Ion exchange chromatography involves the separation of ionizable molecules based on their total charge. This technique enables the separation of similar types of molecules that would be difficult to separate by other techniques because the charge carried by the molecule of interest can be readily manipulated by changing buffer pH.
Related Topics: Column Chromatography Methods and Instrumentation, Affinity Chromatography, Size Exclusion Chromatography, Hydrophobic Interaction Chromatography, Multimodal or Mixed-Mode Chromatography
Fig. 1. Protein charge vs. pH. Protein stability and ion exchange media binding vary with total protein charge, which depends on pH.
Ion exchange chromatography is commonly used to separate charged biological molecules such as proteins, peptides, amino acids, or nucleotides. The amino acids that make up proteins are zwitterionic compounds that contain both positively and negatively charged chemical groups. Depending on the pH of their environment, proteins may carry a net positive charge, a net negative charge, or no charge. The pH at which a molecule has no net charge is called its isoelectric point, or pI.
Fig. 2. Ion exchange resin selection.
The pI value can be calculated based on the primary sequence of the molecule. The choice of buffer pH then determines the net charge of the protein of interest.
In a buffer with a pH greater than the pI of the protein of interest, the protein will carry a net negative charge; therefore, a positively charged anion exchange resin is chosen to capture this protein.
In a buffer with a pH lower than the pI of the protein of interest, the protein will carry a positive net charge; thus a negatively-charged cation exchange resin is chosen.
When an ion exchange chromatography column is loaded with a sample at a particular pH, all proteins that are appropriately charged will bind to the resin. For example, if an anion exchange resin is chosen, all proteins that are negatively charged at the loading buffer pH will bind to the positively charged column resin. A good rule of thumb for choosing a buffer pH is the following:
After loading an impure protein sample onto an ion exchange chromatography column, the column is washed to remove undesired proteins and other impurities, and then the protein(s) of interest is eluted using either a salt gradient or a change in pH.
Fig. 3. Salt gradient elution. Elution of proteins (blue trace) with an increasing salt gradient (red trace).
The charged salt ions compete with bound proteins for the charged resin functional groups. Proteins with few charged groups will elute at low salt concentrations, whereas proteins with many charged groups will have greater retention times and elute at high salt concentrations.
Although less common, a pH gradient can also be used for elution. Here, a pH gradient is chosen that approaches the protein of interest’s pI. Proteins will elute when the pH gradient reaches their pI, because they will no longer carry a net charge that allows them to interact with the column resin.
To elute proteins from an anion exchange resin, a decreasing pH gradient is chosen, while an increasing pH gradient is chosen for elution from cation exchangers.
Fig. 4. Elution of protein (blue trace) with an increasing pH gradient (red trace).
Since it is very difficult to generate reproducible and accurate linear pH gradients, a step-gradient is generally chosen when pH is used for elution.
Lastly, pH can be used to refine elution when using a salt gradient. Altering the pH of the elution buffer can affect the resolution of the method:
Fig. 5. pH can alter resolution of a method. Three overlaid chromatograms showing how changing pH from 6.5 to 8.5 shifts the elution profile when eluting using a salt gradient.
Note: Some proteins fall out of solution at a pH equal to their pI. For these proteins, elution with a pH gradient may not be possible.
Ion exchange chromatography resins are composed of positively or negatively charged functional groups that are covalently bound to a solid matrix. Common matrices are cellulose, agarose, polymethacrylate, polystyrene, and polyacrylamide. The latter three matrices allow higher flow rates.
Several factors inform resin choice:
For many protein purification workflows, protein folding and stability is a concern. In these scenarios, the selection of an anion or a cation exchanger depends on the protein of interest’s stability.
Some proteins are stable both above and below their pI. These proteins can be purified with either an anion or cation exchanger. Other proteins are stable only above or below their pI. For these proteins, stability dictates resin choice; if, for example, a protein is stable only above its pI, an anion exchange resin should be chosen. When protein stability is not of concern, either an anion or cation exchanger can be used.
Ion exchange resins come in two types: strong and weak.
The number of charges on a strong ion exchanger remains constant regardless of the buffer pH. These types of resins retain their selectivity and capacity over a wide pH range. Examples of strong ion exchangers are quaternary ammonium (Q), sulfonate (S), and sulfopropyl (SP) resins.
Weak ion exchangers, in contrast, display pH-dependent function and so deliver optimal performance over only a small pH range. When the pH of the buffer no longer matches the acid dissociation constant (pKa) of the resin functional group, these resins suffer significant capacity loss. Weak anion exchangers function poorly above a pH of 9 and weak cation exchangers begin to lose their ionization below pH 6. When working with weak ion exchange resins such as diethylaminoethyl (DEAE) or carboxymethyl (CM) resins, it is important to work within the supplier-provided working pH range.
