*1.3.1. Noncovalent immobilization technique*

The simple adsorption of ligand to surface, binding to a secondary ligand, or ligand immobi‐ lization through a coordination complex can be classified as this type of immobilization. This technique can be subdivided as follow;

primary amines, but the cyanate ester is more reactive than the imidocarbonate. The CNBr method utilizes relatively mild conditions for ligand attachment, making it suitable for many sensitive biomolecules. But one problem with this approach is that the isourea linkages obtained by the reaction of CNBr with the support are positively charged at a neutral pH. This means that these groups can act as anion exchangers and nonspecific binding can be occur. Other problems with this method include the toxicity of CNBr, requiring the use of adequate safety precautions during the activation process, and the

Ligand-NH2

Ligand-NH2

pH 7.5 - 8.5

OH

Affinity Chromatography and Importance in Drug Discovery

O O

O-C-NH-Ligand

C = NH-Ligand

+NH

http://dx.doi.org/10.5772/55781

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leakage of ligands that can result from CNBr-activated supports (Figure 12).

O-C=N

pH 11 pH 7.5 - 8.5

O O

Imidocarbonate

C = NH

**b.** Reductive amination (also known as the Schiff base method); Reductive amination couples ligands with amine groups to activated periodate is used to oxidize diol groups on the support's surface to give aldehydes. This can be performed directly on carbohy‐ drate-based supports like dextran or cellulose. However, materials like silica or glass must first be treated to place diols on their surface. This can be accomplished by reacting the silica or glass with γ-glycidoxypropyltrimethoxysilane, followed by acid hydrolysis. When an amine-containing ligand reacts with the aldehyde groups, the resulting product is known as a Schiff base. Since this is a reversible reaction, the Schiff base must be converted into a more stable form. This is achieved by including sodium cyanoborohy‐ dride in the reaction mixture. Cyanoborohydride is a weak reducing agent that converts the Schiff base into a secondary amine without affecting the aldehydes on the support. After the coupling reaction is completed, the remaining aldehyde groups can be removed by treating the support for a short period of time with a stronger reducing agent (i.e., sodium borohydride) or by reacting these groups with an excess of a small aminecontaining agent (e.g., ethanolamine). The Schiff base method is relatively easy to perform

OH

Hydroxyl-containing

OH OH

**Figure 12.** Cyanogen bromide immobilization method patway [8]

matrix Cyanate ester

CNBr

