High performance capillary electrophoresis - T.E.A.M.

Modes
3.4 Capillary
isoelectric focusing
Capillary isoelectric focusing (CIEF) is a “high resolution”
electrophoretic technique used to separate peptides and
proteins on the basis of pI. CIEF can be used to separate
proteins that differ by 0.005 pI units and less. Similar to
CGE, this is a well-established gel electrophoretic technique
recently adapted to the CE format.
In CIEF a pH gradient is formed within the capillary using
ampholytes. Ampholytes are molecules that contain both
an acidic and a basic moiety (that is, they are zwitterionic)
and can have pI values that span the desired pH range of
the CIEF experiment (pH 3 to 9, for example). After filling
the capillary with a mixture of solute and ampholytes, the
gradient is formed. With a basic solution at the cathode and
an acidic solution at the anode, upon application of the
electric field the charged ampholytes and proteins migrate
through the medium until they reach a region where they
become uncharged (at their pI). This process is known as
“focusing”. The protein zones remain narrow since a
protein which enters a zone of different pH will become
charged and migrate back. The overall separation process
was depicted in figure 21b.
The status of the focusing process is indicated by the
current. Once complete, a steady-state is reached and
current no longer flows. After focusing, the solutes and
ampholytes are mobilized and the zones passed through the
detector. Mobilization can be accomplished by either application
of pressure to the capillary or by addition of salt to
one of the reservoirs.
The zone width, s (standard deviation), in CIEF is given by
1/2
D
s = (26)
dm dpH
dpH dx
( )( )
75