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What is ion exchange chromatography?
Ion exchange chromatography (IEX) is a form of adsorption chromatography, which separates molecules on the basis of charge.
IEX is one of the most widely used chromatography techniques for the purification of proteins. However, it also lends itself to the separation of peptides and oligonucleotides.
IEX is useful at all stages of a purification scheme and is eluted either by step elution or gradients.
The load capacity of step-eluted IEX is very high, which makes it excellent as a capture step as well as for concentrating samples in general.
The high resolution obtainable makes IEX very useful also for intermediate purification and for polishing.
Results can be controlled in a predictable way by controlling running pH, salt concentration, and by selecting the type of ion exchanger.
When run within the stability window for the sample molecules, recoveries achieved are typically better 90-100%.
 | Basis for selectivity in IEX:
Charged amino acids on the surface of a protein can bind to oppositely charged ligands of the ion exchanger.
Arrows indicate some charged regions of lysozyme. |
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The Separation Mechanism
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Ion exchange chromatography (IEX) separates molecules according to net charge.
Matrix-bound charged groups reversibly adsorb oppositely charged sample molecules like proteins, peptides and nucleotides (Fig 1.1).
Fig 1.1. Charged sample molecules adsorb to ion exchangers of the opposite sign. The interaction is a dynamic equilibrium that can be influenced by pH or salt concentration.
Desorption is brought about either by a change in the pH or by an increase in the salt concentration of the eluent.
Adsorption
Proteins carry charged amino acids on their surfaces and can thus be adsorbed to ion exchangers. Proteins with net negative charges (excess of negative charges) adsorb to anion exchangers, while those with net positive charges (excess of positive charges) adsorb to cation exchangers. The strength of the adsorption increases with increased net charge (Fig 1.2).
Fig 1.2. The larger the net charge, the stronger is the adsorption. The size of the arrow to the right represents the adsorption strength.
Charged amino acids contain either weak acid or amino groups, whose degree of dissociation depends on pH. Consequently the net charge will vary with pH in a way that is fairly specific for each individual protein.
The main way of influencing selectivity in IEX is consequently by varying the running pH.
Desorption
Essentially two possibilities exist to desorb sample molecules from the ion exchanger:
- Reducing the net charge by changing pH.
- Adding a competing ion to "block" the charges on the ion exchanger.
Varying the pH is a powerful way of influencing the net charges of the sample molecules and is therefore normally used to control the selectivity (elution order and distance between eluted peaks).
Adding a competing ion will not influence the selectivity, but provide a means of desorbing the sample molecules in order of increasing net charges (Fig 1.3).
Fig 1.3. The higher the net charge, the higher the salt concentration required for desorption.
Most IEX experiments use a neutral monovalent salt such as NaCl as the desorbing agent, mainly because NaCl has little or no effect on the running pH.
The higher the net charge, the stronger the adsorption and the higher the salt concentration needed to desorb the sample molecule.
Desorption curves
The adsorption reaction is a dynamic equilibrium between free and adsorbed molecules and is controlled by adding a neutral salt. It can be described in terms of a desorption curve obtained by plotting the relative amount of free sample molecules as a function of the salt concentration as shown in Figure 1.4. (Desorption curves have little practical value and are used here only to demonstrate the working principles of IEX.)
Fig 1.4. The desorption curve reflects the distribution of the sample between the mobile and the stationary phase. Within the partition zone this distribution varies as a function of the salt concentration and the elution velocity varies accordingly.
In a column all transport of a sample down the column is carried out by the mobile phase (the eluent) and acts only on the molecules present in the mobile phase.
When a sample travels down the column, its velocity is proportional to the portion of sample molecules present in the mobile phase.
The desorption curve thus represents the velocity of a sample zone as a function of the salt concentration. The salt concentration interval corresponding to the desorption curve will be referred to as the
partition zone.
The position of the desorption curve along the salt concentration axis is governed by the net charge of the sample. An increase of the net charge will shift the desorption curve to the right and a decrease will shift it to the left (Fig 1.5).
Fig 1.5. The desorption curve is shifted to the right with increasing net charge.
When a sample zone travels down the column under continuous re-partitioning, a certaindeviationfrom equilibrium is created. This, however, is unavoidablyassociated with broadeningof the sample zone. |
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Type of ion exchangers
Ion exchange (IEX) ligands can either be negatively or positively charged. Negatively charged ion exchangers adsorb cations while positively charged ones adsorb anions. Hence they are named cation exchangers (CIEX) and anion exchangers (AIEX) respectively.
Depending on the pKa value of the charged ligand, the ion exchangers are further divided into strong and weak.
