Affinity Chromatography – Principle, Components, Procedure, Applications & Advantages

Introduction

• Chromatography is a laboratory technique used to separate components of a mixture based on their interactions with a stationary phase (fixed) and a mobile phase (moving).
• Affinity chromatography is a specialized type of liquid chromatography that uses specific and reversible biological interactions between the target molecule and a ligand attached to the stationary phase.
• Discovered in 1968 by Pedro Cuatrecasas and Meir Wilchek.
• Falls under adsorption chromatography, but is much more specific and selective.
• Commonly used to purify proteins, enzymes, antibodies, and other biomolecules.

Principle of Affinity Chromatography

• Based on the lock-and-key mechanism:
  • Lock = ligand attached to stationary phase.
  • Key = specific site on the target molecule that binds to the ligand.
• The target molecule binds specifically and reversibly to the ligand, while non-target molecules pass through.
• Binding is due to:
  • Hydrogen bonds
  • Electrostatic forces
  • Hydrophobic interactions
  • Van der Waals forces
• Elution (release of bound molecules) is done by:
  • Adding a competing ligand to the mobile phase
  • Changing pH, ionic strength, or polarity

Key Features

• Highly selective – isolates only the desired molecule from a mixture.
• Can be single-step (fast, high purity) or multi-step (for complex purification strategies).
• Works best when a specific ligand for the target molecule is available.

Components of Affinity Chromatography

1. Matrix

• Inert solid support that holds the ligand.
• Must be:
  • Chemically and physically stable
  • Insoluble in solvents and buffers
  • Have a large surface area for maximum ligand attachment
  • Good flow properties for smooth column operation
• Examples: agarose beads, polyacrylamide beads, polystyrene, cellulose.

2. Spacer Arm

• Short chemical link that connects ligand to matrix.
• Prevents steric hindrance (crowding) so the target molecule can bind easily.
• Examples: 1,6-diaminohexane, 6-aminohexanoic acid.

3. Ligand

• Molecule that binds specifically to the target molecule.
• Can be:
  • Biological (e.g., antibodies, enzymes, glycoproteins)
  • Synthetic (e.g., biomimetic dyes, metal chelates, boronates)
• Selection of ligand depends on target molecule:
  • Antigen → Antibody isolation
  • Substrate/inhibitor → Enzyme isolation
  • Metal ions → Histidine-tagged proteins
• Types:
  • Monospecific ligands – bind to one or few molecules (e.g., enzyme inhibitors, vitamins).
  • Group-specific ligands – bind to a broad class of molecules (e.g., boronic acid derivatives, biomimetic dyes).

Examples of Ligand-Target Pairs

Ligand	Target Molecule
Antigen	Antibody
Substrate	Enzyme
Lectin	Glycoprotein, polysaccharide
Complementary DNA sequence	Nucleic acids
Metal ions	His-tagged proteins
Protein A or G	Immunoglobulins
Phenyl boronate	Glycoproteins
Poly(A)	Poly(U) RNA

Types of Affinity Chromatography

1. Boronate & Phenyl Borate Affinity

• Uses boronate ligands.
• Commonly used for glycoprotein purification and HbA1c (glycated hemoglobin) analysis.

2. Lectin Affinity

• Uses lectins (carbohydrate-binding proteins) as ligands.
• Separates polysaccharides, glycopeptides, and carbohydrate-containing cells.

3. Dye-Ligand Affinity

• Uses synthetic dyes as ligands.
• Purifies blood proteins, enzymes, and pharmaceuticals.

4. Immunoaffinity

• Uses antibodies as ligands to purify hormones, viruses, peptides, and enzymes.

5. Immobilized Metal Ion Affinity Chromatography (IMAC)

• Uses immobilized metal ions (e.g., Ni²⁺, Co²⁺) to bind proteins with histidine residues.
• Widely used for recombinant His-tagged protein purification.

6. Analytical Affinity Chromatography

• Used for quantitative analysis and measurement of target molecules.

Sample Preparation

• Remove particulates using filtration or centrifugation.
• Buffer should be optimized for:
  • pH
  • Ionic strength
  • Compatibility with ligand-target binding.
• Avoid components that disrupt interactions (e.g., detergents, denaturing agents).

