Affinity chromatography Principle, Types, Steps, Applications


Table of Contents

Chromatography is a technique used to separate and analyze mixtures of compounds. There are several different types of chromatography, including gas chromatography, liquid chromatography, and paper chromatography, each of which uses a different method to separate the components of a mixture. For example, in gas chromatography, a mixture is vaporized and then passed through a column packed with a stationary phase, while in liquid chromatography, the mixture is passed through a column containing a liquid stationary phase. Chromatography is widely used in many fields, including chemistry, biochemistry, and forensic science, and is a powerful tool for the analysis and purification of complex mixtures of compounds.

Affinity chromatography is unique in purification technology since it is the only technique that allows biomolecules to be purified according to their biological function or particular chemical structure. The approach was created by P. Cuatrecasas, M. Wilchek, and M. Meir Wilchek, for which they were given the 1987 Wolf Prize in Medicine.


Affinity chromatography separates proteins based on the reversible interaction between a protein (or set of proteins) and a specific ligand bound to the chromatographic matrix. Affinity chromatography makes purifications that would otherwise be time-consuming, difficult, or even impossible to accomplish with other techniques. The method can be used to separate active biomolecules from denatured or functionally distinct versions, to isolate pure compounds present at low concentration in huge amounts of crude sample, and to eliminate particular impurities. The approach gives excellent selectivity, and thus high resolution, as well as typically high capacity for the target protein. Purification can be several thousand-fold, and active substance recoveries are typically quite high.

Biological interactions between ligand and target molecule may result via electrostatic or hydrophobic contacts, van der Waals forces, or hydrogen bonding. To elute the target molecule from the affinity medium, the contact can be reversed either specifically with a competing ligand or non-specifically by adjusting pH, ionic strength, or polarity. The linked ligand must maintain its unique binding affinity for the target molecules, and after washing away unbound material, the binding between the ligand and target molecule must be reversible so that the target molecules can be removed in an active state. Any component may be utilised as a ligand to purify its corresponding binding partner. A single run of a serum or cell-lysate sample through an affinity column can yield higher than 1000-fold purification of a specific protein, resulting in the detection of a single band upon gel electrophoresis (e.g., SDS-PAGE) analysis.

Affinity Chromatography
Affinity Chromatography | Image Source:

What is Affinity Chromatography?

The majority of affinity chromatography techniques are comprised of a stationary phase (solid phase) and the mobile phase. Mobile phase refers to your cells lysate, or any other mixture that is made up of biomolecules. A ligand which binds to the targeted molecule is covalently attached with the solid. The interactions between the solid and mobile phase is exploited through affinity chromatography to produce the desired substance in pure form. 

The target molecule is bound to the ligand while the majority of other molecules pass through. The biomolecule of the target is degraded through changing conditions (pH or concentration of salt) or through the competition with a free ligand.The most crucial property the solid phase should possess is the ability to immobilize ligand. Many materials, such as acrylates and silica gels work well. 


To stop steric interference of the target molecule with the ligand the inhibitor is bonded in the phase of solid. This inhibitor is referred to by the name of spacer. The most common spacer can be described as an inhibitor with an Hydrocarbon Chain (CH2 spacer). Chemicals such as cyanogen bromide and epoxy can functionize the solid phase using hydrocarbon chains that result in different lengths of the carbon chain according to the chemical.

Some typical biological interactions, frequently used in affinity chromatography, can be given as;

  • Enzyme. ↔ substrate analogue, inhibitor, cofactor.
  • Antibody ↔ antigen, virus, cell.
  • Lectin ↔polysaccharide, glycoprotein, cell surface receptor, cell.
  • Nucleic acid ↔ complementary base sequence, histones, nucleic acid polymerase, nucleic acid binding protein.
  • Hormone, vitamin ↔receptor, carrier protein.
  • Glutathione ↔glutathione-S-transferase or GST fusion proteins.
  • Metal ions ↔Poly (His) fusion proteins, native proteins with histidine, cysteine and/or tryptophan residues on their surface.
Affinity chromatography 3 steps
Affinity chromatography 3 steps
Affinity chromatography 3 steps
Affinity chromatography 3 steps

Principle of Affinity chromatography

The stationary phase of affinity chromatography consists of a support medium (e.g. cellulose beads) to which the substrate (or occasionally a coenzyme) has been covalently attached, exposing the reactive groups that are needed for enzyme binding. As the protein mixture is passed through the chromatography column, those proteins that contain a binding site for the immobilised substrate will bind to the stationary phase, while all other proteins will be eluted in the column’s empty volume.

Once all other bound proteins have been eluted, the bound enzyme(s) can be eluted in a number of different ways:

  • by increasing the ionic strength of the buffer, such as with a sodium chloride gradient, so weakening interactions between the enzyme and the immobilised substrate;
  • by altering the buffer’s pH; and
  • by adding a high concentration of substrate (or a substrate analogue) to the elution buffer, so that there is competition between the free and immobilised substrate for the enzyme protein;
Principle of Affinity chromatography
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Linking the substrate to the support medium

  • There are numerous activated agarose gels that can be used to attach ligands; CNBr-agarose is simple to use for the attachment of amines, but it lacks a lengthy spacer between the gel beads and the bound ligand, which may sterically inhibit protein binding.
  • The relatively long methylene chains of aminohexanoic acid-agarose (CH-agarose, for reaction with amines) and diaminohexane-agarose (AH-agarose, for reaction with carboxylic acids) retain the ligand at a large distance from the gel beads.
  • Carbonyldiimadazole-agarose is an alternate reagent for attaching amines, while epoxy-activated agarose is utilised for alcohols.
Linking the substrate to the support medium
Image Source:
Illustration of a variety of molecules that can be purified by using affinity chromatography. (Affinity Chromatography Principles and Methods, 18-1022-29)
Illustration of a variety of molecules that can be purified by using affinity chromatography. (Affinity Chromatography Principles and Methods, 18-1022-29) | Image Source:

Components of affinity chromatography / Affinity Chromatography Instrumentation

1. Matrix 

The matrix is an inert support to which ligands can be attached either directly or indirectly. The features listed below are essential for an efficient and successful chromatographic matrix.