Strong ion exchangers are fully charged over the total pH range normally applicable to proteins and peptides. With weak ion exchangers, on the other hand, the charge displayed is a function of the eluent pH.
For protein and peptide purification, the weak ion exchangers are nowadays less frequently used since they provide no essential advantage over the strong ones.
Note that strong or weak in this sense does not refer to the binding strength provided but to the IEX ligand as a strong or weak acid or base.
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Elution modes
Isocratic elution
Isocratic elution is seldome used in IEX.
Gradient elution
The most frequently used elution mode in high resolution applications of IEX is gradient elution.
Gradients of a neutral salt are formed by mixing two eluents, one containing a low concentration of the neutral salt (buffer A) and one containing a high concentration of this salt (buffer B). But for their salt contents, the two eluents are identical.
Chromatography systems usually control the gradient formation by the use of two pumps, one for buffer A and one for buffer B Fig 3.1.
Figure 3.1. The gradient system needs a gradient forming
device i.e. an extra pump to control "buffer B".
To understand how gradient elution works, consider a protein with the desorption curve shown in Figure 3.2.
Figure 3.2. In gradient elution the salt concentration sweeps the partition zone and causes the sample component to accelerate from zero speed to that of the mobile phase. Once at 100% desorption the sample component has reached a fixed position in the gradient.
The protein is applied to the column at a salt concentration well below the partition zone and is thus adsorbed at the top of the column. When the gradient starts, it will remain fully adsorbed until the salt concentration reaches the lower end of the partition zone.
As the salt concentration passes through the partition zone, the protein becomes increasingly desorbed and move down the column with a velocity reflecting its relative desorption.
When the salt concentration reaches the upper end of the partition zone, all protein molecules are desorbed. The protein now moves with the same velocity as the eluent and its position within the gradient becomes fixed.
The time (and thus the required distance of travel) needed to reach this fixed position depends on one hand on the slope of the desorption curve and on the other hand on the slope of the gradient.
The steeper the desorption curve, the shorter the travel needed to reach the fixed position in the gradient.
The shallower the gradient, the longer the travel distance needed to reach the fixed position in the gradient.
Now consider the four proteins used to exemplify isocratic elution above.
As pointed out, no isocratic conditions could separate all four proteins in one single run.
A gradient embracing all the partition zones of the respective proteins will, however, separate all four proteins in one run (Fig 3.3), provided the column is long enough to allow all four proteins to reach their respective final positions in the gradient.
Fig 3.3. Gradient elution will sort the sample components according
to their respective 100 % desorption points.
Another important difference compared to isocratic elution is that the volume of the sample applied will not influence the final results.
Since the gradient starts at a salt concentration well below the partition zones of all the proteins, these will all adsorb at the top of the column during sample application. When eluted they will all appear as narrow zones, regardless of the original sample volume.
In contrast to isocratic elution, gradient elution does not require a lot of test runs to find a suitable elution strength.
The distance between peaks is controlled by the slope of the gradient (Fig 3.4).
Fig 3.4. Resolution can be increased by lowering the gradient slope.
The shallower the gradient, the greater the distance between the peaks.
However, the sample peaks broaden as a result of the re-partitioning mechanism when the gradient passes through the partition zone and the longer the required distanse of travel to reach full desorption, the broader the peaks.
The gain in distance between the peaks is larger than the peak-broadening effect and the net result is that resolution increases with decreased gradient slopes.
To obtain reproducible results it is important that all gradient-eluted components have reached 100% desorption before they leave the column.
The column should be long enough to allow this and the shallower the gradient, the longer the column needed.
Protein desorption curves are normally quite steep and gradient volumes of 20 column volumes and more are used without any problems.
Once all sample components have reached their final positions in the gradient, no further increase in separation is possible. On the contrary peaks will broaden due to diffusion if the column is excessively long (Fig 3.5).
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Fig 3.5. The column should be long enough to allow the sample proteins to reach
their final positions in the gradient. However, excessively long columns will
only contribute to peak-broadening.
Step elution
A third way of using desorption curves is to avoid the partition zone by going directly from fully adsorbed conditions to fully desorbed conditions (Fig 3.6). The salt concentration during sample application is set high enough to leave most of the weaker binding sample components unadsorbed, while the target component and those with higher net charges than the target component will adsorb.
Fig 3.6. Step elution is a binding/non-binding technique used to concentrate
and to reduce the complexity of crude samples.
The salt concentration is then abruptly increased to a level just enough to fully desorb the target component.
This type of group separation will not provide the high resolution obtained with gradients or isocratic elution. Sample components with overlapping desorption curves will in part co-elute with the target protein.
Being a binding/non-binding technique rather than based on continuous re-partitioning, zone broadening will not be dependent of mass transfer and several times larger sample loads are accepted.
