3
IEC
IEC
introduction to TSKgel iEC COLUMNS
Elution is usually performed by changing the ionic strength
of the mobile phase by applying a salt gradient. As the
salt concentration of the mobile phase increases, the salt
ions compete with the bound molecules for the functional
groups of the stationary phase. The higher the net charge of
the molecule, the higher the salt concentration needed for
elution. Very tightly bound compounds are removed at the
end of the elution by a wash step with very high salt buffer.
Ion exchange resins are classified as weak or strong ion ex-
changers. The terms strong and weak do not refer to the
performance of the resins or to the strength of interaction
between resin and target.
‘Strong’, respectively ‘weak’ refers to the extent that the ion
exchange capacity varies with change in pH. Strong ion
exchange groups have a steep titration curve. They show
no variation of their ionization state with the pH and remain
fully charged over a broad pH range.
Typical strong anion exchange groups are quaternary amine
groups (Q & QAE type), typical strong cation exchange
groups are sulfo or sulfopropyl groups (S & SP type).
Carboxymethyl (CM, cationic) and diethylaminoethyl
(DEAE, anionic) are weak ion exchange groups. Figure 2
shows the pKa values for these ligands.
Cation Exchange Groups
Structure
Carboxymethyl (CM)
Weak
-O-CH
2
COO
-
Sulfopropyl (SP)
Strong
-O-R-O-CH
2
-CH
2
-CH
2
-SO
3
-
Anion Exchange Groups
Diethylaminoethyl (DEAE)
Weak
-O-CH
2
-CH
2
-N
+
-(C
2
H
5
)
2
Quarternary Ammonium (Q)
Strong
-O-R-N
+
-(CH
3
)
3
Quarternary Aminoethyl (QAE)
Strong
-O-CH
2
-CH
2
-N
+
H-(CH
3
)
3
table 1
pK
a
values for Ion Exchange groups
Q, SuperQ, QAE pK
a
= 12.2
CM pK
a
= 4.7*
*pK
a
Toyopearl GigaCap CM-650M = 3.6
S, SP pK
a
= 1.2
DEAE pK
a
= 11.5
Cation
Anion
7.0
Decreasing resin pK
a
pK
a
values for ion exchange groups
figure 2
The binding buffer pH is selected between the pI of the protein and
the pKa of the stationary phase.
