Affinity Chromatography - Department of Molecular and Cellular ...

100
Coupled ligand µmoles/g conjugate
75
50
25
20 mg/ml
5 mg/ml
0
9 10 11 12 13
14
pH
Fig. 68. pH dependence of coupling N-acetyl-D-galactosamine to Epoxy-activated Sepharose 6B. Carbonate/bicarbonate
buffers were used in the range pH 9–11, sodium hydroxide solution in the range pH 12–14.
Ligand concentrations: 5 mg/ml and 20 mg/ml.
When a ligand contains more than one kind of group (thiol, amino and hydroxyl), the
coupling pH will determine which of these groups is coupled preferentially. As a general
rule, the order of coupling is e-amino > thiol > a-amino > hydroxyl although the exact
result will depend on the detailed structure of the ligand.
The time of reaction depends greatly on the pH of the coupling solution, properties of the
ligand and the coupling temperature. The stability of the ligand and the carbohydrate chains
of the matrix limit the maximum pH that can be used. Coupling is performed in the range
pH 9–13 as shown in Figure 68 and the efficiency of coupling is pH and temperature
dependent (Figure 69).
100
pH 11.0, +45 °C
pH 10.5, +40 °C
Coupled glycylleucine %
75
50
25
0
0 8 16 24 32
40 48
Time hours
Fig. 69. Efficiency of coupling glycyl-leucine to Epoxy-activated Sepharose 6B.
118