Procedure – Steps in Affinity Chromatography

1. Column Preparation
   • Pack column with chosen matrix.
   • Attach ligand to matrix via spacer arm.
   • Equilibrate column with binding buffer.
2. Sample Loading
   • Apply sample under conditions that favor binding between target molecule and ligand.
   • Non-target molecules pass through.
3. Washing
   • Remove weakly bound or non-specific molecules using wash buffer.
4. Elution
   • Release target molecules using:
     • Competitive ligand
     • pH change
     • Ionic strength change
     • Chaotropic agents
5. Re-equilibration
   • Prepare column for reuse by washing with buffer.

Factors Affecting Efficiency

• Specificity of Ligand – must match target molecule.
• Binding Strength – too weak → loss of target; too strong → hard to elute.
• Matrix Properties – high surface area and appropriate pore size.
• pH & Buffer Composition – maintain stability of ligand and target.
• Flow Rate – slower rates improve binding but increase time.

Applications of Affinity Chromatography

• Purification of enzymes, proteins, antibodies.
• Isolation of active biomolecules from crude extracts.
• Separation of functional proteins from denatured forms.
• Detection of biomarkers for disease diagnosis.
• Purification of monoclonal antibodies in pharmaceutical manufacturing.

Advantages of Affinity Chromatography

• Extremely specific – isolates only target molecule.
• Produces highly pure products in a single step.
• Can process large volumes.
• Matrix is reusable.

Limitations of Affinity Chromatography

• Ligands can be expensive and may not be available for all targets.
• Time-consuming preparation.
• Non-specific binding can occur.
• Sensitive to pH changes – risk of denaturing proteins.

Troubleshooting Tips

• Use fresh, filtered samples to avoid clogging.
• Degas buffers to remove air bubbles.
• Store columns in 20% ethanol to prevent microbial growth.
• Avoid extreme pH unless required for elution.

Recent Innovations

• Use of magnetic beads and monolithic supports for faster separation.
• Development of nanobody-based ligands for higher specificity.
• Miniaturized, automated chromatography systems for precise control.

Affinity chromatography is a powerful, highly selective technique for purifying biomolecules.
Its ability to exploit specific ligand-target interactions makes it a key method in biochemistry, biotechnology, and pharmaceutical industries.
With advancements in ligands and automated systems, it continues to become faster, more efficient, and more widely applicable.

References

1. Labrou, N. (2003). Design and selection of ligands for affinity chromatography. Journal of Chromatography B, 790(1–2), 67–78. https://doi.org/10.1016/s1570-0232(03)00098-9
2. Introduction to affinity Chromatography. (n.d.). Bio-Rad Laboratories. https://www.bio-rad.com/en-np/applications-technologies/introduction-affinity-chromatography?ID=LUSMJIDN
3. Hage, D. S., Anguizola, J. A., Bi, C., Li, R., Matsuda, R., Papastavros, E., Pfaunmiller, E., Vargas, J., & Zheng, X. (2012). Pharmaceutical and biomedical applications of affinity chromatography: Recent trends and developments. Journal of Pharmaceutical and Biomedical Analysis, 69, 93–105. https://doi.org/10.1016/j.jpba.2012.01.004
4. Banjara, M.R. and Thapa Shrestha, U. (2021). Instrumentation in Microbiology. Garuda Publications.
5. Wilson, K. and Walker, J. (Ed.). (2010). Principles and Techniques of Biochemistry and Molecular Biology. Seventh Edition. Cambridge University Press.
6. Jason, J. C. (Ed.). (2011). Protein Purification: Principle, High-Resolution Methods, and Applications. Third Edition. John Wiley & Sons Inc.
7. GE Healthcare. A handbook on Affinity Chromatography: Principles and Methods.
8. Pan, S. (2025, February 10). Affinity chromatography – Principle, Types, Steps, Applications – Biology Notes Online. Biologynotesonline.com. https://biologynotesonline.com/affinity-chromatography-principle-types-steps-applications/