Agriculture Reference
In-Depth Information
9.5.
ELECTROPHORESIS
As with other chromatographic methods, there are an number of elec-
trophoretic methods, including paper, gel, and capillary. Electrophoresis uses
an electric current to move ionic species, either simple ions, amino acids, or
complex proteins, through a medium or a capillary [i.e., capillary electrophore-
sis (CE)]. During this process, the ionic species typically move at different rates
and are thus separated (CE is somewhat different, as discussed below).
9.5.1.
Sample Application
In paper or gel electrophoresis, a sample may be applied with a syringe or a
micropipette similar to the application of samples to thin layer plates. In some
cases there may be “wells” in the gel that accept the solution containing the
species to be separated. In capillary electrophoresis samples may be applied
using electromigration, hydrostatic, or pneumatic injection.
In all cases the ions to be separated must be soluble in and compatible with
the stationary phases and buffers used.
9.5.2.
Movement of Species
For electrophoresis the paper or gel is saturated with the required buffer at
the desired pH. The ends of the paper or gel are placed in a buffer reservoir
that contains the buffer with which the paper or gel is saturated and that also
have electrodes connecting one end to the positive DC terminal and the other
to the negative terminal of the power source. It is the electrical current that
causes the movement of ionic species through the medium.
In capillary electrophoresis a high voltage is used to produce electroosmotic
flow, and both electricity and the buffer flow through the capillary, with the
buffer flowing toward the cathode. Both carry the sample through the capil-
lary, and because of the flow of the buffer, both charged and uncharged species
are separated.
9.5.3.
Stationary Phases
Electrophoresis can be carried out using paper or a gel as the supporting
medium. Typically it can be carried out only in media compatible with water
since buffers or salt solutions are required to carry the electric current
required for separation. Capillary electrophoresis is carried out in a fused-
silica capillary filled with buffer.
9.5.4.
Detection
Once an electrophoretic separation has been accomplished, the paper or gel
is sprayed or dipped in a visualizing solution similar to that used in the visu-
