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What exactly is affinity chromatography?

Chromatography has seen several developments throughout the 20th century. The term chromatography comes from the Greek words chroma, meaning “color”, and graphos, which means “to write”. This is because the technique was primarily used to separate plant pigments such as chlorophyll during its first years of existence.

New chromatography techniques have been developed since then. The technique was drastically improved by Archer John Porter Martin and Richard Laurence Millington Synge, who won the 1952 Nobel Prize in Chemistry for their contribution. Thanks to their research, technology has rapidly advanced, leading to the world of today and modern high-performance liquid chromatography methods.


What is affinity chromatography?

Affinity chromatography is one of the most potent chromatographic methods. The purpose of affinity chromatography is to separate target molecules out of a complex mixture to observe and conduct research on their physical, chemical, or biological properties.

This particular separation procedure works with biological molecules. The term “affinity chromatography” references the fact that a protein affinity tag will be used for purification. In contrast, other chromatography methods rely on physical-chemical properties like magnetism and heat.


Components of affinity chromatography

The affinity chromatography method contains three main components: The mobile phase, the stationary phase, and the chromatography column.

The mobile phase

The mobile phase is a fluid that is meant to flow through the chromatography system. The mobile phase interacts with the stationary phase, where the molecules of interest are meant to adhere.

A liquid or gaseous solution is used for this purpose. When a liquid is used, then the process is known as liquid chromatography. In turn, a chromatography method using a gaseous mobile phase is known as gas chromatography. An affinity chromatography procedure is an example of liquid chromatography.

The stationary phase

The stationary phase is made out of a solid material. Typically a stationary phase is composed of a porous solid such as silica, glass, or alumina.

The chromatography column

The chromatography column will act as a vessel for the procedure to take place in. The stationary phase is placed within the chromatography column and the mobile phase is poured through it.

In modern liquid chromatography, the preferred materials for the manufacturing of affinity columns are acrylic glass, borosilicate glass, and stainless steel. A mechanism made out of ceramic, stainless steel, or a polymer is used to prevent the stationary phase from falling out.

In some affinity chromatography procedures, two columns are used. Known as periodic counter-current chromatography (PCC), it is meant to run affinity chromatography in a semi-periodic manner. This process sees use in research and development, as well as in the manufacturing of biopharmaceuticals.

Learn more about testing procedures


What is affinity chromatography used for?

Due to affinity chromatography taking advantage of the biological properties of proteins, it is a useful technique for a variety of purposes. The procedure is effective for the observation of the biological interaction and properties of molecules, as well as to provide value to the healthcare industry.

Examples of applications of affinity chromatography include:

  • Purification of recombinant proteins
  • Custom affinity media
  • Immunoaffinity
  • Specialty media

Purification of recombinant proteins

The purification of recombinant proteins is affinity chromatography’s most common application. Recombinant proteins are encoded by a cloned gene (recombinant DNA) that exists in an environment that supports its expression and the translation of messenger RNA.

Custom affinity media

The affinity chromatography method counts with a wide variety of media. This gives scientists the opportunity to customize the procedure depending on their intended purpose. These media help eliminate the handling of toxic reagents, as functional spacers, or as a support affinity matrix.

The following are examples of affinity media:

  • Amino acid media: This affinity media is used in serum proteins, peptides, and enzymes. It is also useful to study the chemical properties of rRNA and dsDNA.
  • Carbohydrate bonding: Typically implemented with glycoproteins, lectins, and other substances containing carbohydrates. For example, a generalized affinity ligand known as Heparin can be used for the separation of plasma coagulation proteins, lipases, and nucleic acid enzymes.
  • Hydrophobic interactions: The most common use for this type of media is to target free proteins and carboxyl groups.
  • Nucleic acids: These can be used to gather DNA, mRNA, rRNA, and other nucleic acids and oligonucleotides.

mmunoaffinity chromatography

The antibodies from blood serum can also undergo the process of affinity chromatography. For instance, blood serum gathered from an organism that has been immunized against an antigen can be used to study that antigen.

Specialty media

Specialty media is designed for the specific binding of a protein or coenzyme. An example of this is the purification of E. coli β-galactosidase. The selected affinity matrix contains an enzyme that serves as a good analog to the protein it targets. Together with a higher salt concentration in the column, the media is able to accomplish elution.

