King Abdulaziz University Faculty of Science Chemistry department

Research report
Course Name: Methods of Chromatographic Separation - Chem313

Affinity Chromatography

Prepared By: Reem Farooq Ahmed

Under the supervision of : Dr. Zainab M. Saigl

2nd Semester
2022-2023

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Contents
1. Introduction.....................................................................................................3
2. History of affinity chromatography......................................................……..5
3. Principle of affinity chromatography ............................................….....……7
4. Components of affinity medium .............................................….....……… 9
5. Applications of affinity chromatography .....................................................12
6. Advantages and Disadvantages ...........................……………….................16
7. Limitations of affinity chromatography.…………………….……………..17
8. Reference…...............................................…………………………………18

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1-Introduction:
Affinity chromatography advanced technique has supplanted conventional
purification methods based on pH, ionic strength, or temperature since the
invention of affinity chromatography 50 years ago (Cuatrecasas et al, 1968). Its
been reported that affinity chromatography is used in more than 60% of all
purification methods (Lowe, 1996). Any biomolecule that one intends to purify
typically has an intrinsic recognition site through which it can be bound by a
natural or synthetic molecule, which accounts for the method's broad application.
Therefore, we can conclude that the main mechanism behind affinity
chromatography is the chemical recognition of a target molecule by a molecule
coupled to a column.[1]
Affinity chromatography is the sole method in purification technology that allows
the purification of a biomolecule based on its specific chemical structure or
biological function. This method frequently makes purification possible that would
otherwise be time-consuming, challenging, or even impossible using other
techniques. The technique can be used to separate pure chemicals present in huge
quantities of crude sample at low concentrations as well as to remove particular
impurities. It can also be used to separate active biomolecules from denatured or
functionally different versions.[4]
Chemical separation is a crucial part of modern research and is widely frequently
employed to handle complex samples. Examples range from the large-scale
separation of a recombinant protein to the trace analysis of a medication and
hormone in blood. Because it may be used with a variety of chemicals, the liquid
chromatography method has grown particularly popular for these separations. This
technique can be used in high-performance separations for chemical detection and
measurement or in systems intended to purify a desired product when paired with
the proper support materials. This method is very flexible in terms of the kinds of
chemical or physical qualities that can be used as the foundation for these
separations due to the large variety of stationary phases and mobile phases that can
be used in liquid chromatography. Affinity chromatography, which is generally
referred to as a liquid chromatographic technique that uses a particular binding
agent for the purification or analysis of sample components, is one of the most
adaptable types of liquid chromatography. In several biological systems, there are
selective and reversible interactions that take place, such as when an enzyme binds
to a substrate or an antibody binds to an antigen. By inactivating one of a pair of
interacting molecules onto such a solid support and putting it into a column,
affinity chromatography makes use of these interactions. The affinity ligan is the name
given to the immobilized molecule. The stationary phase of the affinity column is
makes up of this.[2]

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Since its development by Cuatrecasas and coworkers, who isolated acetylcholine’s
tears by using affinity chromatographic technique by attaching a competitive
inhibitor that resembled the normal substrate onto porous agarose, affinity
chromatography has gained widespread acceptance as a potent method for the
isolation and purification of biological macromolecules. This process results in a
product with a significantly higher specific activity and a 6-fold higher yield in a
single step.[7]
Affinity chromatography is a practical and effective separation technique that has
seen widely used. Instead than focusing on the fundamental idea, most research has
solely focused on fixing specific problems, such as purifying a particular enzyme
or antigen. Affinity chromatography's fundamental concepts were discovered at the
same time the technology was first used, in the 1950s, and the "recipe" has since
been widely adopted. The main properties and concepts of affinity chromatography
will be covered in the review that follows, with a focus on those from an engineer's
perspective. [7]
One of the most effective methods for protein purification is affinity
chromatography. Many great works and reviews of the theory of affinity
chromatography have been written throughout the years. Since there are so many
proteins that have been separated using this technique, this res won't go over the
theory underlying it or all of the proteins that have been isolated using it. The main
topics of discussion will be general principles and useful applications, and some of
the more typical advantages and issues that come up when using this method.[6]
Three major steps are involved in affinity purification:
a. Coating an incomplete sample with the affinity support and incubating it
there to allow the target molecule to attach to the immobilized ligand.
b. Washing the support of any non-bound sample components.
c. Elution of the target molecule from the immobilized ligand by altering the
buffer conditions so that the binding interaction no longer occurs.
Since the beginning of this method, the phrase "affinity chromatography" has
generated considerable debate among scientists. According to others, the name
hydrophobic affinity or bioaffinity chromatography (O'Carra et al.,1974) would be
more appropriate (Shaltiel, 1974). However, the definition of affinity
chromatography has been broadened to include a prospective technique for
dissolving biomolecule mixtures based on particular biological interactions.[1]

