Page 10 - Subyek Encyclopedia - Encyclopedia of Separation Science

Sepsci*1*TSK*Venkatachala=BG
I / AFFINITY SEPARATION 5

The beaded agaroses have captured over 85% of
the total market for biological macromolecule separ-
ations, and are regarded as the industry standard to
which all other supports are compared. They have
achieved this position by providing many of the desir-
able characteristics needed, and are also relatively
inexpensive. Beaded agaroses do have one severe lim-
itation } poor mechanical stability. For analytical
applications speed and sensitivity are essential, de-
manding mechanically strong, very small particles.
Beaded agaroses are thus of limited use analytically,
Figure 1 Schematic diagram of affinity chromatography.
a gap Rlled by high performance liquid chromatogra-
phy (HPLC) using silica matrices. For preparative and
large scale operations other factors are more impor-
Matrices
tant than speed and sensitivity. For example, mass
By deRnition matrices must be inert and play no part transfer between stationary phase and mobile phase is
in the separation. In practice most play a (usually) much less important when compared to the contri-
negative role in the separation process. To minimize bution from the chemical kinetics of the binding
these disadvantages matrices have to be selected with reaction between stationary phase and protein.
great care. There is a theoretically perfect matrix, Band spreading is also not a serious problem. When
deRned as consisting of monodispersed perfectly combined with the highly selective nature of the
shaped spheres ranging from 5 to 500 m in dia- afRnity mechanism, these factors favour the com-
meter, of high mechanical strength, zero nonspeciRc mon use of large sized, low mechanical strength par-
adsorption and with a range of selectable pore sizes ticles.
from 10}500 nm, a very narrow pore size distribution In recent years synthetic polymeric matrices have
and low cost. This idealized matrix would then pro- been marketed as alternatives. Although nonbiodeg-
vide the most efRcient separation under all ex- radable, physically and chemically stable, with good
perimental conditions. As always, a compromise has permeabilities up to molecular weights greater than
7
to be reached, the usual approach being to accentuate 10 Da, the advantages provided are generally off-
the most attractive characteristics while minimizing set by other quite serious disadvantages, exempliRed
the limitations, usually by manipulating the experi- by high nonspeciRc adsorption. Inorganic matrices
mental conditions most likely to provide the optimum have also been used for large scale protein separ-
result. ations, notably reversed-phase silica for large scale
The relative molecular masses of proteins vary recombinant human insulin manufacture (molecular
from the low thousands to tens of millions, making weight approximately 6000 Da), but are generally
pore size the most important single characteristic of not preferred for larger molecular weight pro-
the selected matrix. Very large molecules need very ducts. A very slow adoption of synthetic matrices is
open and highly porous networks to allow rapid and
easy penetration into the core of the particle. Struc-
tures of this type must therefore have very large pores, Table 2 Support matrices
but this in turn indicates low surface areas per unit
volume, suggesting relatively low numbers of surface Support matrix Operational
pH range
groups to which ligands can be covalently attached.
The matrix must also be biologically and chemically Agarose 2}14
inert. A special characteristic demanded from biolo- Cellulose 1}14
gical macromolecular separations media is an ability Dextran 2}14
to be sanitized on a routine basis without damage. Silica (8
This requires resistance to attack by cleansing re- Glass (8
Polyacrylamides
3}10
agents such as molar concentrations of strong alkali, Polyhydroxymethacrylates 2}12
acids and chaotropes. In contrast to analytical separ- Oxirane}acrylic copolymers 0}12
ations, where silica-based supports are inevitably Styrene}divinylbenzene copolymers 1}13
used, silica cannot meet these requirements and is Polyvinyl alcohols 1}14
generally not favoured for protein separations. N-Acryloyl-2-amino-2-hydroxy-1, 2-propane 1}11
Unaffected
PTFE
Table 2 contains examples of support matrices used
in afRnity separations. PTFE, polytetrafluoroethylene.