Pseudo-affinity Chromatography: Definition, Principle, Application

In biochemistry and molecular biology, pseudo-affinity chromatography is a method used to separate and purify biomolecules based on their reversible interaction with immobilized ligands on a chromatographic substrate. The term “pseudo-affinity” describes the target biomolecule’s selective binding to the ligand; this binding may or may not be characterized by a high-affinity relationship, as in the case of conventional affinity chromatography.

Pseudo-affinity Chromatography
Pseudo-affinity Chromatography

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What is Pseudo-affnity Chromatography?

  • The process of separating compounds from a mixture using chromatography is based on the characteristics of the individual components. These characteristics include the compounds’ charge and size as well as their interactions with the solid and mobile phases.
  • In affinity chromatography, a particular ligand is added to the solid phase to capture the target compound, such as a certain protein. The protein of interest can be distinguished from a mixture of proteins due to the particular character of this interaction. By altering the chromatographic conditions, the target protein can then be liberated from the ligand.
  • Similar to this, dyes are used as ligands in pseudo-affinity chromatography to target proteins. Although the dyes utilized are ligand-mimicking, their selectivity is low. As a result, different proteins can be captured by these dyes.
  • The ligands immobilized on the chromatographic matrix in pseudo-affinity chromatography replicate some of the characteristics of the target molecule’s natural ligands or substrates. As a result, the target molecule can be selectively bound to and eluted from the mixture.
  • Unlike traditional affinity chromatography, where the interaction is typically strong and specific, pseudo-affinity chromatography exploits reversible interactions, often with moderate affinity, to capture and release the target molecule.
  • When specific ligands for the target molecule are unknown or challenging to find, this approach is especially helpful.
  • It provides a way to separate biomolecules from one another according to their functional qualities as opposed to just their structural features.
  • Pseudo-affinity chromatography is a quick and effective way to separate target biomolecules from complicated mixtures while also allowing for flexibility in experimental design.
  • Applications for pseudo-affinity chromatography include drug discovery, protein purification, and investigations of biomolecular interactions.

Principle of Pseudo-affinity Chromatography

  • The principle of pseudo-affinity chromatography is the use of reversible binding interactions between an immobilized ligand that has an affinity for the target molecule and the target molecule itself.
  • Pseudoaffinity chromatography depends on somewhat weaker interactions such as hydrogen bonding, hydrophobic interactions, or electrostatic forces, in contrast to true affinity chromatography, where interactions mimic natural ligand-receptor binding with high specificity and strength.
  • A ligand is immobilized onto a solid support, a sample containing the target molecule and other biomolecules is applied, the target molecule is selectively bound to the immobilized ligand, unbound or weakly bound molecules are washed away, and the target molecule is eluted by changing the conditions to interfere with the binding interactions.
  • Pseudo-affinity chromatography is a useful method for isolating and purifying target molecules from complex mixtures based on their selective interactions with the immobilized ligands, however it is not as strong or specific as natural affinity interactions.

How Does Pseudo-affinity Chromatography Work?

Here is an example of a study that describes the working mechanism of pseudo-affinity chromatography:

  • Three enzymes from rabbit muscles were separated using pseudo-affinity chromatography in a work by Tulsani et al. The enzymes were aldolase, pyruvate kinase, and lactate dehydrogenase.
  • In this case, different triazine dyes were immobilized using cross-linked guar and cross-linked pectin. The cross-linked guar and cross-linked pectin were first rinsed in deionized water, and then they were suspended in new deionized water to immobilize the dye. After that, the gel was mixed with the dye for approximately five minutes. After adding salt, this mixture was stirred for a further thirty minutes. After that, sodium hydroxide was added, and the mixture was agitated for 16 to 18 hours. Ultimately, deionized water was used to wash this combination.
  • The gel was now put into 2 ml columns with the dye bound.
  • The crude protein sample was put at the top of the column after the phosphate buffer had gone through it. After the sample went through, the unattached proteins were removed from the column by washing it with the buffer. This was confirmed by using a spectrophotometer to measure the absorbance of the buffer exiting the column at 280 nm. After being cleaned, the bound protein was freed by adding salt, sodium pyruvate, or NAD+ to the same buffer.
  • Aldolase, pyruvate kinase, and lactate dehydrogenase were all able to be separated by the procedure.
  • Lactate dehydrogenase was discovered to be kept by dyes 1014 and 1015, while pyruvate kinase was also held by dye 1015 when it was bound to cross-linked pectin. Nevertheless, none of the ten dyes examined in this investigation were able to maintain aldolase.