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History of Affinity Chromatography:
The earliest type of liquid chromatography is affinity chromatography, despite the
fact that many people think of it as a more recent technique. For instance, Michael
Tswett reported the first known application of column liquid chromatography only
seven years prior to the earliest usage of this method. When Emil Starkenstein
examined the binding of insoluble starch to the enzyme -amylase in1910, this did
take place. Additionally, this is the first known case in which liquid
chromatography has been employed for a protein separation.[2]
In 1910, the German scientist, Emil Starkenstein presented an article which
described the concept of resolving macromolecule complexes via their interactions
with an immobilized substrate. The article addressed how chloride affects the
enzymatic activity of liver a-amylase and provided the foundation for various
researchers to use this strategy in the early stages (Arsenis & McCormick, 1966;
Bautz & Hall, 1962; Campbell et al, 1951; Sander et al, 1966). Later, the
terminology affinity chromatography was first used in an essay written in 1968 by
Pedro Cuatecasas, Chris Anfinsen, and Meir Wilchek to describe the process of
purifying enzymes using immobilized substrates and inhibitors (Cuatrecasas et al,
1968). Other early articles described the use of a spacer arm to reduce steric
hindrance and the activation of a Sepharose matrix using a cyanogen bromide
(CNBr) reaction (Axen et al., 1967). (Cuatrecasas et al, 1968).[1]
Affinity chromatography is continuously being developed. It has been critical to
numerous "Omics" technologies, including metabolomics, proteomics, and
genomics. Researchers are now able to investigate areas that were previously
impossible to study, such as protein-protein interactions, post-translational
changes, and protein breakdown, due to the revolutionary development of affinity
liquid chromatography. Finally, the discovery of protein biomarkers has been
helped by the combination of reversed phase affinity chromatography and mass
spectrometry.[1]
The original studies with affinity chromatography all made use of insoluble
materials that served as both the stationary phase and support material. Since that
this is the most basic type of affinity separation, this is not surprising. For instance,
Starkenstein's insoluble starch served as both a substrate and a support for amylase,
which tends to result in the binding and retention of this enzyme. Other researchers
conducted similar work using starch and amylase from the 1920s to the 1940s,
with one of these tests yielding a 300-fold purification. Other examples include the
purification of pepsin using the crystalline protein edestin and the isolation of
porcine elastase using powdered elastin, in addition to the use of polygalacturonase
as a substrate and ligand for the adsorption of alginic acid.[2]

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This suggests that the purification of enzymes was a major focus of most of the
early work using affinity supports. However, research on the selective purification
of antibodies using biological ligands were also being done at this time. This was
inspired by Landsteiner's research, which showed in 1920 how antibodies could
recognize and bind molecules with a certain structure, or "antigens.".[2]
In the 1930s, immune precipitation had become a significant method for antibody
purification due to this type of binding and polyclonal antibodies' capacity to form
insoluble complexes with antigens. For example, Kirk and Sumner employed this
approach to identify antibodies against urease and show that these antibodies were
proteins. This method similar the work being done to purify enzymes in that a
ligand (i.e., the antigen) was employed to generate a particular biological
interaction with the target of interest (the antibodies). However, rather than having
the ligand be the same as the solid used for this isolation, this solid was created as
a byproduct of the binding process.[2]
Even though it has been applied as an experimental separation method for many
years, affinity chromatography as a bio specific technology didn't start until
roughly 20 years ago. One or more biological characteristics of the molecule(s)
being purified are used in this procedure. The isoelectric point (pI),
hydrophobicity, or size of the molecule are not responsible for these interactions.
The really specific reversible interactions between biomolecules are used in this
separation technique.[6]