  • Importantly low non-specific adsorption, as the effectiveness of affinity chromatography is dependent on particular interactions.
  • The sugar residues’ hydroxyl groups are readily derivatized for covalent ligand attachment, offering a suitable platform for the construction of affinity media.
  • Since the inside of the matrix is available for ligand attachment, an open pore structure offers excellent binding capacity for even big biomolecules.
  • Excellent flow characteristics for quick separation.
  • Stability in many experimental conditions, including high and low pH, detergents, and dissociating agents.
  • Sepharose, an agarose bead, provides many of the qualities.

In affinity chromatography, particle size and porosity are optimised to increase the available surface area for coupling a ligand and binding the target molecule. High porosity and a small mean particle size improve the surface area. Increasing the matrix’s degree of crosslinking increases its chemical stability, allowing it to endure potentially harsh elution and wash conditions, and provides a matrix that is stiff enough to sustain high flow rates. Although these high flow rates are not typically utilised during separation, they save a great deal of time during column equilibration and cleaning.


Materials utilized in Matrix;

Agarosehydrophilic, almost no unspecific bonds, the gold standard for protein purification
Silica gelnanoporous (leads to unspecific bonds), functionalized via silane, silanes are washed away by alkaline buffer → reduced stability, applications: bound nucleic acids chaotropic
Aluminium oxideacidic surface, binds amines irreversibly, used to reduce the amount of specific substances
Acrylatepartially hydrophobic (unspecific bounds possible), monodisperse particles, used for cell separation
Organic polymerspartially hydrophobic (unspecific bounds possible), monodisperse particlescan be used for ligand coupling, not recommended for protein purification because of unspecific bounds

2. Spacer arm

It is employed to enhance the interaction between ligand and target molecule by overcoming any steric hindrance. An affinity medium generated by attaching tiny ligands, such as enzyme cofactors, directly to Sepharose may exhibit limited binding capacity due to steric interference, i.e. the ligand is unable to access the binding site of the target molecule. The spacer arm consists of a carbon chain positioned between the ligand and the supporting phase.

  • Used when the active site is buried within the sample molecule.
  • It can interact with sample species on its own if it is too lengthy (London interactions)
  • If ligand is too short, it cannot reach the active site on the sample molecule.

The most commonly used coupling systems include:

ChemicalChain length
Cyanogen bromideC1
Epoxide with C6 acidC10

3. Ligand

The ligand is a molecule which binds reversibly with an individual chemical or group of molecules, making it possible to purify the sample using affinity chromatography. The choice of a specific ligand to be used for affinity chromatography can be influenced by two aspects:

  1. the ligand has to show a an irreversible and specific binding affinity for the targeted substance(s)
  2. It should possess chemically modifiable groups that permit it to be bonded to the matrix without damaging the binding function.

Any element can be utilized as a ligand for purification of the binding partner of its choice. A few of the most common biological interactions often used in affinity chromatography are as follows:

  • Enzyme used for substrate analog and inhibitors, as well as cofactor.
  • Antibody used for antigen, virus, cell.
  • Lectin is a polysaccharide glycoprotein cell surface receptor cell.
  • Nucleic acid is used to complement base sequences, histones nucleic acid polymerase and nucleic acid-binding protein.
  • Hormone vitamin, used to receptors, carriers protein.
  • Glutathione used for glutathione-S-transferase or GST fusion proteins.
  • Metal ions that are used in Poly (His) fusion proteins and native proteins that contain histidine, cysteine or tryptophan residues on their surface.

Most commonly used ligands include;

Iron-, aluminium-ionsPhosphoproteins
Chelator + Ni-, Co-ionsHis-tagged proteins

Affinity Chromatography Ligand and Conditions

Protein to PurifyLigandElute With
Antibody (antigen-specific)Antigenic peptideFree peptide
Polyhistidine-tagged proteinNi2+ or Co2+Imidazole or free histidine
FLAG-tagged proteinFLAG-specific antibodyFLAG peptide or low pH
GST-tagged proteinReduced glutathioneFree glutathione
Myc-tagged proteinMyc-specific antibodyLow pH
Antibody (class-specific)Protein A , G, or L or protamineExtremes in pH
DNA-binding proteinHeparinHigh ionic strength

4. Wash Buffer

Conditions that remove unattached compounds from the column without eluting the target molecules or that return the column to its initial conditions (in most cases the binding buffer is used as a wash buffer).

What is Ligand Coupling?

Typically, ligands are immobilised or “coupled” directly to solid support material through the creation of covalent chemical interactions between specific functional groups on the ligand (such as primary amines, sulfhydryls, carboxylic acids, and aldehydes) and reactive groups on the support. Nevertheless, indirect coupling methods are also conceivable. Using the glutathione-GST affinity interaction as an example, a GST-tagged fusion protein can be trapped to a glutathione support and then chemically crosslinked to immobilise it. The chemical reactivity of groups on the stationary phase (typically agarose) requires activation. The most common activation approach is cyanogen bromide activation.

Pre-activated matrices: matrices that have been chemically changed to enhance the coupling of particular ligand types.

Ligand Coupling
Ligand Coupling | Image Source:

What is Elution?

In order to elute the target molecules from the column, the buffer conditions are altered to reverse (weaken) the interaction between the target molecules and ligand.