Column dimensions are less important and both larger bead sizes and higher flow rates may be used without any detrimental effects on the separation. |
The typical Ion exchange experiment
IEX is the most frequently used in gradient mode and the experiment consists of four phases as shown below.
Fig 4.1. The separation of four proteins with different negative net charges using gradient mode.
Charge properties of proteins and peptides
Protein and peptide net charges depend on their contents of charged amino acids. These carry weak acidic or amino groups and the net charge is therefore a function of pH.
At low pH values the dissociation of the acidic groups is suppressed and they loose their negative charges in accordance with their respective pKa values (see table to the right!). Amino groups on the other hand are protonated and positively charged at low pH values.
At high pH values the acidic groups dissociate and become negatively charged while amino groups loose a proton and become neutral.
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Below a certain pH value the protein obtains a positive net charge.
Above this pH value the protein obtains a negative net charge.
At a certain intermediate pH value, the number of positive charges equals that of the negative ones, making the net charge zero. This pH value is called the isoelectric point (pI) and is specific for each individual protein.
Titration curves (Fig 5.1) reflect the dependence of net charge on pH and can be of great help in finding the optimal pH for an IEX separation of proteins or peptides.
Fig 5.1. The titration curve reflects how the net charge of a protein or peptide varies with pH.
Electrofocusing in gels produces stable pH gradients that can be used to investigate protein titration curves. After having created the pH gradient, normal electrophoresis is run at right angles to the gradient. The sample is applied in the middle of the gel so that it can migrate in either direction depending on net charge. Figure 5.2 shows the result obtained when a meat extract was analyzed in this way. It demonstrates the variation of charge properties between the different proteins of this sample.
Fig 5.2. Electrophoretic titration curves. |
Effect of running pH
Titration curves and resolution
Consider three proteins (red (R), blue (B), and green (G) curves in Fig 6.1), each with its own unique titration curve
pH is by far the most effective parameter to control selectivity in IEX. |
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At low pH all the proteins are positively charged in the order R>G>B.
Cation exchange will elute them in the order
B, G, R.
Anion exchange. Being positively charged they will all elute unseparated in the wash-through fraction.
At less acidic pH R and G are still positively charged (R>G), while B is negatively charged.
Cation exchange will elute B in the flow-through fraction, while R and G elute in the order: G, R but are better separated.
Anion exchange will elute B by the gradient, while R and G, being positively charged are found in the flow-through fraction being positively charged. | 
Fig 6.1. The effect of varying pH in anion exchange and cation exchange chromatography. | At high pH all the proteins carry negative net charges, B>G>R.
Cation exchange will elute them all in the flow-through fraction.
Anion exchange will elute all the proteins by the gradient and in the order: R, G, B.
At slightly alkaline pH G too has switched to negative net charge. R, however, is still positively charged.
Cation exchange will elute B and G in the wash-through fraction and R by the gradient.
Anion exchange will elute B and G by the gradient, while R is found in the wash-through fraction. |
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Finding the best pH
The test tube method (Fig 6.2) is a simple method for finding the pH value at which the target protein binds to an ion exchanger. Once determined, this pH value can then be used as running pH for the first separation.
Fig 6.2. Test tube method for estimating the pH at
which the target protein binds to an anion exchanger.
The example in Figure 6.2 used an anion exchanger and the target protein was found to bind completely at pH 7.5.
The test tubes contain a small quantity of an anion exchanger suspended in buffers of different pH values and a small amount of the sample. After agitation and centrifugation the supernatants are assayed for target protein content. In the example in Figure 6.2 the protein binds completely from pH 7.5 and upwards.
Electrophoretic titration curves (Fig 6.3) provide much more detailed information than the test tube method, since all proteins in the sample are analyzed in the same run and at a broad pH span.
Fig 6.3. Electrophoretic titration curves of proteins from
a meat extract showing their net charges as a function of pH.
With access to a chromatography system equipped for automatic buffer preparation, automated pH scouting is a very effective way to determine the optimum running pH .
A practical example of pH scouting is the separation of pancreatin with ÄKTAexplorer, shown in Figure 6.4. The change in the separation by altering the buffer pH is clear.
Fig 6.4. Automatic pH scouting performed on ÄKTAexplorer 100.
Sample: 2 mg crude pancreatin.
Column: RESOURCE Q; 6 ml
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Resolution in ion exchange chromatography
In IEX, selectivity (distance between and order of eluted peaks) depends on the charge properties of the individual sample components.
With proteins and peptides the charge properties are heavily influenced by the running pH.
Gradients will influence peak-spacing but not elution order.
