Biochemical Techniques and Instrumentation

2.4.7. Elution techniques

The ligand bound molecule can be recovered by different elution techniques. Elution technique involves mainly competitive elution, in which the ligand bound molecule is recovered in the presence of a competing agent.

Elution agents

1. Ligand competition: Addition of free ligand to displace the adsorbed molecule form the immobilized matrix. This is a highly specific technique but highly expensive. Use of co-substrate has therefore proven to be the most efficient and inexpensive type of elution.
2. pH change: pH manipulation is the most popular technique for dissociation of ligand bound molecule. The pH of the running buffer are changed to acid range of 1-1.5 in a gradient manner using citric, formic and acetic acids. Silica support is sensitive to sharp pH changes. Alkaline treatment is used for low pressure affinity but has greater denaturing effects.
3. Chaotropic ion elution: There are several salts that have chaotropic characteristics. They are CCl3COO-, SCN-, CF3COO-, ClO4-, I-, Cl- and used in concentrations between 1.5 and 8 M. The most widely used chaotropic ion is sodium thiocyanate at 3 M concentrations.They cause some denaturation to protein ligands and substrates.

Elution techniques:

There are two basic techniques for recovering the molecules bound to ligands. They are step gradient and linear gradient elution.

1. Step Gradient Elution: In step gradient elution, the recovery of bound molecule is achieved by a series of changes. It is easy to perform but causes loss of activity in the recovered molecule.

2. Linear Gradient Elution: This is the most efficient method for the recovery of bound molecules from the ligands. Gradient making instrument that introduce the elution buffer in a linear concentration gradient is used.

Another classification of elution technique is specific and non-specific elution.

1. Non-specific elution: Change of pH or ionic strength or polarity of the elution buffer recovers the bound molecules form the ligands. Change in pH elution using dilute acetic acid or ammonium hydroxide causes elution due to the ionization of groups in ligand and molecule. Change in ionic strength with or without concomitant change in pH causes elution due to a disruption of the ligand-molecule interaction.

2. Specific elution: Specific elution is achieved by the use of a soluble competitive ligand. A preliminary knowledge on the structure and biological specificity of the molecule is required to choose the competitive ligand. eg. In case of an enzyme, the ligand can be a competitive reversible inhibitor or an allosteric modifier.