Affinity Chromatography
Affinity chromatography- Introduction, theory,
instrumentation and applications.
Ligand Mixture of compounds in
the sample
- The sample is
loaded under ideal
binding
Matrix conditions.
- The target
molecules bind
specifically to the
affinity ligands,
while all other
sample
components, are
not adsorbed.
Introduction to Affinity Chromatography
• Affinity chromatography is a liquid chromatographic technique that separates a mixture of
substances based on their specific and reversible interactions with a stationary phase.
• The stationary phase contains a ligand, a molecule that binds specifically to the target
molecule of interest in the mixture.
• The target molecules bind to the ligand, while other molecules pass through the column
unbound.
• The bound target molecules can then be eluted from the column using a specific buffer or
solution that disrupts the ligand-target interaction.
Theory of Affinity Chromatography
1. Specific Binding: The target biomolecule in the sample mixture binds specifically to the
ligand attached to the stationary phase due to interactions such as enzyme-substrate, antigen-
antibody, receptor-ligand, or metal-chelate binding.
2. Washing: After binding, the column is washed with buffer to remove non-specifically
bound molecules, ensuring that only the target biomolecule remains bound to the ligand.
3. Elution: The bound molecules are then eluted by altering the conditions of the mobile
phase, such as changing the pH, ionic strength, or introducing a competitive ligand. This disrupts
the interactions between the target molecule and the ligand, releasing the target into the elution
buffer.
4. Regeneration: The column is returned to its original state to be used again by washing
away the eluted target molecule and any leftover compounds.
Instrumentation for Affinity Chromatography
Instrumentation for Affinity Chromatography
1. Pump
• Ensures a consistent and controlled flow of the mobile phase (sample solution and elution
buffer) through the affinity column. Modern systems utilize high-pressure pumps for faster
runs.
2. Sample Injector
• This allows for the precise introduction of the sample into the chromatography system.
• Auto-samplers can load multiple samples sequentially for high-throughput.
3. Affinity Column
• The core of the system, it contains the immobilized ligand specific for the molecule of interest.
• The column material typically consists of agarose, Sepharose, or other polymeric beads
covalently linked with the ligand.
4. Detectors: These monitor the eluted compounds from the column. Common detectors include:
o Ultraviolet (UV) Detector: Detects compounds based on their absorbance at specific wavelengths,
useful for most organic molecules.
o Fluorescence Detector: For molecules that fluoresce or can be labeled with fluorescent tags.
o Conductivity Detector: Monitors changes in solution conductivity, useful when elution involves
changes in ionic strength.
5. Fraction Collector: Collects separated compounds in distinct fractions for further analysis or use.
6. Temperature Control Unit: Some procedures require the temperature to be maintained at specific
levels, either for stability or to optimize binding/elution conditions.
7. Recorder/Data System: Modern systems interface with computers that allow for real-time data
acquisition, analysis, and visualization. This is essential for peak identification, quantification, and
data storage.
Pharmaceutical Applications of Affinity Chromatography
1. Employed for the isolation and concentration of immunoglobulins (antibodies).
1. - Utilized to extract and separate IgM from hybridoma cell cultures.
2. - Applied in the extraction and enrichment of clotting factors.
3. - Used for isolating recombinant microbial proteins such as Protein-A, Protein-G, and Protein-
L.
4. - Applied in the extraction and refinement of serine proteases.
5. - Facilitates the extraction of calmodulin-binding enzymes such as ATPases, protein kinases,
and phosphodiesterases.
6. - Involved in the extraction and separation of nucleic acids.
7. - Employed for the extraction and characterization of polysaccharides.
8. - Used to isolate and concentrate antigens.
