Download Dr. Atiya Abbasi Lecture 04_ IEC_ 16 Jan.ppt

Commonly used separation techniques
Size exclusion
Ion exchange
Affinity
Reversed phase,
Hydrophobic interaction
Ion Exchange Chromatography
Basic principles in ion exchange chromatography
1. The Separation Mechanism
- Adsorption
- Desorption
- Desorption curves
2. Type of ion exchangers
3. Elution modes
- Isocratic elution
- Gradient elution
- Step elution
4. The typical IEX experiment
5. Charge properties of proteins and peptides
6. Effect of Running pH
- Titration curves and resolution
- Finding the best pH
7. Resolution in IEX
8. Optimization of IEX experiments
It is a form of adsorption chromatography in which ionic
solutes display reversible electrostatic interaction with the
charged stationary phase.
Ion exchange chromatography (IEX) separates bio-molecules
according to differences in the net surface charge.
IEX for the separation of bio-molecules was introduced in the
1960s and continues to play a major role in the separation and
purification of bio-molecules.
Molecules vary considerably in terms of charge characteristics
and exhibit different degrees of interaction with the matrix
according to differences in the overall charge, charge density
and surface charge distribution.
The charged groups within a molecule that contribute to the net
surface charge possess different pKa values depending on their
structure and chemical microenvironment.
The technique is capable of separating molecular species that
have only minor differences in their charge properties, for
example two proteins differing by one charged amino acid.
All molecules with ionizable groups can be titrated and as
such the net surface charge is highly pH dependent.
Proteins, in particular, are built up of many different amino
acids containing weakly acidic and basic groups, and thus the
net surface charge changes gradually as the pH of the
environment changes i.e. proteins are amphoteric.
As a result each protein has its own unique net charge versus
pH relationship which can be visualized as a titration curve.
IEX chromatography takes advantage of the relationship
between net surface charge and pH which is unique for a
specific protein.
In IEX chromatography reversible interactions between
charged solute molecules and oppositely charged stationary
phase are controlled in order to favor binding or elution of
specific molecules and achieve separation.
Thus if a protein has no net charge at a certain pH (also known
as isoelectric point pI) it will not interact with the charged
medium. However, at a pH above its isoelectric point, a
protein will bind to a positively charged medium or anion
exchanger and, at a pH below its pI, the protein will behind to
a negatively charged medium or cation exchanger.
In addition to the ion exchange interaction, other types of
binding may occur, but these effects are very small and mainly
due to van der Waals forces and non-polar interactions.
The stationary phase or matrix comprises of spherical particles
substituted with ionic groups that are negatively (cationic) or
positively (anionic) 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.
Factors affecting the matrix
Inertness
 An inert matrix minimizes non-specific interactions with
sample components.
Porosity
 High porosity offers a large surface area covered by
charged groups and so ensures a high binding capacity.
High porosity is also an advantage when separating large
bio-molecules.
 Non-porous matrices are preferable for extremely high
resolution separations when diffusion effects must be
avoided.
Physical Stability
 High physical stability ensures that the volume of the packed
medium remains constant despite extreme changes in salt
concentration or pH thus improving reproducibility and
avoiding the need to repack columns.
 High physical stability and uniformity of particle size facilitate
high flow rates, particularly during cleaning or re-equilibration
steps, to improve throughput and productivity.
Chemical Stability
 High chemical stability ensures that the matrix can be cleaned
using stringent cleaning solutions if required.
 Modern IEX media use either polymer or agarose-based
matrices to fulfill not only the requirements for high binding
capacity, chemical and physical stability, but also generate
media with suitable particle sizes for a range of applications
Types of Ion Exchanger
Anion Exchanger
Cation Exchanger
Sulphonic and quaternary amino groups are used to form strong
ion exchangers; the other groups form weak ion exchangers. The
term strong and weak refers to the extent of variation of
ionization with pH and not the strength of binding.
Strong ion exchangers are completely ionized over a wide pH
range whereas with weak ion exchangers, the degree of
dissociation and thus exchange capacity varies much more
markedly with pH.
Some properties of strong ion exchangers are:
Sample loading capacity does not decrease at high or low pH
values due to loss of charge from the ion exchanger.