Efficiency (counteraction of zone broadening) depends on bead size, quality of the packed bed and flow rate in isocratic and gradient modes.
Best resolution is theoretically obtained in isocratic mode.
For practical reasons, however, gradient mode is the most frequently used elution technique.
With large volumes of complex samples, the first purification step often aims at concentrating the sample and removing the bulk of the contaminants by a capture step. Here, step elution mode is to be preferred because of its high loading capacity and that high flow rates can be applied.
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Optimisation of ion exchange chromatography experiments
Optimizing running pH is by far the most effective way of obtaining satisfactory results with protein or peptide samples. The choice of ion exchanger type (anion or cation exchanger) may also influence selectivity quite considerably.
Optimising the gradient slope only influences distances between the peaks and will not change their elution order.
Using an optimal flow rate is important in isocratic experiments in order to keep zone broadening at a minimum. Provided an IEX medium with a suitable bead size (10 - 30 mm for high resolution experiments) is employed, the flow rate is less important in gradient-eluted experiments.
Based on this, the following optimization measures are recommended in order of priority:
- Select the type of ion exchanger that provides best resolution under standard conditions ( e.g. pH 5,0 for cation exchangers and pH 8 for anion exchangers).
- Scout for the running pH that provides best resolution.
- Select the steepest gradient that provides acceptable results.
- Scout for the flow rate that provides the narrowest peaks.
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Ion exchange chromatography
Use of ion exchange chromatography
High resolution mode
(gradient elution) | Group separation mode
(step elution) |
| Separates proteins, peptides, and oligonucleotides according to net charge. | Concentrates dilute samples of proteins, peptides and oligonucleotides. |
| Suitable for intermediate and polishing steps in multi step purification protocols. | Suitable for capture steps in multistep purification protocols. |
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| High resolution mode
(gradient elution) | Group separation mode
(step elution) |
IEX medium | Use strong IEX media only
Use media with 10 - 30 mm beads for purification work.
Use media with 3 - 5 mm beads for analytical work. | Use strong IEX media only.
Use media with 30 mm beads or larger to allow higher flow rates. |
Column | Typical column lengths are 1 - 10 cm.
Short columns may prevent the use of very shallow gradients (large gradient volumes). | Column length is less important. Short and "fat" columns, however, will allow higher flow rates. |
Eluents | Buffering ion should be of the same charge sign as the ion exchanger.
Buffer pKa should not deviate more than 0.5 pH units from the running pH.
A buffer concentration of 20 - 50 mM is usually sufficient. | Buffering ion should be of the same sign as the ion exchanger.
Buffer pKa should not deviate more than 0.5 pH units from the running pH.
A buffer concentration of 20 - 50 mM is usually sufficient. |
Sample volume | With a properly designed gradient the target molecule will bind and concentrate at the top of the column.
The sample volume is thus not important. | The sample volume is not important since the sample will bind to the column. |
Sample amount | 5 - 10 % of the total loading capacity of the column used can be applied without loss of resolution. | Around 40 % of the total loading capacity of the column used can be applied. |
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Buffer preparation tips
- Buffering ion should have the same charge sign as the ion exchanger.
- Adjust pH with HCl and NaOH when NaCl is used to produce the gradient.
- Adjust pH of buffer B after the salt has been added.
- Adjust buffer pH at the temperature intended for the experiment.
Sample preparation tips
- Filter or spin the sample to remove any particulate matter.
- Especially with large sample volumes, adjust sample pH and salt content to match those of "buffer A".
- Buffer exchange on Sephadex™ G-25 is a rapid and mild way to adjust sample conditions.
- Especially with large sample volumes, adjust sample temperature to match that of "buffer A".
Ion exchange chromatography profile
| Working principle |
- Separates molecules according to charge
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| Used for |
- Separation of complex samples.
- Sample concentration.
- Removal of charged contaminants.
- Separation of complex samples.
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| Suitable stage in a purification protocol |
- Capture:
  
- Intermediate:
  
- Polishing:
  
(Number of stars indicates suitability.) |
| Separation characteristics |
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| High |
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| - General separation: High
- Group separation: Very High
- Sample concentration: Very High
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| - General separation: High
- Group separation: Very High
- Sample concentration: Very High
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| High |
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| High |
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| Main optimizing parameter |
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| Special features |
- Selectivity can be varied over a wide range.
- Wide applicability to proteins and peptides.
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| Further reading |
- Applications
- Media selection guide
- Purification protocols
- Purification strategies
- (Handbooks etc.)
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Ion Exchange chromatography
Selection guide
Ion Exchange columns and media - Selection guide pdf
Handbooks
Ion Exchange Chromatography & Chromatofocusing: Principles & Methods pdf
Protein Purification Handbook pdf
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