  • pH elution:  A change in pH modifies the extent of ionisation of charged groups on the ligand and/or the bound protein. This shift may directly affect the binding sites, decreasing their affinity, or indirectly alter affinity through conformational changes. Most commonly, bound compounds are eluted by a stepwise drop in pH. The maximum pH allowed is determined by the chemical stability of the matrix, ligand, and target protein.
  • Ionic strength elution: The precise process of elution by variations in ionic strength is determined by the specific interaction between the ligand and target protein. This is a gentle elution utilising a buffer with increased ionic strength (often NaCl), administered linearly or in stages.
  • Competitive elution: Competitive elution is frequently employed to separate compounds on a group-specific media or when the binding affinity of the ligand/target protein interaction is particularly strong. Either for binding to the target protein or to the ligand, the eluting agent competes.
  • Reduced eluent polarity: Conditions that lessen the polarity of the eluent facilitate elution without deactivating the eluted compounds. Dioxane (up to 10 percent) or ethylene glycol (up to 50 percent) are typical components of this type of eluent.
  • Chaotropic eluents: If alternative elution techniques fail, deforming buffers that modify the structure of proteins, such as guanidine hydrochloride or urea, can be utilised. Chaotropes are likely to denature the eluted protein and should be avoided wherever possible.

Procedure of Affinity Chromatography (affinity chromatography steps)

Steps of Affinity Chromatography 

Step: 1 Attach ligand to column matrix

The binding of the ligand with the matrix requires that a covalent bond is created between both. This is accomplished through derivatization of the sugar-based”hydroxyl groups. It is essential to understand that the substrate may not be able to access the active site of the ligand, in the event that it is hidden inside the ligand. The majority of ligands are connected to spacer arms that are then attached onto the matrix. The matrix-ligand gel is loaded into an column of elution.

Step: 2 Load Protein Mixture onto the Column

After the column is made, the mix containing isolate is then poured into the column for elution. Gravity pulls the solution into the gel because most of the proteins don’t attach to the ligand-matrix. If ligand that is recognized as substrate moves through the gel it bonds to the ligand-matrix complex, stopping its flow within the gel. Certain impurities pass through the gel because of gravity, however the majority remain unbound in the gel column.

Step 3: Proteins Binds to the ligand

To remove these impurities which are not bound the wash must be of high pH or salt concentration or temperature is passed across the gel. It is crucial to make the most powerful wash possible to ensure all impurities are eliminated. After the impurities have been removed then the only thing left of the protein mixture must be the specific isolates.

Step 4: Wash the column to remove the unwanted Materials

To finally collect the an isolate that is attached to the ligand matrix in the gel second wash is run over the column.

Step 5: Wash off the Proteins that are loosely bind

The second wash is based on the reversible properties of binding of the ligand. This lets the bound protein separate from its ligand in the presence of the more powerful wash.

Step 6: Elute proteins that are tightly bound to ligand and collect purified protein and interest

The protein then has the freedom to pass through the gel before being removed.

Steps of Affinity Chromatography 

Elution methods of Affinity Chromatography

1. pH elution

A variation in pH alters the amount of ionization by charged groups on the ligand as well as proteins bound. The alteration could affect site of binding directly decreasing its affinity or trigger an indirect change in affinity due to modifications in the conformation.

A gradual decrease by a step in pH can be the most popular method of eluting bound substances. Chemical stabilities of the matrix the ligand, and the target protein determines the limits of pH which can be used. If a low pH is required then you should collect the fractions into neutralization buffers like 1 M Tris-HCl pH 9 (60-200 mg ul for each ml fraction eluted) to bring the fraction back to an equilibrium pH. The column should be returned in the neutral pH immediately.

2. Ionic strength elution

The exact mechanism behind elimination through changes in the strength of ionic depends on the specific relationship between the ligand and the target protein. This is a moderate elimination using a buffer that has higher Ionic strength (usually NaCl), applied in a linear manner as well as in steps. Enzymes typically elute with a concentration of 1 million NaCl and less.

3. Competitive elution

They are typically used to distinguish substances in an individual medium or when the attraction of the target protein interactions is quite high. The eluting agent is competing with the protein of interest or for binding to the binding ligand. Substances can be eluted by the concentration gradient of one Eluent, or by pulse elimination.

In the case of competitive elution the concentration of the competing compound should be equal to that of the ligand that is coupled. If, however, the competing compound is able to bind more easily than the ligand targeted molecule, you should use an amount that is ten times greater than the ligand.

4. Reduced polarity of eluent

Conditions are employed to reduce the polarity of the eluent and allow for elution but not inactivating the substances that are eluted. Dioxane (up to 10 10%) as well as Ethylene glycol (up to 50 percent) are common to this kind of liquid eluent.

5. Chaotropic eluents

If other methods of elution fail the deforming buffers which alter the protein’s structure can be employed, e.g. Chaotropic agents like the guanidine hydrochloride and urea. Avoid chaotropes whenever possible as they can cause to cause denature of the protein eluted.

Types of Affinity Chromatography

The kind of ligand can be used to classify affinity procedures into subcategories such as lectin, immunoaffinity, and dye ligand, among others. These methods are listed as follows.

