Page 166 - Instant notes

Physical chemistry 152

This field is usually sufficient to overcome the electrostatic force between the ion and its
diffuse double-layer and cause shearing (or movement in different directions) at the
outside of the Stern layer. The large ion with its Stern layer therefore moves towards one
electrode at a rate governed by its effective charge and its solvated ion size and the
diffuse double-layer (of opposite charge) moves towards the other electrode. The
mobility of the ion, complete with its Stern layer, can therefore be varied by judicious
choice of counterion type and concentration.
For further separation of large ions such as biomolecules that have similar size and
charge, a two-stage electrophoresis method is often used (Fig. 2). This takes advantage of
the fact that these large molecules often have many ionizable groups such as
and where R is the
biomolecule. This means that the position of these equilibria, and hence the degree of
ionization and the charge on the ion, depend upon the solution pH. If, after separation by
electrophoresis, two or more different biomolecules with similar size and charge have not
separated and remain together in a particular spot, a second electrophoresis experiment is
then carried out. This involves applying the field to the separated spots at an orientation
of 90° to the first field in a solution of different pH. The biomolecules within the same
spot, each of which contains its own characteristic number and

Fig. 2. The two-stage electrophoresis
method. (a) Initial spot application; (b)
after first electrophoresis separation;
(c) application of second
electrophoresis field; (d) after second
electrophoresis separation.

distribution of ionizable groups, will now have a different effective charge and separation
of the spot into separate spots, each containing an individual biomolecule, will occur.