Page 11 - Subyek Encyclopedia - Encyclopedia of Separation Science

6 I / AFFINITY SEPARATION / Derivatization

indicated as improvements are made to current ma- coupled directly to the matrix, but most require coup-
terials and the prices of synthetics begin to approach ling via a previously activated matrix. The afRn-
those of agarose beads. Other factors resist any signif- ity matrix selected must have an adequate number of
icant movement towards synthetic matrices. Most appropriate surface groups onto which the ligand can
installed processing units are designed for low perfor- be bonded. The most common surface group is hy-
mance applications. Higher performance matrices droxyl. The majority of coupling methods involve the
would need reinstallation of new, much higher cost activation of this group by reacting with entities con-
high performance plant; plant operators would need taining halogens, epoxy or carbonyl functional
retraining; operating manuals would need rewriting; groups. These surface residues are then coupled to
and plant and factory would need reregistration with ligands through primary amines, hydroxyls or thiol
the FDA. In combination, the implication is that groups, listed in Table 3.
penetration of high performance systems for large Polysaccharides, represented by agarose, have
scale applications will be slow, and agarose beads will a high density of surface hydroxyl groups. Tradition
continue to dominate the market for protein separ- still dictates that this surface is activated by cyanogen
ations. bromide, but it is well established that this reagent
forms pH-unstable iso-urea linkages, resulting in
a poorly performing product. Furthermore CNBr-
Covalent Bonding
activated agarose needs harsh coupling conditions
A basic requirement of all chromatographic media is if high yields of Rnal media are to be obtained,
the need for absolute stability under all operational suggesting high wastage of often expensive ligands.
conditions through many cycles of use. Consequently This factor is particularly evident with fragile entities
all ligands must be covalently bonded onto the such as the very-expensive-to-produce antibodies,
matrix, and various chemistries are available to and yet many workers simply read previous literature
achieve this. and make no attempt to examine alternative far
A number of factors are involved: superior coupling methods. The advantages of mild
coupling regimes are demonstrated in Figure 2, where
1. The performance of both ligand and matrix are the use of a triazine-activated agarose is compared to
not impaired as a result of the coupling process. CNBr-activated agarose. Yield is signiRcantly in-
2. Most of the coupled ligand is easily accessible to creased, largely by coupling under acidic rather than
the ligate. alkaline conditions.
3. Charged or hydrophobic groups are not generated
on the matrix, so reducing nonspeciRc adsorption. Intermolecular Binding Forces
4. The immobilized ligand concentration is optimal
for ligate bonding. Almost all chromatographic separations rely upon
5. There is no leakage of immobilized ligand from the interaction of the target molecule with either
the matrix. a liquid phase or a covalently bonded molecule on the
solid phase, the exceptions being those relying upon
Some ligands are intrinsically reactive (or can be molecular size, e.g. molecular sieves and gel Rltra-
designed to be so) and contain groups that can be tion. In afRnity separations ligates are inevitably

Table 3 Activation materials
Activating reagent Bonding group on ligand

Cyanogen bromide Primary amines
Tresyl chloride Primary amines, thiols
Tosyl chloride Primary amines, thiols
Epichlorohydrin Primary amines, hydroxyls, thiols
1,4-Butanediol diglycidyl ether Primary amines, hydroxyls, thiols
1,1 -Carbonyldiimidazole Primary amines, hydroxyls
Cyanuric chloride Primary amines, hydroxyls
Divinylsulfone Primary amines, hydroxyls
2-Fluro-1-methylpyriinium-toluene-4-sulfonate Primary amines, thiols
Sodium periodate Primary amines
Glutaraldehyde Primary amines