Purification of monoclonal antibodies

The vast majority of monoclonal antibodies have been purified with affinity chromatography based on proteins derived from bacteria. Thanks to Protein G and Protein A, chromatography empowers researchers to find better treatments for conditions such as cancer.

What are monoclonal antibodies?


How does affinity chromatography work?

The affinity column is prepared so that the binding partners or immobilized ligands located in the stationary phase appropriately interact with the mobile phase. This creates stable covalent bonds and keeps the target molecules attached to the stationary phase.

The ligands used in affinity chromatography can have either an organic or inorganic origin. Biological examples of useful ligands include serum proteins, lectins, and antibodies. On the other hand, inorganic sources for ligands are moronic acid, metal chelates, and triazine dyes.

Why is reversed-phase chromatography used?


How does affinity chromatography separate proteins?

Affinity chromatography achieves protein purification by taking advantage of the possible biological interactions between molecules. Typically, the ligand is meant to attach to a solid, insoluble matrix. Examples of such matrixes include polymers like agarose and polyacrylamide in a chemically-modified form.

What are the steps of affinity chromatography?

The term affinity chromatography typically refers to a process that follows three main steps: binding, washing, and elution.

1. Binding

The column, mobile, and stationary phases must be prepared. Next, the mobile phase must be poured onto the stationary phase inside the affinity column in order for a binding interaction to be observed. A successful attachment is dependent on the interaction between the affinity tag and the matrix.

2. Washing

Additional amino acids and other proteins are sometimes captured by the stationary phase alongside the targeted molecule. This is known as nonspecific binding. Due to this phenomenon, a suitable buffer must be used to “wash” away the irrelevant material by altering the binding site.

3. Elution

Elution is the process of extracting one material from another. To elute specifically bound proteins from the affinity column, one can implement a similar molecule that binds competitively. Afterwards, the purified molecular sample can be taken for further study.

The elution of an affinity column can be accomplished by inducing changes in salt concentration, pH, pI, charge, and ionic charge. Ionic strength can also be used, either directly or through the use of a gradient.


Applications of affinity chromatography

Laboratory applications of affinity chromatography include:

  • Magnetic beads
  • Fast protein liquid chromatography (FPLC)
  • Batch chromatography
  • Drip columns

Magnetic beads

When ligands are bound to magnetic beds, they increase the efficiency of the protein purification process. A magnetic bead separator is used for this purpose.

Fast protein liquid chromatography (FPLC)

FPLC is the main affinity chromatography method for the purification of proteins. Its advantages include a buffer flow rate that can be controlled via a positive-displacement pump, keeping the total flow of the buffer at a constant rate. FPLC offers highly reproducible results.

Batch chromatography

This method is performed at ambient pressure. It is a very straightforward method for the fast purification of proteins.

Drip columns

These are small disposable plastic columns that have chromatography affinity resin inside. In contrast to FPLC, the control pressure and flow rate can’t be controlled using this method.


Advantages of affinity chromatography

Because it uses the biological structure or function of a protein for purification, affinity chromatography offers improved selectivity, resolution, and capacity in most protein purification schemes. A major benefit of the affinity chromatography method is the speed at which it achieves the purification of proteins.

Performing affinity chromatography is relatively simple compared to other particle separation methods. Its high selectivity allows for the development of single-step affinity purification strategies that can hasten the chromatographic process.

Affinity chromatography can be integrated into a high-pressure liquid chromatography (HPLC) procedure. When this happens, the speed and sensitivity of HPLC are boosted by the high selectivity of affinity chromatography. Potentially, this process can be used to isolate biological molecules such as growth factors being produced in low concentrations.

Chromatographic ultra-performance liquid chromatographic systems 


Valentia Analytical services

If your professional goals are related to HPLC affinity Chromatography, then a partnership with Valentia Analytical may be just what your organization requires.  Thanks to access to the best minds and equipment in the market, Valentia Analytical can help you achieve successful ultra high-performance liquid chromatography (UHPLC). This method offers superior resolution and quality than traditional chromatography procedures.

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