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Principle of Affinity Chromatography:
Affinity chromatography depends on reversible interactions between the desired
protein to be separated and the affinity ligand attached to the chromatographic
matrix. As stated earlier, the majority of proteins have a built-in recognition site
that can be exploited to choose the right affinity ligand. The chosen ligand and the
target protein must bind with specificity and reversibility.[1]

Fig. 1. Typical affinity chromatography purification

Figure 1 shows a typical affinity purification process, which has numerous phases.
In the begin, samples are administered in a way that maximizes binding with the
affinity ligand. After the application of the sample, unbound substances are
removed using a washing phase, leaving the targeted (bound) molecule still bound
to the affinity support. A desorption step is usually carried out to release and elute
the bound molecules, either1) selectively employing a competing ligand or2) non-
specifically by altering the media environment (for example, by changing the ionic
strength, pH, or polarity) (Zachariou, 2008). As the elution is carrier out , the
purified protein can be collected in a concentrated form.[1]

As unlike to most other types of chromatography and methods like electrophoresis

and centrifugation, affinity chromatography does not rely on variations in the
physical properties of the analytes to separate and purify the analytes. Instead,
separation and purification are accomplished by taking advantage of a special
property of extremely specialized biological interactions. As a consequence,
affinity chromatography has the potential to provide absolute purification in a
single step, even from complex mixtures. Originally designed to purify enzymes,
the technique has since been applied to nucleotides, nucleic acids,

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immunoglobulins, membrane receptors, even whole cells and cell fragments,
nucleic acids, and membrane receptors. The substance to be separated must be able
to bind potentially to a specific ligand that is coupled to an insoluble matrix in
order for the process to work.[5]
Only that compound will bind to the immobilized ligand, which is generally found
in a traditional chromatography column, under the proper experimental
circumstances when a complex mixture containing the particular molecule to be
isolated is added. Therefore, all other compounds may be washed away, and the
compound may then be retrieved by displacement from the ligand (Fig. 11.10). The
method needs a thorough understanding of the structure and biological specificity
of the chemical to be purified in order to properly select the separation conditions
that have the best chance of being effective. A competitive reversible inhibitor, an
allosteric modulator, or the substrate all could be considered ligands in the context
of an enzyme. Normally, the conditions chosen would be ones that promote the
best enzyme-ligand binding.[5]
As the enzyme is gradually added to the insolubilize ligand in a column, the
enzyme molecules will be stimulated to bind, and a dynamic situation will develop
in which the concentration of the complex and the strength of the binding increase.
The success of the technique depends on the reversible formation of the complex
and on the numerical values of the first-order rate constants k1 and k-1.[5]

8
 Fig. 2 .Principle of purification of an enzyme by affinity chromatography.

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Components of affinity medium:
When affinity chromatography is used for the purification and separation of large
biomolecules from complex mixtures, the support (matrix), spacer arms, and ligand
must be considered.[1]

 Affinity supports (matrix):

An inert support, the matrix, can be coupled to by a ligand either directly or
indirectly. Many of the qualities required for an efficient and effective
chromatography matrix are highlighted in the list below.[4]
 Very little non-specific adsorption is necessary since affinity
chromatography relies on particular contacts for success.
 • Sugar residues' hydroxyl groups could be easily derivatized for ligand
covalent attachment, making them the perfect building block for affinity
media.
 Since the interior of the matrix is open to ligand attachment, an open pore
structure ensures high capacity binding even for large biomolecules.
 Good flow properties allow for rapid separation.
 Stability under a range of experimental conditions, including high and low
pH, detergents, and dissociating agents.[4]

 Spacer arms:
A spacer arm is frequently placed between the matrix and ligand to aid efficient
binding and provide a more effective and better binding environment because the
target molecule's binding sites can occasionally be deeply located and difficult to
access due to steric hindrance. See Figure 3.[1]