A very simple mechanism of interaction exists between the
ion exchanger and the solute.
Ion exchange experiments are more controllable since the
charge characteristics of the media do not change with
changes in pH. This makes strong exchangers ideal for
working with data derived from electrophoretic titration
curves.
Factors affecting Separation
Capacity
The capacity of an ion exchanger is a quantitative measure of
its ability to take up exchangeable counter-ions and is therefore
of major importance.
The capacity may be expressed as:
Total ionic capacity i.e. the number of charged substituent
groups per gram dry ion exchanger or per ml swollen gel. Total
capacity can be measured by titration with a strong acid or
base.
Available capacity i.e. the actual amount of protein which can
be bound to an ion exchanger under defined experimental
conditions.
Dynamic capacity i.e. the actual amount of protein which can
be bound to an ion exchanger under defined experimental
conditions including the flow rate at which the gel was
operated.
Available and dynamic capacities depend upon:
The properties of the protein.
The properties of the ion exchanger.
The chosen experimental conditions.
The properties of the protein which determines the available or
dynamic capacity on a particular ion exchange matrix are its
molecular size and its charge/pH relationship.
The capacity of an ion exchanger is thus different for different
proteins.
Available capacity is restricted to the charged substituents present
on the surface of the gel.
Similarly, since the interaction is ionic, the protein’s charge/pH
relationship must be such that the protein carries the correct net
charge, at a sufficiently high surface charge density, to be bound
to a particular ion exchanger under the chosen buffer conditions
Factors which affect the observed capacity include pH, the
ionic strength of the buffer, the nature of the counter-ion, the
flow rate and the temperature.
Porous matrix exhibits a higher available capacity as small
molecules can enter the pores compared to larger molecules
which are restricted to the charged substituents present on the
surface of the gel.
Non-porous matrices have considerably lower capacity than
porous matrices, but higher efficiency due to shorter diffusion
distances.
Available capacity of porous matrix is dependent on
a) its exclusion limit and
b) the type and number of the charged substituent.
High available capacity is obtained by having a matrix which
is macroporous and highly substituted with ionic groups which
maintain their charge over a wide range of experimental
conditions.
Elution modes
Biomolecules like proteins/peptides usually have different
affinities for the ion exchanger and so variations in the pH
and ionic strength of the eluent can cause elution at different
times and thus their separation from each other.
One can choose to use either continuous or stepwise
gradients of pH or ionic strength.
- Isocratic elution
- Stepwise gradient
- Continuous gradient
Isocratic elution
In all forms of isocratic elution, a limiting factor with
regards to achievable resolution is zone broadening as a
result of longitudinal diffusion.
Stepwise Elution
pH gradient
Easier to produce and are more reproducible than linear pH
gradients.
In the case of weak ion exchangers the buffer may have to
titrate the ion exchanger leading to a short period of reequilibration before the new pH is reached.
Stepwise Elution (contd.)
Ionic strength gradient
Produced by the same buffer at different ionic strengths.
Technically simple and offers the potential of high resolution
in preparative applications.
Care must be taken in the design of the steps and the
interpretation of results since substances eluted by a sharp
change in pH or ionic strength elute close together.
Peaks tend to have sharp fronts and pronounced tailing since
they frequently contain more than one component.
Tailing may lead to the appearance of false peaks if a buffer
change is introduced too early.
Continuous Gradients
pH
Difficult to produce at constant ionic strength, since
simultaneous changes in ionic strength, although small, also
occur.
Linear pH gradients cannot be obtained simply by mixing
buffers of different pH in linear volume ratios since the
buffering capacities of the systems produced are pH
dependent.
A relatively linear gradient can be produced over a narrow
pH interval (Max. 2 pH units) by mixing two solutions of
the same buffer salt adjusted, respectively, to 1 pH unit
above and 1 pH unit below the pKa for the buffer.
Continuous Gradients (contd.)
Ionic strength
Most frequently used type of elution in ion exchange
chromatography.
Easy to prepare and very reproducible.
Two buffers of differing ionic strength, the start and limit
buffers, are mixed together and if the volume ratio is changed
linearly, the ionic strength changes linearly.
