Covalent Bonding

A basic requirement of all chromatographic media is the need for absolute stability under all operational conditions through many cycles of use. Consequently all ligands must be covalently bonded onto the matrix, and various chemistries are available to achieve this. A number of factors are involved:

1. The performance of both ligand and matrix are not impaired as a result of the coupling process.

2. Most of the coupled ligand is easily accessible to the ligate.

3. Charged or hydrophobic groups are not generated on the matrix, so reducing nonspecific adsorption.

4. The immobilized ligand concentration is optimal for ligate bonding.

5. There is no leakage of immobilized ligand from the matrix.

Some ligands are intrinsically reactive (or can be designed to be so) and contain groups that can be coupled directly to the matrix, but most require coupling via a previously activated matrix. The affinity matrix selected must have an adequate number of appropriate surface groups onto which the ligand can be bonded. The most common surface group is hy-droxyl. The majority of coupling methods involve the activation of this group by reacting with entities containing halogens, epoxy or carbonyl functional groups. These surface residues are then coupled to ligands through primary amines, hydroxyls or thiol groups, listed in Table 3.

Polysaccharides, represented by agarose, have a high density of surface hydroxyl groups. Tradition still dictates that this surface is activated by cyanogen bromide, but it is well established that this reagent forms pH-unstable iso-urea linkages, resulting in a poorly performing product. Furthermore CNBr-activated agarose needs harsh coupling conditions if high yields of final media are to be obtained, suggesting high wastage of often expensive ligands. This factor is particularly evident with fragile entities such as the very-expensive-to-produce antibodies, and yet many workers simply read previous literature and make no attempt to examine alternative far superior coupling methods. The advantages of mild coupling regimes are demonstrated in Figure 2, where the use of a triazine-activated agarose is compared to CNBr-activated agarose. Yield is significantly increased, largely by coupling under acidic rather than alkaline conditions.

Solar Panel Basics

Solar Panel Basics

Global warming is a huge problem which will significantly affect every country in the world. Many people all over the world are trying to do whatever they can to help combat the effects of global warming. One of the ways that people can fight global warming is to reduce their dependence on non-renewable energy sources like oil and petroleum based products.

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