Fig. 3. Chromatogram showing better ligation and elution when spacer arms are introduced between the ligand and matrix

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These spacer arms' length is crucial. A binding failure or even non-specific binding
may result from arms that are either too short or too long. Typically, when
coupling molecules less than 1000 Da, the spacer arms are used.[1]

 Ligand:
The chemical nature of a ligand is dictated by the biological specificity of the
substance that requires to be purified. In actuality, it is sometimes possible to
choose a ligand that exhibits absolute specificity, binding just to a single
compound. More commonly, it is feasible to choose a ligand that exhibits group
selectivity, which means that it will bind to a family of chemicals that share a
common chemical specificity. The latter type of ligands includes compounds like
5'AMP, which can bind reversibly to a variety of NAD*-dependent
dehydrogenases due to its structural resemblance to a component of the NAD*
molecule. The ligand must have a suitable chemical group that can be used to
connect it to the matrix but won't be involved in the reversible binding of the
ligand to the macromolecule. These groups most common members are NH,
COOH, SH, and OH (phenolic and alcoholic).[5]
It is typically preferable to interpose a spacer arm between the ligand and the
matrix in order to prevent the ligand's attachment from interfering with its capacity
to bind the macromolecule. This spacer arm's ideal length is six to ten carbon
atoms, or their equivalent. In some cases, the chemical composition of this spacer
is crucial to the separation's success. Most commonly, methylene (CH) groups
make up the hydrophobic spacers, whereas carbonyl (CO) or imide (NH) groups
make up the hydrophilic spacers. In general, macromolecular ligands (such as
those used in immu-noaffinity chromatography) do not require spacers since their
binding site for the mobile macromolecule is adequately displaced from the matrix.
However, spacers are crucial for small immobilized ligands. Commercially
available supports of the agarose, dextran, and polyacrylamide types come with a
range of pre-attached spacer arms and ligands that are ready for use immediately
Table 11.5 lists some ligand examples. Any GST-tagged cloned protein can be
isolated using the agarose support Glutathione Sepharose High Performance.
PreScissionTM Protease, that will be coupled to the column and remove the tag as
the protein is eluted, can be used to remove the GST tag off the protein in a single
step by adding it to the matrix.[5]

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Applications of Affinity Chromatography:
Affinity chromatography has been used to purify many proteins, including
immunoglobulins, receptor proteins, and enzymes. The only restriction on the
technique's use is the lack of immobilized ligands. The ideas were expanded to
include nucleic acids and significantly aided advancements in molecular biology.
For instance, messenger RNA is frequently isolated by selective hybridisation on
poly(U)-Sepharose 4B by taking use of its poly(A) tail. Complementary RNA and
DNA can be isolated using single-stranded DNA which has been immobilized.
While columns can be used to achieve this separation, it is typically carried out
using single-stranded DNA immobilized on nitrocellulose filters. For the isolation
of proteins involved in nucleic acid metabolism, immobilized nucleotides are
helpful.[5]
Initially developed for the purpose of isolating and purifying proteins, affinity
chromatography's potential in this area is now understood. There are certainly
more applications for this method outside the purification of proteins. To purify
other nucleic acids, DNA and RNA have both been attached to insoluble matrices.
Immobilized lectins have been used to extract carbohydrates and glycoproteins.
Affinity chromatography was also used to purify cell organelles and even entire
cells.[7]
Affinity chromatography is often used for preparative applications, but it is also
used for investigative purposes. It has been discussed how this technique can be
used to quantitatively study protein-ligand interactions. Affinity chromatography
may have been most helpful in mechanistic investigations, where it helped validate
the findings of previous kinetic studies; For example, O'Carra and Barry s0,154)
have shown how to apply the technique to get highly precise information regarding
the compulsory-ordered kinetic mechanism of lactate dehydrogenase (LDH) in the
binding with NADH and its substrates..[7]
Additionally, they stated that the "AMP component" of the dinucleotide NADH is
mostly in charge of causing NADH to bind to LDH. There are many examples in
the literature that show the existence of such a dinucleotide binding domain or the
necessity of a certain moiety at a specific position in order to alter the binding.
Thus, affinity chromatography offers a practical method for investigating active-
site binding mechanisms.[7]
Most bindings in experimental affinity chromatography require at least two
macromolecular molecules, such as an enzyme, an antibody, or a hormone, and
take place between these molecules. The macromolecule determines the affinity
adsorption's specificity. However, the potential for applying the concept of
selective binding to small-molecule systems has not yet been fully realized.
Whitesides and Nishikawa's review was brief. [7]