The limit buffer may be of the same buffer salt and pH as the
start buffer, but at higher concentration, or the start buffer
containing additional salt e.g. NaCl
Generally leads to improved
sharpening occurs during elution.
resolution
since
zone
The leading edge of a peak is retarded if it advances ahead of
the salt concentration or pH required to elute it.
The trailing edge of the peak is exposed to continuously
increasing eluting power. Thus the trailing edge of the peak
has a relatively higher speed of migration, resulting in zone
sharpening, narrower peaks and better resolution
Gradient elution also reduces zone broadening by
diminishing peak tailing due to non-linear adsorption
isotherms.
Choice of Gradient Shape
Linear
Strongly recommended in initial experiments with a new
separation problem.
The results obtained can then serve as a base from which
optimization can be planned.
If better resolution is required then the separation can be
improved by altering the shape or slope of the gradient.
Concave
Used to improve resolution in the first part of the gradient or to
shorten the separation time when peaks in the latter part of the
gradient are more than adequately separated.
Convex
Used to improve resolution in the last part of the gradient or to
speed up a separation when the first peaks are well separated and
the last few need to be adequately separated.
Complex gradients
Can be generated to use the maximum resolution offered
by isocratic resolution when required combined with
steeper gradient portions where resolution is adequate or
unnecessary.
Complex gradients offer the maximum flexibility in terms
of combining resolution with speed during the same
separation.
A knowledge of the chromatographic behavior of the
sample obtained from previous separations using simpler
gradients is essential.
Resolution
 Extent of separation between the
peaks eluted from the column (the
selectivity of the medium),
 Ability of the column to produce
narrow,
symmetrical
peaks
(efficiency)
 Amount (mass) of sample applied.
These factors are influenced by
practical issues such as matrix
properties, binding and elution
conditions, column packing, flow
rates
A single, well resolved peak is not necessarily a pure
substance, but may represent a series of components which
could not be separated under the chosen elution conditions.
Efficiency
Is dependent on the ability to elute
narrow, symmetrical peaks from a
packed bed and is related to the
zone broadening effect.
Zone broadening can be minimized
if the distances available for
diffusion are minimized.
Columns that are packed unevenly,
too tightly, too loosely or that
contain air bubbles will lead to
channeling, zone broadening and
hence loss of resolution.
Rapid exchange of counter- Buffer flow
ions, typically Na+ or Cl-, and
solute molecules
Rapid diffusion
Small bead size
Uniform pore size distribution
Even buffer flow distribution
Uniform packing
Narrow particle size distribution
Narrow, symmetrical peaks
Minimal zone broadening
Resolution increase by decreasing particle size, however small
particle size creates high back pressure
Viscosity of samples
influence resolution.
also
Selectivity
Nature and number of the functional groups on the matrix
The experimental conditions, such as pH (influencing the protein
charge), ionic strength and elution conditions
Good selectivity is a more important factor than high efficiency
in determining resolution
Effect of pH on protein binding and elution patterns.
Most acidic pH: all three
proteins are below their
isoelectric point, positively
charged, and bind only to a
cation exchanger. Proteins are
eluted in the order of their net
charge.
Most alkaline pH: all three
proteins are above their
isoelectric point, negatively
charged, and bind only to the
anion exchanger. Proteins are
eluted in the order of their net
charge.
Less acidic pH: blue protein is
above its isoelectric point,
negatively charged, other
proteins are still positively
charged. Blue protein binds to
an anion exchanger and can
be separated from the other
proteins which wash through.
Alternatively, red and green
proteins can be separated on a
cation exchanger and the blue
protein washes through.
Less alkali pH: red protein
below its isoelectric point,
positively
charged.
Red
protein binds to cation
exchanger and can be
separated from the other
proteins which wash through.
Alternatively, blue and green
proteins can be separated on
an anion exchanger and the
red protein washes through.
The pH and ionic strength of the equilibration buffer are
selected such that proteins of interest bind to the ion exchanger
matrix whereas most of the impurities pass through without
any interaction.
The optimally charged proteins bind effectively with the
matrix and thus get concentrated onto the column while other
proteins with out surface charge pass through the column and
elute in the flow through volume.
Conditioning of the sample is very important to achieve the
most effective high resolution or group separations and make
the most of the high loading capacity.
Ideally, samples should be in the same conditions as the
starting buffer.
