be inert to prevent analyte from interacting nonspecifically with matrix itself. Economical Eg. Agarose Cellulose dextran sepharose is a bead formed of agarose gel.

Sample introduction

Adsorption

Washing

Elution

Elution Methods
(1) pH elution method (2) Ionic strength changing method (3) Competitive agents elution (4) Reduced polarity of eluent

(1) pH elution method

most common techniques that are used to remove bounded protein from the ligands. A change in pH alters the charged groups on the ligands and/or the bound protein. altering in conformation of proteins. A sudden decrease in pH is one of the most common methods to elute bounded proteins. The chemical stability of the ligand and target proteins determines the limitation of pH change.

(2) Ionic strength changing method

Changing ionic strength of buffer solution will alter the specific interaction between the ligand and target protein. This method is a mild elution using a buffer with increased ionic strength.

(3) Competitive agents elution

The eluting agent competes either for binding to the target protein or for binding to the ligand. This is an example of competitive inhibitors that occur in nature. Selective eluents are often utilized to separate substances on a specific medium

(4) Reduced polarity of eluent

Conditions are used to lower the polarity of the eluent promote elution without inactivating the proteins. Dioxane or ethylene glycol are typical of this type of eluent.

Applications

Antibodies :- antigen is immobilized and used to isolate and purify the appropriate antibody. Receptor protein:- hormones act on cells of specific membrane receptor and can be used to purify receptor from cell homogenates. Isolation of insulin receptor of liver cell. Enzymes:- Substrate inhibitors can be identified. Purify and concentrate a molecule from a mixture into a buffering solution Reduce the amount of a molecule in a mixture Discern what biological compounds bind to a particular molecule, such as drugs

Advantages

Specific adsorption can increase the purity by 1000 folds. No. of purification steps are reduced. The species are concentrated. The species are stabilized on binding. Rapid process. No loss of protein due to denaturation. Suitable for large scale purification processes.