Affinity Chromatography
By
Dr. Mehboob Peeran
After activation it can be derivatised by a large
number of different groups creating different immobilised ligands
on the matrix. The following scheme illustrates this feature.
The Ligand
The ligand selected, naturally depends on the substance
to be purified. The conditions used while attaching the ligand
and later while elution should be mild and should not damage either
the solid support, the ligand itself, or denature the component
being separated. The coupled gel must be washed thoroughly.
Most importantly there must be an accurate assay system available for the ligand
to find out how much of the material is bound to the ligand.
An example: The NH2 groups on the beads may be detected very easily
by the conventional diazotisation test. However for quantitative estimation the
following method is used. A known amount of the dye which binds to he amino groups
(picryl sulphamic acid) is added to a known amount of the beads. Some dye binds
and the excess unreacted dye is estimated through absorbance measurement(540
nm). Thus the capacity of the beads is expressed in terms of the number of millimoles
of the dye that is bound. |
