Intermolecular Binding Forces

Almost all chromatographic separations rely upon the interaction of the target molecule with either a liquid phase or a covalently bonded molecule on the solid phase, the exceptions being those relying upon molecular size, e.g. molecular sieves and gel filtration. In affinity separations ligates are inevitably

Table 3 Activation materials

Activating reagent

Bonding group on ligand

Cyanogen bromide Primary amines

Tresyl chloride Primary amines,

Tosyl chloride Primary amines,

Epichlorohydrin Primary amines,

1,4-Butanediol diglycidyl ether Primary amines,

1,1 '-Carbonyldiimidazole Primary amines,

Cyanuric chloride Primary amines,

Divinylsulfone Primary amines, 2-Fluro-1-methylpyriinium-toluene-4-sulfonate Primary amines,

Sodium periodate Primary amines

Glutaraldehyde Primary amines thiols thiols hydroxyls, thiols hydroxyls, thiols hydroxyls hydroxyls hydroxyls thiols

Figure 2 Triazine coupling. (A) Coupling of human serum albumin (HSA) to ready-acitivated supports as a funciton of pH. (B) Time course of coupling of human IgG to ready-activated supports at 4°C. O, CNBr-activated agarose 4XL; ■, triazine-activated agarose 4XL.

complex biological macromolecules or assemblies, mostly or exclusively consisting of amino acids entities linked together in a specific manner. This complexity of structure provides many opportunities to exploit the physicochemical differences between the target molecule and the ligand to be used. Each structure contains the four basic intermolecular binding forces - electrostatic, hydrogen bonding, hydrophobic and van der Waals interactions - spread throughout the structure in an exactly defined spatial manner. The degree of accessibility and spatial presentation within the pore of the medium, and the strength of each force relative to each other, dictate whether these forces are utilized to effect the separation. The biological recognition between species is a reflection of the sum of the various molecular interactions existing between them, and this summation is fixed for the ligate. However, various ligands may be found that emulate some or all of the available binding forces to various degrees.

Affinity adsorbents are therefore assigned to one of three broad ligand categories: nonspecific, group specific or highly specific. Nonspecific ligands have only a superficial likeness to biological ligands and binding is usually effected by just one of the four binding processes described above. Although ion exchange materials can be used in a similar manner to affinity adsorbents, they only exhibit the single force of electrostatic binding. They are thus limited to relatively indiscriminate binding. In this case the only criterion for binding is that of an overall charge.

Fortunately there are a vast number of biological ligands that can interact with more than one macro-molecule and consequently group-specific ligands are commonplace. Since group-specific adsorbents retain a range of ligates with similar binding requirements, a single adsorbent may be used to purify a number of ligates. Group-specific ligates can be used when the desired ligate is present in high concentration, but this implies that some preprocessing has taken place and a concentration step interposed. The use of non/ group-specific adsorbents can only offer partial separations. This results in having to apply several stages in series, each only capable of removing a proportion of the impurities. In contrast a highly selective ligand can exclusively remove the target in one step, but often the resulting complexes are very tightly bound, have low binding capacities, are easily denatured and are expensive to produce. Until recently these adsorbents were restricted to technically difficult isolations. Today the use of computerassisted molecular modelling systems provides opportunities to investigate relationships between designed ligands and relevant protein structures. For the first time logical design approaches can be applied and consequently stable inexpensive ligands have now become available.

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