Page 326 - Biosystems Engineering

Bioseparation Pr ocesses 303

they can only be used at maximum capacity over a narrow pH range.
The extent of binding the ions on the resin depends on the nature of the
resin and temperature, ionic strength, and composition of the solvent.
In the case of weakly ionized resins, ion uptake also depends on the
degree of ionization of the resin and the materials to be separated. The
preparation of ion-exchange material is done by washing the ions with
+
an appropriate solution. For example, the Na form is prepared by
washing the resin with sodium hydroxide and then with water until the
+
washings are neutral. Similarly, H forms of a cation-exchange resin are
obtained by washing the material with hydrochloric acid and then with
water until the washings are neutral. Generally, isolation of immuno-
globulin (IgG) can be done by ion-exchange chromatography.

Gel Filtration Chromatography
Gel filtration chromatography is the technique where the molecules can
be separated on the basis of their size by passing them down a column
containing swollen particles of an uncharged gel. Typically, the gels
consist of macromolecules with great affinity for the solvent used.
These have been cross-linked to form a three-dimensional network,
thereby making them insoluble. Instead of dissolving the liquid, the
macromolecules swell, taking up large amounts of liquid. Small mole-
cules can enter the gel, but larger molecules are completely separated,
leaving the column first (Fig. 9.8). The separation of proteins with
different molecular weights can be done by gel filtration.

Affinity Chromatography
Affinity chromatography is a single-step method that can be applied
to purify an enzyme from a very complex mixture. This method is based
on a biological property of some enzymes, that is, their capacity for
specific binding another molecule called a ligand. In the enzyme, the
ligand attached to the matrix is usually a powerful inhibitor with a
high affinity constant that will bind only one enzyme in a complex mix-
ture of proteins. Choice of the proper matrix is very important parameter

Gel particles
Large molecules
Small molecules

FIGURE 9.8 Gel fi ltration chromatography.