1. Lectin Affinity Chromatography

  • Plants, vertebrates, and invertebrates generate lectins, which are non-immunogenic proteins. High numbers of lectins are produced by plant seeds, in particular.
  • Due to the ability of all lectins to identify and bind certain types of carbohydrate residues, this approach can be used to separate them.
  • The most common lectins employed in affinity columns are concanavalin A, soybean lectin, and wheat germ agglutinin. Concanavalin A binds to -D-mannose and -D-glucose residues, whereas wheat germ agglutinin binds to D-N-acetylglucosamine.
  • In Table are listed lectins commonly employed for the extraction of carbohydrate-containing compounds, such as polysaccharides, glycoproteins, and glycolipids, by affinity chromatography. .
  • Bioaffinity chromatography types can also utilise enzymes, inhibitors, cofactors, nucleic acids, hormones, and cell chromatography as ligands. These techniques include DNA Affinity Chromatography and Receptor Affinity Chromatography.
LectinSourceSugar specificityEluting sugar
Con AJack bean seedsα- D-mannose, α- D -glucoseα – D -methyl mannose
WG AWheat germN-acetyl-β- D -glucosamineN-acetyl-β- D -glucosamine
PSAPeasα- D -mannoseα- D -methyl mannose
LELTomatoN-acetyl-β- D -glucosamineN-acetyl-β- D -glucosamine
STLPotato tubersN-acetyl-β- D -glucosamineN-acetyl-β- D -glucosamine
PHARed kidney beanN-acetyl-β- D -glucosamineN-acetyl-β- D -glucosamine
ELBElderberry barkSialic acid or N-acetyl-β- D -glucosamineLactose
GNLSnowdrop bulbsα -1→→ 3 mannoseα -methyl mannose
AAAFreshwater eelα – L-fucoseL -fucose

Applications of Lectin Affinity Chromatography

  • Purification of glycoproteins, specifically the membrane-receptor proteins.
  • Lectins are a class of proteins made by animals and plants that are able to bind glycoproteins and carbohydrates.
  • Useful to separate cells into different types, making use of saccharide component of their outer membranes.
  • The most commonly used lectins include: ConcanavalinA Soyabean lectin, etc..

2. Immuno Affinity chromatography

  • Immunoaffinity chromatography is one of the most widely used affinity-derived procedures, and it permits the production of ligands when the needed ligand is unavailable.
  • In this method, the stationary phase consists of an antibody or an agent linked to antibodies. Due to the high specificity of antibodies, it is possible to isolate varied substances using this method. There are reports that immunoaffinity chromatography can be used to detect natural food pollutants such aflatoxins, fumonisins, and ochratoxins.
  • In order to use antibodies as ligands for the purpose of purification, antibodies are initially immobilised on a support. To adequately bind the ligand to the surface of the support, protein A and G are typically employed as a bridge that offers sufficient space for the ligand-protein interaction.
  • In immunoaffinity application, which is a non-covalent, irreversible purification technique based on highly specific interactions between analyte and antibody, columns, dialysis membranes, capillaries, or beads may be utilised.
  • Before preparing the immunoaffinity column, the antibodies should initially be purified. Purification of antibodies may be accomplished using precipitation with ammonium sulphate, ion-exchange chromatography, gel filtration chromatography, or affinity chromatography.
  • As a support material, activated beads coated with bacterial proteins A or G may be utilised. For the elution of the sample solution, some parameters may be altered, such as the ionic conditions of the mobile phase or the use of chaotropic buffers.
  • IAC can detect both small and big analytes utilising direct detection. Additionally, this technique can be used independently or in conjunction with other chromatographic techniques. If this procedure is implemented as part of an HPLC system, it is known as high performance immunoaffinity chromatography.
  • Immunoaffinity chromatography is perhaps the most specific bioaffinity chromatography technique. However, this technology has significant limitations, including a relatively high cost, the possibility of ligand leaking from the column, and a partial denaturation of the attached protein during the desorption phase.


  • It is used in the isolation and elimination of a variety of proteins such as antigens, membrane proteins that are of viral origin.
  • It is used to purify antibodies.
  • Ligands used include the Protein A as well as protirn G.

Protein A or protein G affinity chromatography

  • Protein A is generated by Staphylococcus aureus and protein G by Streptococcus group G. These ligands can bind to a variety of immunoglobulins at a pH close to neutral and dissociate in a buffer with a lower pH.
  • Protein A interacts with great specificity and affinity to the immunoglobulin G (IgG) derived from human and other mammalian species. In certain instances, protein G may be substituted for protein A.
  • The ability of these two ligands to attach to antibodies of different species and classes differs between them. Protein A and protein G are advantageous ligands for the separation of immunoglobulins due to their high immunoglobulin-binding specificity and affinity.
  • In immunoaffinity applications, protein A and protein G are utilised as secondary ligands for the adsorption of antibosies onto the support material. This technique can also be utilised if high antibody activity or replacement of the antibodies in affinity chromatography is required.

3. Metal-chelate affinity chromatography (Immobilized-metal (Ion) affinity chromatography)

  • In the 1970s, metal-chelate affinity chromatography, also known as “immobilized-metal (ion) affinity chromatography” (IMAC), was applied for the first time.
  • Metal-chelate chromatography technology harnesses selective interactions and affinity between transition metal immobilised on a solid substrate (resin) and amino acid residues that act as electron donors in the target protein.
  • In addition to aromatic and heterocyclic chemicals, proteins including histidine, tyrosine, tyriptophane, and phenylalanine have an attraction for transition metals that form complexes with electron-rich molecules.
  • Histidine is the most frequently utilised amino acid among those listed. Attachment of histidine tags to the polypeptides of recombinant proteins is the most well-known advancement in the field of IMAC. Protein purification commonly employs histidine and other metal affinity tags.
  • By binding chelators to the surface and metals to the chelators, one can create adsorbents. To bind to metal ions, the analyte requires free coordination sites of the metal ions.
  • Zn2+, Ni2+, and Cu2+ are the metal ions that are most frequently employed. Basic groups on protein surfaces, particularly the side chain of histidine residues, are attracted to metal ions, which then form weak coordinate bonds.

