Affinity chromatography is one of the most selective kinds of chromatography. Chromatography generally is used to separate and identify different components present in a mixture. But when encountered with a task to isolate a single, specific compound from a complex mixture, the chemist would prefer a special type of chromatography called affinity chromatography. It is particularly useful for protein purifications.
This article provides an overview on affinity chromatograph. We will learn here its working principle, stationary phase development, mobile phase composition and the many different applications of affinity chromatography.
What is affinity chromatography
Affinity chromatography is a type of liquid column chromatography. The stationary phase is packed into the column, with special functional moieties called affinity ligands immobilized on it. Buffer solutions are used as a mobile phase to sweep the sample components through the column.
Historical background of affinity chromatography
Affinity chromatography was developed and performed for the first time back in 1951 by three immunochemists: Campbell, Luescher and Lerman. They retrieved antigen (a type of protein) from a complex mixture by adsorbing it onto a solid matrix called an immunosorbent. The technique was then used for an enzyme isolation in 1968 by Cuatrecasas, Wilchek and Anfinsen. It was then that this term ‘affinity chromatography’’ was introduced for the first time.
Working principle of affinity chromatography
Affinity chromatography is based on highly specific, reversible biological interactions between the analyte component and the molecules attached to the stationary phase. Other chromatographic analyses depend upon the physical and chemical properties of analyte molecules for their retention onto the stationary phase. Contrarily, affinity chromatography employs biospecific interactions.
For instance, in case of protein purification, special ligands are bonded to the stationary phase material. Targeted proteins from the sample with appreciable affinity towards the ligand will be strongly retained while all other components pass out of the column with the mobile phase buffer.
Stationary phase in affinity chromatography
The stationary phase in affinity chromatography mainly consists of the following three components:
- Affinity matrix
- Spacer arm
- Affinity ligand
The affinity matrix is the principal solid support packed into the column. It is ideally a macroporous substance with high chemical, mechanical and physical stability. The affinity matrix should have uniform particle and pore sizes. The porous texture of the matrix allows a high surface area for ligand immobilization. Meanwhile the pore size should be large enough to prevent the targeted protein molecules from getting trapped into the matrix itself. An ideal pore size for the affinity matrix is 300 Å for a targeted protein of size 60 Å.
Agarose, cellulose, dextran, silica and polyacrylamide etc. are some commonly used affinity matrices for stationary phase packing in affinity chromatography. The incorporation of magnetic beads as an affinity matrix in affinity chromatography is a rising sensation in the modern scientific world. A spacer arm also sometimes called a linker is then attached to the affinity matrix. The spacer is usually a hydrophobic carbon chain composed of 5-6 C-atoms, with specific functional groups attached on both terminals.
Possible functional groups could be a hydroxyl (-OH) functional group, a thiol (-SH) group, an amino (-NH2) group and/or a carboxylic acid (-COOH) functional group. Chemicals such as a cyanogen bromide (C1) or an epoxide (C3) can be used for functionalizing the matrix core with these hydrocarbon spacers. Spacer arms provide a biphasic interaction. One terminal functional group binds the spacer with the affinity matrix while the other functional terminal binds the affinity ligand, as shown in the figure below.
A complete knowledge about the targeted protein and the nature of interaction governs the type of ligand to be immobilized. In the presence of reactive functional groups, the affinity ligands bind to the spacer via covalent bonding.
If the targeted protein to be analyzed is an antibody, then the affinity ligand will be a corresponding antigen. Substrates, inhibitor or co-factors are immobilized as affinity ligands for capturing enzymes from the sample mixture while receptors are used against hormones.
Mobile phase in affinity chromatography
Buffer or salt solutions of varying pH and ionic strengths are employed as mobile phase in affinity chromatography. For instance, a 1M NaCl solution in a borate, phosphate or carbonate buffer. Acidic buffers such as a glycine-HCl buffer (pH 2.8) or 1 M propanoic acid can also be used. Similarly alkaline buffers for e.g., 0.1M diethyl amine (pH 11) can be employed as per need.
The ionic strength of the mobile phase solutions is varied such that the forces of attraction binding the protein to the ligand can be weakened. Complementary substances from the mobile phase in turn competes with the retained solutes for the binding sites. The targeted solutes are progressively displaced and eluted out of the column while the competing substances get attached to the ligand present onto the stationary phase.
How to perform affinity chromatography
The following step-by-step guide will take you through an affinity chromatographic protocol as performed in the laboratories on a regular basis.
Step I: Column Packing
The column is packed with the stationary phase material as discussed in the section above.
Step II: Sample Loading
The crude sample is passed through the column. A particular protein from the sample gets retained onto the stationary phase by forming a protein-ligand complex. The protein-ligand complex formation typically follows a lock-and-key mechanism. A specific protein binds to a specific ligand just as a particular key fits into its special lock. The protein-ligand binding should be strong but reversible.
Step III: Washing
All the other non-specific sample molecules are washed out of the column with a suitable buffer.
Step IV: Elution
Specifically bound proteins are eluted out of the column by changing the pH, ionic strength of the buffer and/or by-passing competitive protein molecules through the column.
Where do we need affinity chromatography
Affinity chromatography is important because it allows:
- Solute specific separation of protein mixtures: antigens and antibodies. In-vitro antibody-antigen interactions can be studied by affinity chromatography.
- Contaminant removal: Impurities and contaminants present in protein mixtures may include certain detergents necessary for cell lysis and protein solubilization. The protein of interest from a crude mixture is held back while these contaminants are thrown out of the column.
- Enzyme assays: Enzyme binding sites and the substrates that get attached to them can be detected.
- Affinity-tagged purification: A naturally occurring amino acid sequence on a protein can be used as a binding site for the ligand. Other than that, a special amino acid sequence can be engineered into the protein of interest. This method is called tagging while the engineered amino acid sequence is called a ‘tag’’. This tag for e.g., a polyhistadine tag binds to a special metal containing complex present on the affinity matrix. In this way, tagged recombinant proteins can be purified.
- Purification and detection of nucleic acids such as DNA: A customized media can be developed on the affinity matrix using DNA-binding proteins such as polymerases to retain DNA from a sample through a coupling linkage.
- Drug Evaluation: Affinity chromatography can also be used for isolating individual optical isomers from a complex drug formulation. Either a levo or a dextro-rotatory enantiomer binds with a specific ligand on the column while the other is eluted out.
- Affinity chromatography is a powerful chromatographic tool that saves time and provides maximum recovery of a targeted compound via efficient analyte capture in a single step.
- It is primarily used as a protein purification technique.
- It purifies samples upto several hundred folds.
You may like to watch a video tutorial on affinity chromatography here.
Discover the other different types of chromatography in our articles:
1. Hage, D. S. (1999). “Affinity Chromatography: A Review of Clinical Applications.” Clinical Chemistry 45(5): 593-615.
2. Mayers, G. L. and C. J. van Oss (1998). Affinity Chromatography. Encyclopedia of Immunology (Second Edition). P. J. Delves. Oxford, Elsevier: 47-49.
3. Urh, M., D. Simpson and K. Zhao (2009). “Affinity chromatography: general methods.” Methods Enzymol 463: 417-438.