Affinity Chromatography.pdf

Affinity Chromatography involves the covalent attachment of an immobilized biochemical called as affinity ligand to a solid support. When a sample is passed through the column, only solute that selectively binds to the complementary ligand is retained; other sample components elute without retention. The separation exploit the “lock and key” binding that is prevalent in biological systems. The retention solutes can be eluted from the column by changing the mobile phase composition.

1. AFFINITY CHROMATOGRAPHY
Presented By:
Miss. Mohini Tawade
First Year M. Pharmacy,
Department of Quality Assurance,
Dr.D.Y. Patil College of Pharmacy, Akurdi, Pune.

2. INTRODUCTION
Thehigh-selectivityseparationofbiomoleculescanbeachievedbyusingaffinitychromatography
Thisseparationtechniqueisspecialanduniquebecausetheseparationisachievedbasedonthe
biologicalfunctionortheuniquechemicalstructureofabiomolecule
Thisfeaturemakesaffinitychromatographysuitablefortheselectiveseparationoftheactive
biomoleculesandtheirisolationfromtheinactiveordenaturedforms
Thisbiomoleculebindsselectivelytotheaffinityligand,whicharefixedonstationaryphaseandthe
bindingofbiomoleculeswithaffinityligandisreversible.
Thematerialiselutedfromcolumnbychangingthecompositionofmobilephase
Thedifferentbiomoleculesthatcanbeseparatedbyusingthistechniqueareenzymes,proteins.
hormonesetc.

3. AffinityChromatographyinvolvesthecovalentattachmentofanimmobilized
biochemicalcalledasaffinityligandtoasolidsupport.
Whenasampleispassedthroughthecolumn,onlysolutethatselectivelybindstothe
complementaryligandisretained;othersamplecomponentselutewithoutretention.
Theseparationexploitthe“lockandkey”bindingthatisprevalentinbiological
systems.
Theretentionsolutescanbeelutedfromthecolumnbychangingthemobilephase
composition.
PRINCIPLE

4. INSTRUMENTATION
.
1.Stationary Phase
2.Affinity Ligand
3.Sample Preparation
4.Binding of Molecule
5.Elution of Molecules
6.Regeneration

5. Sameas that of high performance liquid chromatography
Only change in the stationary phase
Here to stationary phase, affinity ligand binds and by lock and key mechanism biomolecules (Target
molecules) get attached to ligand and separation takes place

6. Stationary Phase
Ideal properties of stationary phase
Should be sufficiently hydrophilic to avoid nonspecific binding of solutes
Stableto most water soluble organic solvents
Porous in nature to permit a high degree of ligand attachment
Agarose is very popular matrix, porous in nature
other examples Cellulose, dextran, polyacrylamide, combination polymers, microporous
glass beads

7. Affinity Ligand
A short alkyl chain of hexamethylenediamineis inserted between the ligand and stationary
phase to reduce or eliminate steric influence of stationary phase.
2 types
Specific ligands:
Bindsto one particular solute
Advantage
High selectivity
Short column is used for separation
Separation in less than 1 min
Group specific ligands:
Binds to certain groups of solutes Separation
can be done by using isocratic or gradient
elution techniques
For better separation longer columns are used

8. Sample Preparation
Sample must be clear solution free from solid particles
0.45-μm pore size filters can be used for filtration
To maintain the solubility and stability of sample, influence of pH, salt concentration and
presence of any organic solvent need to be considered
Factors affecting interaction of between desired target protein and matrix bound affinity
ligands are pH, salt concentration, temperature
Sample components interfering with affinity ligand should be removedbefore loading onto
the column

9. BindingofTheMoleculeofInterestAndWash-outofThe
UnboundMaterial
Sampleloadingsdependsonthestrengthofinteraction
Forhigh-affinitysampleshighflowratemaybeapplied
Incaseofweakinteractionorslowequilibriumprocess,reducedtherateofsample
loading.
Aftersampleapplication,columnisfurtherwashedwithbindingbuffer(itensures
interactionoftargetmoleculeswithligandsandothermoleculesarewashedthrough
column)untilallunboundcomponentsareremoved

10. Elution Of The Molecules Of Interest By Changing
The Composition Of The Mobile Phase
Elution via pH or ionic strength changes:
Elution achieved by decreasing the interaction
strength between the ligand and target
protein
Change in pH will change the ionization states
of charged groups of ligand and target protein ,
thereby changing the strength of interaction
Increasing ionic strength, reduces the
interaction strength ( done by raising NaCl
concentration)
Competitive Elution:
Here materials are used that react with the
target molecule or Ligand , competing for
pre-existing interaction.

11. Regeneration
Aftersuccessfulcompletionoftheelution,thecolumncanbewashedwith
severalcolumnvolumesofbindingbuffer,anditcanthenbereused.
Forlong-termstorage,onemustensurethatthecolumnisnotexposedto
bacterialorfungalinfection.
Thetoxiccompoundsodiumazidecanbeusedtopreventsuchinfections.

12. Affinity ligands are bound on solid support
Loading of sample containing molecule of interest (Target
Molecules)
Allowsthe target molecule in the sample to bind to the
immobilized ligand (Affinity ligand)

13. Highly selective target molecules binds to
affinity ligand
Washing away non-bound molecules from the
solid support.

14. Elution(dissociationandrecovery)ofthetarget
moleculefromtheimmobilizedligandby
changingthecompositionofmobilephase

15. Advantages:
The technique provides high selectivity, high resolution and generally high capacity
for the desired protein.
Thus the degree of purification of protein is thousands times greaterthan the other
techniques.
Also the isolation of biomolecule can be carried out from the inactive or denatured
forms.
Another significant advantage of the method is that, in many cases, it allows single-
step isolationof the desired biomolecule.

16. Time consuming method
More amounts of solvents are required which may be expensive.
Non-specific adsorption cannot be totally eliminated, it can only be minimized.
Limited availability and high cost of affinity ligands.
Proteins get denaturedif required pH is not adjusted.
Disadvantages:

17. Application:
Used to purify and concentrate a substance from a mixture, this reduces the amount of
unwanted substances in mixture
Used for separation of antibodies, where antigen are bound chemically to the stationary
phase
Analysis of enzymes can also be carried out using affinity chromatography
Used for isolation and purification of biological macromolecules along with nucleic acid,
proteins, enzymes etc
Investigation of binding sites for enzyme

18. Reference:
https://www.slideshare.net/sagarsavale1/affinity-chromatography-56328075
https://www.slideshare.net/rajpalchoudharyjat/affinity-chromatography-71915487
https://www.slideshare.net/ArvindHeer/affinity-chromatography-64645157
UrhM, Simpson D, Zhao K. Affinity chromatography: general methods. Methods
Enzymol. 2009;463:417-38. doi: 10.1016/S0076-6879(09)63026-3. PMID:
19892186.

19. MCQ
In affinity chromatography separation is based on:
a.Molecular size
b.Molecular structure
c.Specificity
d.Stereochemistry
The molecule of interest can be immobilized through:
a.Hydrogen bond
b.Sulphur bond
c.Covalent bond
d.Nitrogen bond

20. Specific ligands bind only to:
a.One particular solute
b.Certain groups of solutes
c.One or certain groups of ligand
d.None of this
When the medium is bound to protein of interest it becomes.
a.Solubilized
b.Partialized
c.Equilibrated
d.Immobilized
Affinity chromatography mostly uses:
a.Antibody coated beads
b.Column with empty beads
c.Only mobile phase
d.Only column

21. THANK YOU!