Some examples of chelating compouns used in IMAC

Chelating CompoundCoordinationMetal Ions
Aminohydroxamic acidbidentateFe(III)
8-Hydroxy-quinoline (8-HQ)bidentateAl(III), Fe(III), Yb(III)
Iminodiacetic acid (IDA)tridentateCu(II), Zn(II), Ni(II), Co(II)
Dipicolylamine (DPA)tridentateZn(II), Ni(II)
ortho-phosphoserine (OPS)tridentateFe(III), Al(III), Ca(II), Yb(III)
Nitrilotriacetic acid (NTA)tetradentateNi(II)
Carboxymethylated aspartic acid (CM-Asp)tetradentateCa(II), Co(II)
N,N,N’-tri(carboxymethyl)ethylenediamine (TED)pentadentateCu(II), Zn(II)

Applications of Metal-chelate affinity chromatography

  • Special type of chromatography which immobilised metal ions such as Cu2+ Zn2+ , Mn2+, Ni2+ etc. are employed.
  • It is used to purify proteins that contain imidazole groups or indole groups.
  • Commonly metal ions are immobilised by attachment to an imino-diacetate or tris(carboxymethyl)ethylenediamine substituted agarose.

4. Dye Ligand Chromatography

  • Observation of the uneven elution properties of certain proteins during fractionation on a gel filtration column containing blue dextran led to the development of the dye-ligand affinity chromatography. Blue dextran is composed of a triazine dye (cibacron blue F3G-A) covalently attached to dextran with a high molecular mass. Some proteins bind triazine dye, allowing its immobilisation as an affinity adsorbent.
  • This approach is particularly popular for purifying enzymes and proteins. Dye-ligand adsorbents are desirable because they are affordable, readily available, and simple to immobilise. These adsorbents can be utilised for analytical, preparative, and large-scale research.
  • In spite of these benefits, the dye-ligand affinity approach for pharmaceuticals has been discontinued due to leakage and toxicity issues.
  • Therefore, proteins purified with this method are suitable for analytical or technical applications. Dye-ligands such as Procion Red HE3b, Red A, and Cibacron Blue F3G-A are employed for purification.

Applications of Dye Ligand Chromatography

  • Utilizes a variety of triazine dyes for ligands.
  • The most popular color is Cibracron Blue F3G-A.
  • It is used to purify interferons and lipoproteins as well as factors that cause coagulation, etc.

5. Covalent chromatography

  • Specially designed to separate proteins containing thiol
  • The most frequently used ligand an adiquate 2′-pyridyl group
  • It is used to purify many proteins, however its application is restricted due to its price and difficult regeneration.

6. Boronate affinity chromatography

  • This approach is known as boronate affinity chromatography when boronic acid or boronates are used as ligands in affinity chromatography.
  • Most boronate derivatives are known to covalently bind substances with cis-diol groups at a pH greater than 8.
  • Due to the cis-diol groups of the sugars, separation of glycoproteins from non-glucoprotein structures is achievable by the boronate affinity method.
  • This method may be used, for instance, to successfully separate glucohemoglabin and normal haemoglobin or to identify distinct types of glycoproteins in a sample.

There are other chromatographic techniques that are similar to conventional affinity chromatography. As an illustration, Analytical Affinity Chromatography (Quantitative Affinity Chromatography or Biointeraction Chromatography) is a technique used to determine solute-ligand interactions. This technique can be used to study a variety of biological systems, including lectin/sugar, enzyme/inhibitor, protein/protein, and DNA/protein interactions, as well as the binding of medicines or hormones to serum proteins.

This approach enables the detection of drug competition with other medicines or endogenous chemicals for protein binding sites. In studies of drug-protein and hormone-protein binding, either immobilised pharmaceuticals or immobilised proteins may be utilised, although protein-based columns that can be used for numerous experiments are more popular. This method can also be used to study the competitiveness between two solutes for binding sites; this technique is known as Frontal Affinity Chromatography. In addition to affinity chromatography, Hydrophobic Interaction Chromatography and Thiophilic Adsorption are similar techniques. Thiophilic Adsorption (Covalent/Chemisorption Chromatography) uses immobilised thiol groups as ligands to separate sulfhydryl-containing peptides or proteins from mercuric polynucleotides.

In Hydrophobic Interaction Chromatography, proteins, peptides, and nucleic acids bind to short non-polar chains, such as those initially utilised as spacer arms on affinity supports. Methods of chiral liquid chromatography can also be considered affinity-based approaches. Utilized extensively in the pharmaceutical industry and clinical chemistry for the isolation of unique chiral forms of pharmaceuticals and the measurement of different chiral forms of medications or their metabolites. Since most affinity chromatography ligands are chiral, they may be preferred as stationary phases for chiral separations. Protein- and carbohydrate-based ligands may be utilised as stationary phases for the HPLC characterization of chiral substances. Orosomucoid (1-acid glycoprotein), bovine serum albumin, and ovomucoid (glucoprotein of egg whites) are examples of protein-based stationary phases, whereas cyclodextrins (particularly -cyclodextrin) are examples of carbohydrate-based stationary phases.

Types of affinity media used in Affinity Chromatography

A variety of affinity media are available to serve a range of applications. Briefly, they are (generalized) activated/functionalized that work as a functional spacer, support matrix, and eliminates handling of toxic reagents.

  • Amino acid media: It is used in conjunction with a variety of proteins from serum enzymes, and peptides in addition to dsDNA and rRNA. 
  • Avidin biotin media: Avidin biotin media is used to purify the process of biotin/avidin, as well as their derivatives.
  • Carbohydrate bonding is most often used with glycoproteins or any other carbohydrate-containing substance; carbohydrate is used with lectins, glycoproteins, or any other carbohydrate metabolite protein
  • The dye ligand medium is nonspecific however it mimics biological substrates as well as proteins.
  • Hydrophobic interaction medium are frequently employed to attack free carboxyl groups and proteins.
  • Immunoaffinity media uses antigens’ as well as antibodies that have high specificity to differentiate immobilized metal affinity chromatography is further described below. It uses interactions between proteins and metal ions (usually specifically labeled) to separate. Nucleotide/coenzyme which helps separate dehydrogenases, kinases and transaminases.
  • Speciality media are made for specific classes or types of co enzyme. This kind of media can only function to isolate a particular type of protein, or coenzyme.

What is Weak affinity chromatography?

