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
Ion exchange chromatography (IEX) separates bio-molecules according to differences in the net surface charge. It is a form of adsorption chromatography in which ionic solutes display reversible electrostatic interaction with the charged stationary phase. 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.
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. 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.
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 bed is then equilibrated with buffer which fills the pores of the matrix and the space in between the particles. The matrix is usually porous to give a high internal surface area. The medium is packed into a column to form a packed bed.
Factors affecting the matrix Inertness Porosity An inert matrix minimizes non-specific interactions with sample components. 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.
High chemical stability ensures that the matrix can be cleaned using stringent cleaning solutions if required. Chemical Stability 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. 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
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. 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.
Some properties of strong ion exchangers are: 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. 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.
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. 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. The capacity may be expressed as:
Available and dynamic capacities depend upon: The properties of the protein. 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. 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 The chosen experimental conditions. The properties of the ion exchanger. 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.
Non-porous matrices have considerably lower capacity than porous matrices, but higher efficiency due to shorter diffusion distances. 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.
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 - Isocratic elution - Continuous gradient - Stepwise gradient 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.
In all forms of isocratic elution, a limiting factor with regards to achievable resolution is zone broadening as a result of longitudinal diffusion. Isocratic 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 re- equilibration before the new pH is reached. Stepwise Elution
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. Stepwise Elution (contd.)
Difficult to produce at constant ionic strength, since simultaneous changes in ionic strength, although small, also occur. pH 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
Most frequently used type of elution in ion exchange chromatography. Ionic strength 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 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. Continuous Gradients (contd.)
Generally leads to improved resolution since zone sharpening occurs during elution. Gradient elution also reduces zone broadening by diminishing peak tailing due to non-linear adsorption isotherms. 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
Linear Choice of Gradient Shape 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 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. 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.
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 A knowledge of the chromatographic behavior of the sample obtained from previous separations using simpler gradients is essential. Complex gradients offer the maximum flexibility in terms of combining resolution with speed during the same separation.
Extent of separation between the peaks eluted from the column (the selectivity of the medium), These factors are influenced by practical issues such as matrix properties, binding and elution conditions, column packing, flow rates Resolution Ability of the column to produce narrow, symmetrical peaks (efficiency) Amount (mass) of sample applied. 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. Small bead size Uniform pore size distribution Rapid exchange of counter- Buffer flow ions, typically Na+ or Cl-, and solute molecules Rapid diffusion Even buffer flow distribution Uniform packing Narrow particle size distribution Narrow, symmetrical peaks Minimal zone broadening 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.
Resolution increase by decreasing particle size, however small particle size creates high back pressure Viscosity of samples also influence resolution.
Good selectivity is a more important factor than high efficiency in determining resolution 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
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. Effect of pH on protein binding and elution patterns.
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 non- binding 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.
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. The column is then re-equilibrated in start buffer before applying more sample in the next run. A wash step in very high ionic strength buffer removes most tightly bound proteins at the end of an elution.
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. pH and ionic strength 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. Separation of impurities in carbonic anhydrase on reducing the pH from 8.0 to 7.5 resulted in decreased retention and better resolution Resolution between proteins can be affected in anion-exchange chromatography using protonation states of histidine between pH 7.5 and 8.5 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 C Hb F Hb A Hb S
Sample Concentration Sample Composition 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. The ionic composition should be the same as that of the starting buffer.
Sample volume Ion exchange thus serves as a useful means of concentrating a sample in addition to fractionating it. 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.
Sample viscosity High sample viscosity causes instability of the zone and an irregular flow pattern. The viscosity may limit the quantity of sample that can be applied to a column.
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.
If turbid solutions are injected onto the column, the column lifetime, resolution and capacity can be reduced. Samples should be clear after filtration and free from visible contamination by lipids. 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 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. Temperature affects ion-exchange chromatography separations through its effect on the structure of the protein. Subtle variations in selectivity with temperature may result from temperature induced changes in protein structure.