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Applications of Chromatographic
Techniques
HPLC TYPE APPLICATIONS‐ Affinity , Ion Exchange, Gel Permeation
Affinity Chromatography
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Affinity Chromatography
• Ligand is an atom, ion, or molecule that generally
donates one or more of its electrons through a
coordinate covalent bond to, or shares its electrons
th
through
h a covalent
l t bond
b d with
ith one or more central
t l
atoms or ions .
• Two types of Ligands are brought into use:
– Specific
– General
• Specific Ligands : Binds only to one species
• Group Specific Ligands: Binds to specific groups on
target species.
Affinity Chromatography
Ligand Types
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Affinity Chromatography
SPACER
• Carbon chain interposed between Ligand and Matrix.
• Used when active site is located deep within the
sample molecule
• If too long it can interact with sample species on its
own ( hydrophobic interactions)
• If too short the ligand is unable to reach the active
sample molecule.
molecule
• Commercial phases have spacers which are optimized
for specific separations.
Affinity Chromatography
• Should be a rigid, stable and high surface
area.
• It must be insoluble in solvents and
buffers employed in the process
• it mustt be
b easily
il coupled
l d to
t a ligand
li d or spacer
arm onto which the ligand can be attached
• must exhibit good flow properties and have a
relativley large surface area for attachment
• Agarose is the most popular however,
cellulose, dextrans and polyacrylamide has
also been used frequently.
• Sepharose is a bead form of agarose gel.
gel
• In general any matrix useful for ion
exchange or gel filtration is also good for
affinity chromatography.
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Affinity Chromatography
FORMATION OF A PHASE
• Ligand should be bound to support to create a stable
phase‐ immobilization.
• Two Steps
–Activation of the support with a reactive
compound.
–Attachment of a ligand
• How the spacer arm is attached to the ligand is
important, as it should not interfere with ligand binding
to the protein?
• But usually the best way to attach a ligand has to be
worked out by trial and error, synthesizing small test
molecules with alkyl groups attached to the ligand in
various ways and determining which bind best to the
protein.
Affinity Chromatography
FORMATION OF A PHASE
• Ligand should be bound to support to create a stable
phase‐ immobilization.
• Two Steps
–Activation of the support with a reactive
compound.
–Attachment of a ligand
• How the spacer arm is attached to the ligand is
important, as it should not interfere with ligand binding
to the protein?
• But usually the best way to attach a ligand has to be
worked out by trial and error, synthesizing small test
molecules with groups attached to the ligand in various
ways and determining which bind best to the target
molecule.
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Affinity Chromatography
FORMATION OF A PHASE
• Ligand should be bound to support to create a stable
phase‐ immobilization.
• Two Steps
–Activation of the support with a reactive
compound.
–Attachment of a ligand
• How the spacer arm is attached to the ligand is
important, as it should not interfere with ligand binding
to the protein?
• But usually the best way to attach a ligand has to be
worked out by trial and error, synthesizing small test
molecules with groups attached to the ligand in various
ways and determining which bind best to the target
molecule.
Affinity Chromatography
Specific Phases
Type Specificity
Protein A- Sepahrose Cl4B Fc region of IgG and related
molecules
Con A- Sepharose Terminal –D- glucopyranosyl ,
D- mannopyranosyl or similar
residues
Blue Sepharose- Cl6B Broad range of enzymes
which
hi h h
have nucleotide
l tid
cofactors, serum albumin etc.
Lysine- Sepharose 4B Plasminogen, ribosomal RNA
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Affinity Chromatography
BASIC STEPS
• Sample Introduction
• Adsorption of the components of interest
• Removal of impurities
• Elution of components
Affinity Chromatography
BASIC STEPS
Sample Introduction
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Affinity Chromatography
BASIC STEPS
Absorption
Use a slow
l fl
flow rate off the
h solvent
l
so that the SAMPLE is allowed to
move through the column.
Affinity Chromatography
BASIC STEPS
Washing
Basically
i ll removing
i the
h impurities
i ii
out of the column by passing fresh
volumes of solvent in a repeated
fashion
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Affinity Chromatography
BASIC STEPS
Elution
Removall off the
h target molecule
l l from
f
the matrix and its collection.
Affinity Chromatography
BASIC STEPS
Elution Methods‐ Biospecific
Inhibitor
hibi ( free
f li d) is
ligand) i added
dd d into
i the
h
eluting buffer ( solvent) where it
competes with the solute (TARGET
MOLECULES)
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Affinity Chromatography
BASIC STEPS
Elution Methods‐ Non‐ specific
A reagent is
i added
dd d that
h denatures
d the
h
solute (TARGET MOLECULE)
(pH, KSCN, Urea, ionic strength etc.)
Affinity Chromatography
Affinity Chromatogram
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Affinity Chromatography
Example 1
Affinity Chromatography
Example 2
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IEC
PRINCIPLE
Ion exchange chromatography retains analyte
molecules based on coulombic (ionic) interactions.
Stationary
S i phase
h surface
f di l
displays i i functional
ionic f i l
groups that interact with analyte ions of opposite
charge
TYPES
Cation exchange : positively charged cations because
the stationary phase displays a negatively charged
functional group such as a phosphonic acid ( NET
NEGATIVE CHARGE)
Anion exchange : negatively charged anions using
positively charged functional group such as a
quaternary ammonium cation ( NET POSITIVE CHARGE)
Column Chromatography
Net Charge on the Ion exchanger‐measure to retain
opposite charge ion clearly depends upon the pH of
the mobile phase with which it is contact.
Net charge is also a function of pKa of the ion group
involved.
(R+ X‐) + P‐ ( R+P‐) + X‐ ( Absorption)
( R+P‐) + S‐ (R+S‐) + P ( Desorption)
Example of Anion Exchanger‐ Diethylaminoethyl
(DEAE) and Cation Exchangers is Carboxymethyl (CM).
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+ ++ + ++
+ ++ + ++
+ ++ ++ ++
++ ++
++
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