  • WAC is a weak affinity technique. (WAC) is an affinity chromatography technique used for testing affinity for drug development.
  • WAC is an affinity-based technique for liquid chromatography which separates chemical compounds according to their weak affinities with the immobilized object.
  • The greater affinity a compound has with the target the longer it will remain within the separator and this is measured as a longer retention time.
  • The measurement of affinity and rank of affinity are accomplished by processing the retention times of the compounds being studied.
  • Affinity chromatography is a part of a wider set of chemoproteomics techniques for the purpose of identifying drug targets.
  • The WAC technology has been demonstrated against various protein targets , including proteases chaperones, kinases, along with protein-protein interaction (PPI) specific targets. WAC has been proven to be more efficient than traditional methods for screening based on fragments.

What is Stationary phase?

The stationary phase in chromatography is the material that is packed into the chromatography column and through which the mixture of compounds is passed. The stationary phase is responsible for the separation of the components of the mixture, and its properties determine which compounds will be retained in the column and which will elute.

There are different types of stationary phases used in chromatography, including:

  • Solid-phase: the stationary phase is a solid, such as a bead or a powder, and the mixture is passed through the column in a liquid or gas form. Examples include silica gel and alumina.
  • Liquid-phase: the stationary phase is a liquid and the mixture is passed through the column in a liquid form. Examples include normal-phase liquid chromatography, where the stationary phase is a non-polar liquid, and reverse-phase liquid chromatography, where the stationary phase is a polar liquid.
  • Affinity-based: the stationary phase is a bead or matrix that is covalently or non-covalently linked to a specific ligand that binds to the target molecule. This is used in affinity chromatography, where the target molecule is separated from the mixture based on its specific interaction with the ligand.

The choice of stationary phase will depend on the properties of the mixture and the target compound. The stationary phase should have a high selectivity for the target compound and minimal interaction with other components of the mixture.

Scientists have various stationary phase options:

  • Porous supports –Different pore diameters can be generated from agarose, cellulose, silica, and polymethacrylate, which are utilised to create porous supports.
  • Nonporous supports – these often have a smaller surface area compared to porous supports, but can result in faster purification
  • Monolithic supports – These monolithic supports combine big and tiny flow-within pores
  • Membranes – these can be employed for faster purifications, however their lack of porosity reduces their surface area.
  • Expanded-bed absorbents – These absorbents are designed to keep the chromatography column from becoming clogged.
  • Perfusion media (flow-through beads) – These perfusion media (flow-through beads) have pores of varying sizes.

Support particle size is important. Smaller molecules can result in a larger surface area, but they also increase the likelihood of pollutant buildup and undesirable odours. Larger particles can counteract these issues and are therefore frequently employed as an alternative.

It is essential that the stationary phase chosen is not appealing to any molecule in the solution besides the one to be purified. It must be chemically stable and incapable of binding to various solutions, such as enzymes, cleaning agents, and elution buffers, that will be passed through it. To endure the numerous purification treatments that are expected to be carried out, the structure itself must be sturdy.

Protein A Sepharose and Protein G Sepharose Affinity Chromatography

  • The high affinity of protein A (cell wall protein obtained from Staphylococcus aureus) and protein G (derived from groups C and G Streptococci) for the Fc region of polyclonal and monoclonal IgG-type antibodies provides the basis for the purification of IgG, IgG fragments, and subclasses. Protein A can be utilised to separate monoclonal and polyclonal IgG from ascites, serum, tissue culture supernatants, and bioreactor supernatants. Human (excluding IgG3; mouse IgG1 may bind only weakly), rabbit, guinea pig, and pig antibodies should be purified with Protein A. At pH 8.0, the antibody is added to a protein A–Sepharose column, followed by elution at a lower pH.
  • Antibodies bind to protein G better at low pH and poorly at high pH, which is the opposite of the pH-dependent binding profile of protein A. Nevertheless, certain antibodies (mouse IgG1, rabbit, and human antibodies) remain attached to protein G at high pH (8 to 10), therefore it is better to bind the antibody at pH 5 and elute at pH 2.8. This approach is applicable to mouse IgG1, rat (most subclasses attach weakly, however IgG2b may not), monkey, rabbit, cow, goat, horse, and sheep antibodies.
  • Similarly to protein A purification, low-pH elution may result in some loss of antibody binding capacity. Protein A SepharoseTM CL-4B is protein A immobilised on Sepharose CL-4B using the CNBr technique. Protein A interacts with the immunoglobulin heavy chain to attach to the Fc region. The binding of protein A to IgG from a range of mammalian species, as well as certain IgM and IgA, has been thoroughly reported. Protein A Sepharose CL-4B has been utilised as a potent technique for isolating and purifying immunoglobulin classes, subclasses, and fragments from biological fluids and cell culture medium. Due to the fact that only the Fc region is engaged in binding, the Fab region is open for antigen binding. Therefore, Protein A Sepharose CL-4B is extraordinarily effective for separating immune complexes.
  • A large selection of prepacked columns, ready-to-use media, and pre-activated media for ligand coupling through various functional groups makes affinity chromatography accessible for a vast array of applications. The HiTrapTM column family is ideal for normal laboratory scale applications in which the risk of sample cross-contamination must be minimised, for purification of crude materials, or for rapid technique development prior to scaling up purification. HiTrap columns are compatible with syringes, peristaltic pumps, and any KTATMdesign chromatography equipment.

