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What is Affinity
Chromatography???
Principle of Affinity
Chromatography
Inject a sample into an initially
equilibrated affinity chromatography
column
Only the substances with affinity for the
ligand will retained in the column.
Other substances with no affinity for the
ligand will eluted from the column.
The substances retained in the column
can be eluted from the column by
changing pH or salt or organic solvent.
Method of Affinity
Chromatography
Binding of the selected ligand to the matrix requires that a
The Chromatogram
Affinity chromatography is not just limited to isolating
one protein; given a sample of similar proteins with
similar binding affinities, a chromatogram can be
generated.
A chromatogram is a plot of absorbance vs. time for
the elution of proteins in affinity chromatography.
Four things may be learned from a chromatogram:
1)The level of complexity of the sample (indicated by
the number of peaks)
2)Qualitative information about the sample composition
(by comparing peak positions with known standards)
3)Quantitative information of the relative component
concentrations (by comparing peak areas)
4)Total column performance (by comparing with known
standards).
Chromatogram
Applications of Affinity
Chromatography
Affinity chromatography can be used to:
Purify and concentrate a substance from
a mixture into a buffering solution
Reduce the amount of a substance in a
mixture
Recognize what biological compounds
bind to a particular substance
Purify and concentrate an enzyme
solution.
Case Studies.