Pseudo-affinity Chromatography for Removing Proteins from Plasma

Human plasma is the liquid part of blood, and it contains proteins. 75% of this extremely complicated mixture consists of immunoglobulin G (IgG), a kind of antibody, and human serum albumin. This indicates that not only are those proteins highly detectable, but they may also mask the presence of other proteins. This could be challenging because those low-abundance proteins could be diagnostic indicators for specific disorders. Consequently, one of the most important steps in preparing serum for diagnostic use is the elimination of serum albumin and IgG.

In a study, Urbas et al. optimized the procedures for isolating serum albumin and IgG from plasma. Here, they combined affinity chromatography with pseudo-affinity chromatography to remove serum albumin and IgG simultaneously, in addition to using the latter method to remove serum albumin selectively. The Mimetic Blue SA A6XL dye employed in the pseudo-affinity chromatography to extract serum albumin was not very specific, even though this was found to be effective. As a result, the amount of plasma put into the column determined how well this technology removed serum albumin. The more serum albumin on the column could push out the other proteins from the dye if more plasma was added.

Application of Pseudo-affinity Chromatography

  • Protein Purification: Pseudo-affinity chromatography finds extensive use in purifying proteins from complex biological samples. By immobilizing ligands that mimic the binding specificity of target proteins, this technique allows for selective capture and purification of the protein of interest from crude extracts or cell lysates.
  • Drug Development: Pseudo-affinity chromatography is used in drug discovery to screen possible candidates for drugs by evaluating how they interact with target proteins or receptors. This facilitates the discovery of lead compounds with high binding affinity and specificity—two essential stages in the creation of new drugs.
  • Diagnostic Assays: In diagnostic assays, pseudo-affinity chromatography is essential for identifying disease-related proteins or biomarkers in biological samples. This method helps with illness diagnosis and monitoring by selectively capturing and detecting the target biomolecule by immobilizing ligands specific to it.
  • To characterize and classify proteins or metabolites in biological materials, pseudo-affinity chromatography is incorporated into proteomic and metabolomic procedures. Through the process of selecting and collecting target molecules according to how they interact with immobilized ligands, this method makes it easier to conduct a thorough investigation of intricate biological systems.
  • Pseudo-affinity chromatography is used in the biopharmaceutical industry to purify medicinal proteins, monoclonal antibodies, and recombinant proteins. It makes it possible to effectively separate the target molecule from complicated fermentation broths or cell culture supernatants, which helps to produce high-quality biopharmaceuticals.
  • Pseudo-affinity chromatography is used by scientists to investigate interactions between proteins, ligands, and receptors. Researchers can study the binding kinetics and thermodynamics of these interactions by immobilizing ligands that resemble natural binding partners. This can provide light on a variety of biological processes.

Advantages of Pseudo-affinity Chromatography

  • Selective binding
  • High recovery and purity
  • Versatility

Limitations of Pseudo-affinity Chromatography

  • Limited binding strength
  • Non-Specific Interactions
  • Ligand Immobilization Challenges


  • Keven Lothert and Michael W. Wolff, “Affinity and Pseudo-Affinity Membrane Chromatography for Viral Vector and Vaccine Purifications: A Review”, Membranes 2023, 13(9), 770;
  • Champagne, J., Delattre, C., Shanthi, C. et al. Pseudoaffinity Chromatography Using a Convective Interaction Media®-Disk Monolithic Column. Chroma 65, 639–648 (2007).
  • Taruna Arora ,Pankaj Patel,and K. Muralidha, “Assessment of Pseudoaffinity Chromatography Using Textile Dyes for Isolation of Buffalo Pituitary Luteinizing Hormone” Volume 2012 | Article ID 639514 |

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Kabita Sharma

Kabita Sharma, a Central Department of Chemistry graduate, is a young enthusiast interested in exploring nature's intricate chemistry. Her focus areas include organic chemistry, drug design, chemical biology, computational chemistry, and natural products. Her goal is to improve the comprehension of chemistry among a diverse audience through writing.

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