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One method for isolating tiny molecules is to utilize an affinity adsorbent that
contains the required antibody (also a macromolecule). However, because of the
complex nature of the processes employed to generate and isolate certain
antibodies as well as the limited capacity of columns holding such immobilized
macromolecular ligands, this approach is not practicable. Utilizing stereo-specific
absorbents to resolve optical isomers according to their stereoselectivity is an
alternative method. Buss and Vermeulen have reviewed and examined this process.
In addition to naturally occurring stereospecific adsorbents like wool, silk, starch,
quartz, lactose, and cellulose, numerous synthetic asymmetric adsorbents have
been created with stereoselectivity on par with enzymes, potentially providing an
alluring alternative for the resolution of optical isomers.[7]

 Immunoglobulin purification (antibody immobilization):

Both covalent and adsorption methods can be used to immobilize antibodies. The
majority of random covalent immobilization techniques use cyanogen bromide, N-
hydroxysuccinimide, N,N'-carbonyldiimidazole, tresyl chloride, or tosyl chloride to
attach antibodies to the solid substrate via their free amine groups. On an activated
support, free amine groups can also react with aldehyde or free epoxy groups.
Since these techniques of immobilization are random, the antibody binding sites
may be blocked as a result of incorrect orientation, multi-site attachment, or steric
hindrance.[1]
By changing the carbohydrate residues in the F region of the antibody to aldehyde
residues which can react with amine or hydrazide supports, site-specific covalent
immobilization of antibodies can be accomplished (Ruhn et al, 1994). The free
sulfhydryl groups of Fab fragments can be used to further immobilize antibodies at
specified sites. Several well-established techniques, such as epoxy, divinylsulfone,
iodoacetyl, bromoacetyl, thiol, maleimide, TNB-thiol, tresyl chloride, or tosyl
chloride, can be used to couple these groups to an affinity support (Hermanson et
al, 1992).[1]
Adsorbing antibodies onto secondary ligands is another technique for immobilizing
them. For example, if hydrazide biotin is used to react with an antibody, the
hydrazide may react with oxidized carbohydrate residues on the antibody's Fc
region. An avidin or streptavidin affinity support can then be used to adsorb the
resulting biotinylated antibody. Using commercially available biotinylation kits,
this method of biotin immobilization enables site-specific antibody immobilization.
[1]
As an alternative, due to the way specifically antibodies bind with protein A and
protein G, they can be directly adsorbed onto these supports. By employing a
powerful eluent, regenerating the protein A/G, and re-applying fresh antibodies,

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immobilized antibodies on the protein A or G support can be easily changed. This
method is generally employed when a high capacity/high activity support is
required. Adsorbed antibodies may be cross-linked to the support material using
carbodimide (Phillips et al.,1985) or dimethyl pimelimidate if a more long-lasting
immobilization is needed (Schneider et al, 1982; Sisson & Castor, 1990).[1]

 Applications of modern affinity chromatography

Affinity chromatography can be applied to a wide range of wavs for chemical or
biological separations, purification, analysis, or characterization. Based on research
done in the United States that has employed this technique, Fig. 4 illustrates
various domains in which affinity chromatography has been used often. Many of
these applications (a combined total of 49.2% for the publications analyzed in
Fig.4) have been in the fields of molecular biology, biochemistry, and biochemical
research. This is understandable considering that these fields were where affinity
chromatography was first developed to address the need for precise separations.
This is not unexpected given that affinity chromatography was created to initially
address the demand for precise separations in these domains. Similar to this, this
separation technique is routinely used in the fields of biotechnology, microbiology,
cell biology, and immunology (a combined total of 22.7%). Along with
pharmacology and pharmaceutical science(4.2%), analytical chemistry and other
branches of chemistry (such as environmental chemistry, clinical chemistry, and
multidisciplinary chemistry) make up another significant group of applications for
affinity chromatography (17.7%). Following the creation of high-performance
affinity methods in the late 1970s and early 1980s, these applications, which first
appeared in the early 1970s, experienced tremendous expansion. Another constant
area of collaboration is the application of affinity chromatography in biophysical
studies of biological systems(4.2%), with work based on this combination initially
appearing in the middle of the 1970s.[10]

