Affinity chromatography technique for separation of biomolecules. It is an topic of MPAT chromatography.

1. Affinity Chromatography
Presented by:
Aniket Murkute
M.Pharm 1 year
(Pharmaceutical chemistry)
Priyadarshini J.L. College of Pharmacy
Electronic Zone Building, Hingna Road
Nagpur-440016
2022-2023

2. CONTENTS
Content Slide Number
Introduction 3
Principle 4-5
Chromatographic Media 6-8
Experimental Procedure 9-11
Detectors 12
Application 13
Summary 14
References 15
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3. Definition: Affinity chromatography is a method of separating
bio-chemical mixtures based on a highly specific interaction, such as
that between Antigen-Antibody, Enzyme-Substrate.
History: Affinity chromatography is a separation technique that
was first developed in the 1960s. The first practical implementation
of affinity chromatography was developed by the Swedish
biochemist ArneTiselius and his colleagues in the 1960s.
INTRODUCTION
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4. • Affinity chromatography is one of the most diverse and powerful
chromatographic methods for purification of a specific molecule or a group of
molecules from complex mixtures
• The affinity of any enzyme to bind with substrate or affinity of antigen to bind with
antibody.
• In this method the stationary phase is Gel Matrix which is made up of linear sugar
molecule obtained from algae(Agarose bead).
• And sample is put in mobile phase solution and run on column.
• When sample is run on column then that enzyme present in sample combine with
ligand molecule, substrate molecule and remaining particle removed out.
Principle:
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6. Chromatographic Media
Stationary Phase:
• It consist of material where column is made from. Beads like Agaros, Silica
are use as stationary phase.
• Legands are bound to these stationary medium.
• It must be insoluble in solvents and buffers employed in the process.
• It should be stable chemically as well as mechanically.
Mobile Phase:
• In mobile phase the sample mixture (eg. blood serum) is present which we
have to separate out particular element or biomolecule. e.g. Antibody to
Antigen, Enzyme to Substrate
• Sample containing enzyme will bind to there specific substrates.
E.g.Hepatoglobin as a substrate to bind with Hemoglobine for it’s
separation
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7. • Immobilized Legand: Immovable complex, made up from legand and
matrix
• Elute: It consist of buffer, which helps to separate out the enzyme from
substrate.
• Buffer: Buffer must be inert (High/low pH) Buffer is used for formation
of complex between a matrix and ligand. As slight change in ionic
concentration weakens the interactions between them.
• Affinity elution: In this method a selective substance added to the
buffer causes selective elution of bound macromolecule-ligand
complex. Resulting in elution of desired macromolecule.
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8. A REACTATNTTOWHICH
ENZYME IS BIND
SUBSTRATE
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9. 1) Column preparation :
• Formation of bead
• We fill the column with gel matrix and legand and leave
it for saturation 12hr.
2) Applying Sample:
• Fill the sample with combination of mobile phase from
upper side of column.
3) Complex formation:
• Here enzyme will bind to there specific substrate and
those who are not coupled will be removed by washing
with buffer solution which is called as wash buffer.
• Complex will stick to there place and extra material like
which are not convert into complex will be wash out.
(Containing particles which we don’t have to test or
have not to find their quantity)
Experimental Procedure:
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10. 4) Wash out: Washing will be done by Elute buffer which
will separate the collected enzyme from its substrate
matrix.
5) Detection of Collected sample molecule: Detection of
sample molecule will be held by various
spectrophotometer
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12. The detector is used to monitor the elution of the target molecule from the column.
• UV-Visible Spectrophotometer: It is used to monitor the absorbance of the eluting fractions at
a specific wavelength.This detector is commonly used for the detection of proteins and nucleic
acids.(For protein 280-300Nm)
• Fluorescence Detector: It is used to monitor the fluorescence of the eluting fractions.This
detector is commonly used for the detection of fluorescent-labeled molecules such as proteins,
peptides, and nucleic acids.
• Radioactivity Detector: It is used to monitor the radioactivity of the eluting fractions.This
detector is commonly used for the detection of radiolabeled molecules.(C-14.K-20)
• Mass Spectrometer: It is used to identify and quantify the eluting fractions based on their mass-
to-charge ratio.This detector is commonly used for the detection of small molecules, peptides,
and proteins.
Detectors use in Affinity Chromatography
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13. • It is used for isolation and purification of all
biological macromolecule.
• It is used to purify nucleic acid, antibodies, enzymes.
• To notice which biological compounds bind to a
particular substance.
• To reduce a amount of substance in a mixture.
APPLICATIONS
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14. Summary
In above slides we have discussed about the affinity chromatography. Affinity chromatography
is a separation technique used to isolate and purify biological macromolecules based on their
highly specific interactions, such as antigen-antibody or enzyme-substrate.The technique
involves using an immobilized complex made up of a ligand and a matrix, and a buffer that
helps to separate the enzyme from the substrate. Affinity elution is a method that involves
adding a selective substance to the buffer to cause the selective elution of the desired
macromolecule.The experimental procedure involves column preparation, applying the
sample, complex formation, and using a detector to monitor the elution of the target molecule
from the column.Affinity chromatography is commonly used for the isolation and purification
of nucleic acids, antibodies, and enzymes, and to reduce the amount of a substance in a
mixture.
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15. References
• Modern experimental biochemistry-rodney f. Boyer
• Chapter 26 Affinity Chromatography: General Methods –
ScienceDirect
• Thomson’s Principle of Instrumental analysis by Douglas A. Skoog
pg.no 848
• Instrumentation method of analysis by Nirali Prakashan.
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