The typical IEX experiment
The pH and ionic strength of the equilibration 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
After loading sample the column
is washed to remove all nonbinding proteins and conditions
are then altered in order to elute
the bound proteins.
Proteins are normally eluted by either 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 matrix 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
eluted first 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. 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
differentially in a purified and
concentrated form.
A wash step in very high ionic
strength buffer removes most
tightly bound proteins at the
end of an elution.
The column is then re-equilibrated in start buffer before
applying more sample in the next run.
Alternatively, conditions can be chosen to maximize the
binding of contaminants and allow the target protein(s) to pass
through the column thus removing contaminants.
pH and ionic strength
Must be compatible with protein stability and activity. The
most suitable pH should allow the proteins of interest to
bind, but should be as close to the point of release (elution)
as possible.
If the pH is too low or too high, elution becomes more
difficult and high salt concentrations may be needed. This
should be avoided since some proteins begin to precipitate at
high ionic strength and high salt concentrations may
interfere with assays or subsequent chromatographic steps.
Optimization by careful adjustment of the pH of the mobile phase.
Resolution between proteins can be affected in anion-exchange
chromatography using protonation states of histidine between pH 7.5
and 8.5
Separation of impurities in carbonic
anhydrase on reducing the pH from 8.0
to 7.5 resulted in decreased retention
and better resolution
The effect of pH on resolution of a
small peak trailing conalbumin. The
trailing peak which is only a shoulder
at pH 7.5, is more completely
separated at pH 8.0.
Protein structure and charge changes as aspartic acid and glutamic acid
become protonated between pH 2.5 and 5.0. Although the acid side
chains are not directly involved in the cation exchange interaction, the
overall affect on the charge, charge density and, possibly, the tertiary
structure of the proteins is more evident.
Extent of resolution of impurities in
lysozyme at pH 4.7 and pH 2.5. At pH
2.5 aspartic and glutamic acid side
chains are protonated.
Separation of Standard Human Hemoglobins F, A, S & C
Hb A
Hb F
Hb S
Hb C
Sample Concentration
The amount of sample depends on the dynamic capacity
of the ion exchanger and the degree of resolution
required. For the best resolution it is not usually advisable
to use more than 10-20% of this capacity.
Sample Composition
The ionic composition should be the same as that of the
starting buffer.
Sample volume
If the ion exchanger is to be developed with the starting buffer
(isocratic elution), the sample volume is important and should
be limited to between 1 and 5% of the bed volume.
If the ion exchanger is to be developed with a gradient,
starting conditions are normally chosen so that all important
substances are adsorbed at the top of the bed. This means that
large volumes of dilute solutions, such as pooled fractions
from a preceding gel filtration step or a cell culture
supernatant can be applied directly to the ion exchanger
without prior concentration.
Ion exchange thus serves as a useful means of concentrating a
sample in addition to fractionating it.
Sample viscosity
The viscosity may limit the quantity of sample that can be
applied to a column.
High sample viscosity causes instability of the zone and an
irregular flow pattern.
Sample preparation
In all forms of chromatography, good resolution and long
column life time depend on the sample being free from
particulate matter.
It is important that “dirty” samples are cleaned by filtration or
centrifugation before being applied to the column.
The “grade” of filter required for sample preparation depends
on the particle size of the ion exchange matrix. Samples may
be filtered using a 1 μm filter for 90 μm matrix.
For 3, 10, 15, 30 and 34 μm matrix, samples should be
filtered through a 0.45 μm filter.
Samples should be clear after filtration and free from visible
contamination by lipids.
If turbid solutions are injected onto the column, the column
lifetime, resolution and capacity can be reduced.
Centrifugation at 10,000 g for 15 minutes can also be used
to prepare samples. This is not the ideal method of sample
preparation but may be appropriate if samples are of very
small volume or adsorb nonspecifically to filters.
Effect of Temperature
Temperature
affects
ion-exchange
chromatography
separations through its effect on the structure of the protein.
Although temperature does not affect the electrostatic
interaction, it often affects the structure of a protein and
therefore the interaction of the protein with the ion-exchange
resin.
Subtle variations in selectivity with temperature may result
from temperature induced changes in protein structure.
Applications