There are a variety of uses for affinity chromatography purification techniques. They are being increasingly used to separate pharmaceutical and biological samples nowadays. Here are several ways that the technique is used.
Boronate and Phenyl Borate Affinity Chromatography
In these methods, boronate or phenyl borate can be used as affinity ligands in combination with agarose or high-performing affinity chromatography (HPAC) methods for the stationary phase. The boronate or phenyl borate bind to molecules that contain cis-diol groups, that can be present in many carbohydrates.
Boronate affinity chromatography can be used in the analysis of hemoglobin A1c (HbA1c) which is a component of glycated hemoglobin. It can also capture glycoproteins, such as lactoferrin, by using a capillary boronate affinity monolith structure.
Similarly, cellufine phenyl borate is an affinity ligand that can be used for the purification of glycoprotein, glycated-protein, and diol compounds. The beads are packed in a spherical cellulose structure and a pH level between 3 and 12 is used.
Principles of Chromatography
Lectin Affinity Chromatography
This form of chromatography is growing in use. Lectins are non-immune system proteins such as glycoproteins. They originate from animals, plants, and microorganisms and have an affinity for carbohydrate residues. For example, they can separate polysaccharides, glycopeptides, and oligosaccharides and cells that contain particular carbohydrate structures.
Lectins can be used with an agarose support. The purification method can also be combined with other types of analysis equipment such as high-erformance liquid chromatography (HPLC) and mass spectrometry. Also, several lectin columns can be combined for the purification of glycoproteins and glycoconjugates in an approach called serial lectin affinity chromatography.
Immobilized Metal Ion Affinity Chromatography
Immobilized metal ion affinity chromatography (IMAC) involves binding with target molecules such as proteins, nucleic acids, amino acids and peptides and metal ions, for example Zn2+, Ni2+, Cu2+, and Fe3+ that have been immobilized in a column. The ions have been introduced to the column with the help of chelating agents such as nitriloacetic acid and iminodiacetic acid. Solutions to be analyzed can include phosphorylated proteins, membrane proteins, and histidine-tagged proteins.
Dye-Ligand Affinity Chromatography
This is a method used to purify blood proteins, protein pharmaceutical agents, some enzymes, and albumin. The dyes, for example, bind at the proteins reactive site. The columns contain synthetic triazine dyes which help remove molecules such as hepatitis B viral particles and prion proteins.
Frontal Analysis Chromatography
A mobile phase containing a known concentration of a target is flushed through an affinity column with an immobilized binding agent which becomes saturated. Readings during the process will help to create a curve which will have a mean position that can contribute to the production of the curve. This curve will provide a guide that helps scientists to derive the kind of interactions that might be happening with other binding agents. This can be used to help determine the binding of drugs.
Immunoaffinity Chromatography
In this process, molecules such as peptides, viruses, hormones, and enzymes can be purified in a column containing antibodies or other similar agents. Agarose or HPAC can be used as the support. The pH is changed or a chaotropic agent or organic modifier is added to trigger the elution stage.
Analytical Affinity Chromatography
This method is used to study the reactions between biomolecules with lectins, antibodies, and aptamers or drug binding with agents such as enzymes, serum proteins, and receptors.
Zonal elution is often employed in this method where a small amount of a drug is introduced to the affinity column. The retention factor is analyzed under different conditions.
The process can also be carried out in combination with HPAC for high throughput analysis of drug interactions. Once again, the conditions such as the temperature, pH, and solvent polarity can be changed to see the effect on retention.
Equipment
Several life sciences companies including AMSBIO provide the component parts for affinity chromatography.
References
Further Reading
Why use light scattering to analyze proteins and viral vectors?