Fraction Collector:

  • A fraction detector is a device that permits regular or specified samples to be collected and stored in a retrievable format from a column eluate. The storage vessels are typically small sample tubes or vials positioned in a rotating disc or on a moving belt; their movement is often governed by a microcontroller.
  • Upon receiving a signal from the microprocessor, the next vial is positioned beneath the column outlet, and the eluate is collected until another signal is received from the computer. After determining the parameters of the chromatogram that describes the separation, the collection programme may be defined. The fractions may be taken at regular intervals or at precise times in order to collect specific peaks.
  • Alternately, fractions can be collected by monitoring the detector output and activating the fraction collector when a peak begins to elute. The peak is then collected in a designated vial. When the peak returns to the base line, the column eluate is discarded until the subsequent peak begins eluting. The majority of liquid chromatographs make use of fraction collectors. They are utilised to gather samples for further purification and subsequent spectroscopic evaluation.

Material Required

  • Ascites fluid or MAb supernatant
  • PBS Buffer
  • Tris base buffer
  • Citric acid
  • Column
  • Dialysis tubing and clamps
  • Magnetic stirrer and Magnetic stir bar
  • Absorbent paper
  • Forceps
  • Glass pipette
  • Pipette pump
  • Refrigerated centrifuge
  • Centrifuge tube
  • 0.45µm filter
  • Syringe
  • Spectrophotometer
  • Pasteur pipette
  • Test tubes

Reagents Preparation

  • PBS (phosphate-buffered saline): 0.23 g NaH2PO4 (anhydrous; 1.9 mM), 1.15 g NaH2PO4 (anhydrous; 8.1 mM), 9.00 g NaCl (154 mM). Increase the volume to 900 ml with water. Adjust the pH to the desired level with 1 M NaOH or 1 M HCl. To achieve a final volume of 1 litre, add water.
  • HCl (1 M): Combine 913.8 ml H2O and 86.2 ml concentrated HCl in the following sequence.
  • NaOH (10 M): Dissolve 400 g NaOH in 450 ml H2O. Add 1 litre of water.


  1. At 4 degrees Celsius, centrifuge the supernatant of monoclonal antibody at 20,000 g x (13,000 rpm in an SS-34 rotor).
  2. Apply a 0.45-μm filter to the supernatant to filter it.
  3. Adjust the pH of the MAb supernatant to 8.0 by dialyzing it against PBS, pH 8.0, using dialysis tubing.
  4. Prepare the protein A–Sepharose column and connect it to the fraction collector.
  5. At either 4°C or ambient temperature, equilibrate column with PBS, pH 8.0.
  6. Antibody solution is applied to a bed of resin.
  7. Wash the column with many litres of PBS at pH 8.0.
  8. For mouse IgG1 use pH 6.5, for IgG2a use pH 4.5, and for IgG2b and IgG3 use pH 3.0.
  9. Collect eluent in vials and dialyze against PBS at a pH of 7.3.
  10. Change the buffer for dialysis twice. Samples should be stored at 4°C in PBS or borate-buffered saline.

Typical biological interactions used in affinity chromatography

Types of ligandTarget molecule
Substrate analogueEnzymes
Nucleic acidComplementary base sequence
AvidinBiotin/Biotin-conjugated molecule
CalmodulinCalmodulin binding partner
GlutathioneGST fusion protein
Protein A or Protein GImmunoglobulins
Nickel-NTApolyhistidine fusion protein

Application of Affinity Chromatography

  • It is used to isolate and purification of all biological macromolecules.
  • It is used to purify nucleic acid,antibodies,enzymes.etc
  • To determine which compounds in the biological world are bound to a specific substance.
  • To decrease the amount of substance present in a mix.
  • Utilized for Genetic Engineering for nucleic acid purification
  • Utilized for the Production of Vaccines – antibody purification from blood serum
  • It is used for Basic Metabolic Research such as the purification of enzymes or proteins from cells free extracts.
  • Affinity chromatography also serves to get rid of particular contaminants, like that of Benzamidine. Sepharose(tm) 6 Fast Flow removes serine proteases like the Factor Xa and thrombin. Figure 2 shows the main phases of an affinity purification.

Advantages of Affinity Chromatography

  • Extremely high-specificity
  • The purest of levels can be achieved
  • The process is highly reproducible.
  • The binding sites of biological molecules could be investigated by simply looking at the binding sites of biological molecules.
  • Single-step purification.
  • The matrix is reusable in a short time.
  • The matrix is solid that is easy to clean and dried.
  • Provide purified products with high yield.
  • Affinity chromatography may also be used to get rid of particular contaminants, for instance proteases.

Disadvantages of Affinity Chromatography

  • Expensive ligands
  • Leakage of the ligand
  • Degradation of the solid support 
  • Relatively low productivity
  • Non-specific adsorption can not be totally eliminated, it can only be minimized.
  • The limited life span and the high cost for immobilized lipdfgands.
  • Proteins are denatured when the necessary pH is not maintained.

Affinity chromatography notes pdf

Affinity chromatography notes by Sourav Pan


What is Affinity Chromatography?

Affinity Chromatography is a method of separating and purifying proteins, nucleic acids, and other biomolecules from a complex mixture by using specific interactions between the biomolecule of interest and an immobilized ligand.

What are the principles of Affinity Chromatography?

The principle of Affinity Chromatography is based on the specific binding interactions between the biomolecule of interest and an immobilized ligand. The immobilized ligand is covalently attached to a solid support, such as beads or a column, and the sample containing the biomolecule of interest is passed through it. The biomolecule of interest will bind to the immobilized ligand while other molecules in the sample will not, allowing for separation and purification of the biomolecule of interest.

What are some common types of Affinity Chromatography?

Some common types of Affinity Chromatography include:
1. Protein A or G Affinity Chromatography: used for purifying antibodies
2. His-tag Affinity Chromatography: used for purifying proteins that have a histidine tag
3. Glutathione Affinity Chromatography: used for purifying proteins that have a glutathione S-transferase tag
4. Nucleic Acid Affinity Chromatography: used for purifying nucleic acids

What are the advantages of Affinity Chromatography?

Affinity Chromatography offers several advantages, such as:
1. High specificity and selectivity
2. High binding capacity
3. High purity of the purified biomolecule
4. Can be used for purifying a wide range of biomolecules

What are the limitations of Affinity Chromatography?

