2
IEC
IEC
HOW IT WORKS
IEC retains molecules based on ionic interactions. The
stationary phase surface displays ionic functional groups
that interact with analyte ions of opposite charge. IEC is fur-
ther subdivided into cation exchange and anion exchange
chromatography.
Anion exchange media carry positively charged groups that
attract negatively charged anions. Cation exchange resins
display negatively charged groups which attract positively
charged cations. Charged target molecules are retained on
the stationary phase but can be eluted by increasing the
concentration of a similarly charged ion that will displace
the analyte or target ions from the stationary phase.
Proteins have numerous functional groups that can have
both positive and negative charges. IEC separates proteins
according to their net surface charge, which is dependent
on the pH and ionic strength of the mobile phase. Accord-
ing to differences in their overall charge and surface charge
distribution, proteins can be separated by IEC.
IEC takes advantage of the fact that the relationship be-
tween net surface charge and pH is unique for a specific
protein. At a pH, equivalent to its isoelectric point, a protein
has no net charge and will not interact with the charged
stationary phase. At a pH above the pI the protein will have
a negative net charge and will therefore bind to a positively
charged anion exchanger. At a pH below its pI it will have
a positive net charge and will consequently interact with a
negatively charged cation exchanger. By adjusting the pH
or the salt concentration of the mobile phase, separation
can be optimized. For loading, the pH and ionic strength
are selected in a way that the targets or analytes bind to the
stationary phase (Figure 1).
Surface net
charge
0
pH
Cation
Anion
+
-
Acidic pH: all three
components are
positively charged.
They bind to cation
exchangers and
elute by applying
a salt gradient.
Low acidic pH: red
and green are
positively charged
and bind to cation
exchangers. Blue
is negatively charged
and flows through.
Low alkaline pH: Blue
and green are
negatively charged
and flow through
cation exchangers.
Red is still positively
charged and binds.
Very alkaline pH: all
three components
are negatively
charged and flow
through cation
exchangers.
Acidic pH: all three
components are
positively charged
and flow through
anion exchangers.
Low acidic pH: red
and green are
positively charged
and flow through.
Blue is negatively
charged and binds
to anion exchangers.
Low alkaline pH: red
is still positively
charged and flows
through, blue and
green are negatively
charged and bind
to anion exchangers.
Alkaline pH: all
three components
are negatively
charged. They
bind to anion
exchangers and
elute by applying
a salt gradient.
Figure 1