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 Fig. 4. Fields in which affinity chromatography is often used in chemical or biochemical separations, isolation,
analvsis, or characterization. These results are based on a literature search made using Scilinder during anuary 2019
for areas with daders in the listed helds that were linked to the term attinity chromatogradhy". The
percentages shown in this graph are based on a total of 46,335 papers that were obtained during this search.

A lot of the applications shown in Fig. 4 make use of affinity chromatography as a

technique for biomolecule purification. This represents the growth of this method's
history. This kind of application makes use of affinity chromatography's ability to
deliver a target with high selectivity and strong binding, typically allowing for the
isolation of the target in just one or two steps even when it is present in a complex
matrix. The use of affinity chromatography for the small-scale and large-scale
purification of enzymes, native proteins, and recombinant proteins, such as by
utilizing ave ligand or biomimetic affinity chromatography, are significant
examples of these applications. IMAC's separation of his-tag proteins and IAC's
isolation of certain antibodies or antigens are two examples of prevalent small-
scale applications.[10]

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Advantages and disadvantages of Affinity Chromatography:[9][6][7]

Advantages
 Extremely high specificity
 High degrees of purity can be obtained
 The process is very reproducible
 The binding sites of biological molecules can be simply investigated
 Affinity chromatography can also be used to remove specific contaminants,
such as
 proteases.
 Single step purification
 The matrix can be reused rapidly.
 The matrix is a solid, can be easily washed and dried.

Disadvantages
 Expensive ligands
 Degradation of the solid support
 Non-specific adsorption
 Relatively low productivity
 Limited lifetime
 Leakage of ligand


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Limitations of Affinity Chromatography:[8]
 Time consuming method.
 More amounts of solvents are required which may be expensive.
 Intense labor.
 Non-specific adsorption cannot be totally eliminated, it can onlv be
minimized.
 Limited availability and high cost of immobilized ligands.
 Proteins get denatured if required pH is not adiusted.

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8- Reference:
1. Magdeldin, Sameh, ed. Affinity chromatography. BoD–Books on Demand,
2012.
2. Hage, David S., and Jack Cazes. Handbook of affinity chromatography.
CRC Press, 2005.
3. Pfaunmiller, Erika L., et al. "Affinity chromatography." Analytical
separation science (2015): 461-482.
4. Biotech, Amersham Pharmacia. "Affinity Chromatography: Principles and
Methods (handbook)." Biotech UK Limited (1999).
5. Wilson, Keith, and John Walker, eds. Principles and techniques of
biochemistry and molecular biology. Cambridge university press, 2010.
6. Ostrove, Steven. "[29] Affinity chromatography: General
methods." Methods in Enzymology. Vol. 182. Academic Press, 1990. 357-
371.
7. Yang, Che-Ming, and George T. Tsao. "Affinity
chromatography." Chromatography. Berlin, Heidelberg: Springer Berlin
Heidelberg, 2005. 19-42.
8. Tesser, Godefridus Ignatius, Hans‐Ulrich Fisch, and Robert Schwyzer.
"Limitations of affinity chromatography: solvolytic detachment of ligands
from polymeric supports." Helvetica Chimica Acta 57.6 (1974): 1718-1730.
9. Lundqvist, Andreas, and Per Lundahl. "Advantages of quantitative affinity
chromatography for the analysis of solute interaction with membrane
proteins." Journal of Biochemical and Biophysical Methods 49.1-3 (2001):
507-521.
10.Rodriguez, Elliott L., et al. "Affinity
chromatography: A review of trends and
developments over the past 50 years." Journal of
Chromatography B 1157 (2020): 122332.

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