UNIT 4

Ion exchange chromatography

SEPARATION affinity chromatography
METHODS
Ion exchange
chromatography
Principles of ion exchange

IEX separates molecules on the basis of differences in

their net surface charge

Molecules vary considerably in their charge properties

and will exhibit different degrees of interaction with
charged chromatography media according to differences in
their overall charge, charge density, and surface charge
distribution.
Ion exchange chromatography
An IEX medium comprises a matrix of spherical particles substituted with ionic groups that
are negatively or positively charged.

The matrix is usually porous to give a high internal surface area.

The medium is packed into a column to form a packed bed. The bed is then equilibrated with
buffer which fills the pores of the matrix and the space in between the particles.
Ion exchange chromatography
Equilibration

The first step is the equilibration of the stationary phase to the desired start conditions.

When equilibrium is reached, all stationary phase charged groups are bound with exchangeable
counterions, such as chloride or sodium.

The pH and ionic strength of the start buffer are selected to ensure that, when sample is loaded,
proteins of interest bind to the medium and as many impurities as possible do not bind.
Ion exchange chromatography
Sample application and wash

The second step is sample application and wash.

The goal in this step is to bind the target molecule(s) and wash out all unbound material.

The sample buffer should have the same pH and ionic strength as the start buffer in order to bind
all charged target proteins.

Oppositely charged proteins bind to ionic groups of the IEX medium, becoming concentrated on
the column. Uncharged proteins, or those with the same charge as the ionic group, pass through the
column at the same speed as the flow of buffer, eluting during or just after sample application,
depending on the total volume of sample loaded.
Ion exchange chromatography
Elution

When all the sample has been loaded and the column washed with start buffer so that all
nonbinding proteins have passed through the column, conditions are altered in order to elute the
bound proteins.

Most frequently, proteins are eluted by increasing the ionic strength (salt
concentration) of the buffer or, occasionally, by changing the pH.

As ionic strength increases the salt ions (typically Na+ or Cl-) compete with the bound components
for charges on the surface of the medium and one or more of the bound species begin to elute and
move down the column.

The proteins with the lowest net charge at the selected pH will be the first ones
eluted from the column as ionic strength increases. Similarly, the proteins with the highest charge
at a certain pH will be most strongly retained and will be eluted last.
Ion exchange chromatography
Elution

The higher the net charge of the protein, the higher the ionic strength that is needed for elution.

By controlling changes in ionic strength using different forms of gradient, proteins are eluted
differently in a purified, concentrated form.
Ion exchange chromatography
Regeneration

A final wash with high ionic strength buffer regenerates the column and removes any molecules
still bound.

This ensures that the full capacity of the stationary phase is available for the next run.

The column is then re-equilibrated in start buffer before starting the next run.

The above describes a typical IEX separation.

Alternatively, conditions can be chosen to maximize the binding of contaminants to allow the
target protein(s) to first pass through the column to be collected.*

* This method is know as flow through method

Ion exchange chromatography
Ion exchange chromatography
Efficiency

Column efficiency (the ability to elute narrow, symmetrical peaks from a packed bed) relates to the
zone broadening which occurs on the column and is frequently stated in terms of the number of
theoretical plates.

One of the main causes of zone broadening is longitudinal diffusion of the solute molecules, that
is, proteins, peptides, or oligonucleotides.

Zone broadening can be minimized if the distances available for diffusion are minimized.

In all situations, a well-packed column will contribute significantlyto resolution.

Columns that are packed unevenly, too tightly, too loosely, or that contain
air bubbles will lead to channeling (uneven passage of buffer through the column), zone
broadening and hence loss of resolution.
Ion exchange
chromatography
Efficiency

Figure illustrates the parameters that contribute to good

column efficiency.

 Obviously, particle size is a significant factor in

resolution and, in general, the smallest particles will
produce the narrowest peaks under the correct elution
conditions and in a well-packed column.
Ion exchange
chromatography

Examples of the influence of

particle size and selectivity on final
resolution
Ion exchange
chromatography
Gradient elution is often used
when starting with an unknown
sample (as many components
as possible are bound to the
column and eluted
differentially to see a total
protein profile) and for high-
resolution separation or
analysis.
Ion exchange
chromatography

Step elution is used in several

ways. When an IEX separation
has been optimized using
gradient elution, changing to a
step elution speeds up
separation times and reduces
buffer consumption while
retaining the required purity
level.
Ion exchange chromatography

Electron micrograph of
MonoBeads showing spherical, Structure of cross-linked agarose media
monodispersed particles. (Sepharose).
Affinity Chromatography
Purification overview

Affinity chromatography separates proteins on the basis of a reversible interaction between a protein (or
group of proteins) and a specific ligand coupled to a chromatography matrix.

The technique is ideal for a capture or intermediate step in a purification protocol and can be used
whenever a suitable ligand is available for the protein(s) of interest.

With high selectivity, hence high resolution, and high capacity for the protein(s) of interest, purification
levels in the order of several thousand-fold with high recovery of active material are achievable.

Target protein(s) is collected in a purified, concentrated form.

Affinity Chromatography
Purification overview

Biological interactions between ligand and target molecule can be a result of electrostatic or hydrophobic
interactions, van der Waals’ forces and/or hydrogen bonding.

To elute the target molecule from the affinity medium the interaction can be reversed, either specifically
using a competitive ligand, or non-specifically, by changing the pH, ionic strength or polarity.

In a single step, affinity purification can offer immense time-saving over less selective multistep
procedures. The concentrating effect enables large volumes to be processed.

Target molecules can be purified from complex biological mixtures, native forms can be separated from
denatured forms of the same substance and small amounts of biological material can be purified from high
levels of contaminating substances.
Affinity Chromatography
Purification overview

Successful affinity purification requires a bispecific ligand that can be covalently attached to a
chromatography matrix.

The coupled ligand must retain its specific binding affinity for the target molecules and, after washing
away unbound material, the binding between the ligand and target molecule must be reversible to allow
the target molecules to be removed in an active
form.

Any component can be used as a ligand to purify its respective binding partner. Some
typical biological interactions, frequently used in affinity chromatography, are listed below:

Enzyme  substrate analogue, inhibitor, cofactor.

Antibody  antigen, virus, cell.

Lectin  polysaccharide, glycoprotein, cell surface receptor, cell.

Affinity Chromatography
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 surfaces.
Affinity Chromatography
Purification overview (Tagged protein)

Histidine-tagged proteins have a high selective affinity for Ni2+ and several other metal ions
that can be immobilized on chromatographic media using chelating ligands.

Consequently, a protein containing a histidine tag will be selectively bound to metal ion-charged media
such as Ni Sepharose High Performance (HP) and Ni Sepharose 6 Fast Flow (FF) while other cellular
proteins will not bind or will bind weakly.

This chromatographic technique is often termed immobilized metal ion affinity chromatography (IMAC).
Affinity Chromatography
Purification overview (Tagged protein)

Purification of histidine-tagged proteins secreted into eukaryotic cell culture, such as insect
cells or Chinese hamster ovary (CHO) cells, often leads to stripping of the immobilized metal
ions because of chelating agents present in
the cell culture medium. Ni Sepharose excel has strongly bound nickel ions and is especially
designed for purifying histidine-tagged proteins from eukaryotic cell cultures or from other
samples that cause nickel stripping.
Affinity Chromatography
Purification overview (Tagged protein)
Affinity
Chromatography
Resin options
Resin options

Affinity
Chromatography
Affinity
Chromatography