Some limitations of Affinity Chromatography include:
1. The need for a specific immobilized ligand for the biomolecule of interest
2. The need for high sample concentrations
3. The possibility of conformational changes in the purified biomolecule
4. The need for stringent washing conditions to remove non-specifically bound molecules.

Is HPLC an affinity?

No, HPLC (High-Performance Liquid Chromatography) is not considered an affinity chromatography method. HPLC uses different principles for separation and purification, such as size exclusion, ion exchange, or reversed-phase. These methods are based on differences in physical or chemical properties of the molecules, rather than specific binding interactions as in Affinity Chromatography. HPLC can be used for purifying biomolecules, but not specifically through the binding interactions of affinity chromatography.

Is TLC affinity chromatography?

No, TLC (Thin Layer Chromatography) is not considered an affinity chromatography method. TLC is a form of planar chromatography, which is based on the separation of molecules on a solid support by differences in their partitioning between a mobile phase and a stationary phase. In TLC, a small amount of sample is applied to a plate coated with a stationary phase, usually silica gel or alumina. The plate is then developed with a mobile phase, usually a solvent, and the separated components are visualized by staining or other detection methods. TLC is a useful tool for qualitative analysis of samples, but it is not a method for purifying or isolating specific biomolecules as it does not use specific interactions like Affinity Chromatography does.

Which buffer is used in affinity chromatography?

The buffer used in Affinity Chromatography will depend on the specific immobilized ligand and biomolecule of interest. In general, the buffer should be compatible with both the immobilized ligand and the biomolecule, and should not interfere with the specific binding interactions.
For example, if the immobilized ligand is a protein and the biomolecule of interest is another protein, the buffer should be a neutral or slightly alkaline buffer, such as phosphate-buffered saline (PBS) or Tris-buffered saline (TBS). If the immobilized ligand is a nucleic acid and the biomolecule of interest is a protein, the buffer should be a low salt buffer, such as TE buffer (Tris-EDTA).
It is important to note that the buffer used for eluting the purified biomolecule from the immobilized ligand should be different from the buffer used for binding, as it should alter the binding interaction to release the biomolecule. For example, a high salt buffer or chaotropic agent can be used to dissociate the binding interaction between the biomolecule and the immobilized ligand.

Which materials is used for affinity chromatography?

There are different materials that can be used for Affinity Chromatography, depending on the application and the biomolecule of interest. Some common materials include:
Solid supports: These are the materials that the immobilized ligand is attached to. They can be beads, such as agarose or sepharose beads, or a column packed with beads. The beads can be made of different materials such as agarose, sepharose, cellulose, or silica.
Immobilized ligands: These are the specific molecules that are covalently attached to the solid support and that interact with the biomolecule of interest. They can be proteins, such as protein A or G, antibodies, or enzymes, or small molecules such as glutathione or biotin.
Buffers and reagents: Buffers and reagents are used to maintain the appropriate conditions for the binding interaction between the biomolecule of interest and the immobilized ligand. These include buffers for binding, washing, and elution, as well as reagents for detection and analysis of the purified biomolecule.
Detection and analysis reagents: These are used to detect and quantify the purified biomolecule. They can be stain, dyes, or antibodies for visualizing the protein or nucleic acid on gel or plate or mass spectrometry for identification.
It is important to note that the choice of materials should be based on the specific properties of the biomolecule of interest and the immobilized ligand, as well as the downstream applications of the purified biomolecule.

How is the immobilized ligand in Affinity Chromatography prepared?

The immobilized ligand in Affinity Chromatography can be prepared by covalently attaching it to a solid support, such as beads or a column. This can be done through chemical reactions, such as amine coupling or epoxy activation. The solid support can also be pre-coated with the immobilized ligand.

How is the sample prepared for Affinity Chromatography?

The sample for Affinity Chromatography should be prepared in a buffer that is compatible with the immobilized ligand and the biomolecule of interest. The sample should also be of a high enough concentration to ensure efficient binding to the immobilized ligand. In some cases, it may be necessary to denature the sample or add a detergent to solubilize the biomolecule of interest.

How is the purified biomolecule eluted from the immobilized ligand?

The purified biomolecule can be eluted from the immobilized ligand by altering the conditions of the binding interaction, such as by changing the pH or temperature, or by adding a competitive ligand. The purified biomolecule can also be eluted by using a dissociating agent, such as a high salt or chaotropic solution.

How can I know if my purification is successful?

Successful purification can be determined by analyzing the purity of the purified biomolecule using techniques such as SDS-PAGE or HPLC. A high percentage of the total protein or nucleic acid in the sample should be in the purified fraction. Additionally, the presence of other contaminants can be checked using a mass spectrometer or by running a western blot.

Can Affinity Chromatography be used for downstream applications?

Yes, Affinity Chromatography can be used as a step in downstream applications such as protein crystallography, enzymatic assays, and structural biology. The high specificity and selectivity of the method makes it an ideal choice for purifying biomolecules for these applications.


  • Urh M, Simpson D, Zhao K. Affinity chromatography: general methods. Methods Enzymol. 2009;463:417-38. doi: 10.1016/S0076-6879(09)63026-3. PMID: 19892186.
  • Mayers, G. L., & van Oss, C. J. (1998). Affinity Chromatography. Encyclopedia of Immunology, 47–49. doi:10.1006/rwei.1999.0012
  • GODING, J. W. (1996). Affinity Chromatography. Monoclonal Antibodies, 327–351. doi:10.1016/b978-012287023-1/50059-6
  • Bailon, P., Ehrlich, G. K., Fung, W.-J., & Berthold, W. (Eds.). (2000). Affinity Chromatography. Methods in Molecular Biology. doi:10.1007/978-1-60327-261-2 

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