* Check manufacturer’s instructions for pH range for each individual resin. Proteins of interest may not be stable over the full pH range.
Strong ion exchangers are often preferred resins for many applications because their performance is unaffected by pH. However, weak ion exchangers can be powerful separation tools in cases where strong ion exchangers fail because the selectivities of weak and strong ion exchangers often differ.
The ionic form of a support refers to the counterion that is adsorbed onto the resin’s functional groups. This ion can be changed by swapping the column equilibration buffer. Common counterions for anion and cation exchangers are Na+ and Cl-, respectively.
The strength of the interaction with a given resin varies for different counterions. The lower the selectivity of a counterion for the support, the more readily it can be exchanged for another ion of like charge (for example, the protein of interest). Similarly, elution buffer containing a counterion with a relatively lower selectivity for the support will displace proteins from the column resin less readily during elution. In some cases, this difference can be exploited, and counterions such as Li+, Br-, and SO42- are often used to improve resin selectivity.
Resin particle size refers to the size of the resin solid support. Particle size does not affect the selectivity of the resin but it does impact resolution.
Fig. 6. Relationship between resin particle size, pressure, and resolution.
Smaller particles provide higher resolution but typically also require lower flow rates. High-resolution media are commonly used for analytical and small-scale work as well as for the final polishing steps of preparative chromatography. Very viscous samples such as cleared E. coli lysates or samples containing glycerol often cannot be separated using small-particle IEX resins due to the increased backpressure of small-particle resins, which can exceed the column’s operating pressure limit.
Larger particles permit higher flow rates but yield lower resolution. A method that yields sharp, distinct peaks using a small-particle IEX resin will yield broader, less defined peaks when a larger-particle resin is used. Large-particle IEX resins are a great choice for large-scale and preparative work. Larger particle sizes are also the best choice when samples are viscous, such as when IEX is used as a first step in a protein purification workflow.
Flow rate refers to how fast buffer is being passed over a resin. The flow rate therefore determines the amount of time in which proteins can interact with the column resin, which is called the residence time of a particular column at a given flow rate. Unlike particle size, flow rate affects both resolution and capacity: longer residence times increase both the capacity and the resolution of a resin.
Fig. 7. Effect of flow rate on IEX resolution. Separation of a 5 ml sample of myoglobin (peak 1), ribonuclease A (peak 2), and cytochrome c (peak 3) on a 1 x 13 cm (8.7 ml) Macro-Prep® High Q cation exchange column.
Flow rates are not only limited by the loss of resolution and capacity at higher flow rates but also by the resin itself. As flow rates increase, pressure on the resin increases. If the backpressure is too high, it can crush the column resin. Manufacturers thus provide a pressure limit for all of their resins.
Generally, the fastest flow rate that still renders the desired capacity and resolution is chosen. Although slower flow rates may provide even better resolution and capacity, this is often at the expense of protein activity, as many proteins lose activity with time under the conditions in the chromatography system.
The dynamic binding capacity of a resin refers to the amount of protein the resin can bind at a given flow rate; it is generally reported as mg/ml of protein bound at a certain flow rate. This value varies from resin to resin and can be important when fast flow rates are required to maintain protein activity.
The composition of loading, wash, and elution buffers is an important consideration for ion exchange chromatography. When a buffer contains the wrong counterion, it can prevent binding of the protein of interest to the column resin. The charged species in buffers used for ion exchange chromatography should thus generally have the same sign as the charged species of the IEX resin. For example, although phosphate buffers are commonly used for protein purification, they are not appropriate for anion exchange chromatography because the phosphate ion interacts strongly with positively charged anion exchange resins.
Common buffers for anion and cation exchange chromatography are:
Ion exchange chromatography is a very powerful separation technique that is used not only for preparative chromatography but also for analytical chromatography. However, like all other chromatography modes, IEX does have some limitations.
One of the main disadvantages of ion exchange chromatography is its buffer requirement: because binding to IEX resins is dependent on electrostatic interactions between proteins of interest and the stationary phase, IEX columns must be loaded in low-salt buffers. For some applications, this restriction may require a buffer exchange step prior to ion exchange chromatography.
Conversely, its requirement for loading samples in buffers of low ionic strength makes ion exchange chromatography an excellent second purification step after hydrophobic interaction chromatography (HIC).
Ion exchange chromatography, unlike some other chromatography methods, also permits high flow rates, which in some cases can be crucial to the recovery of active protein. Finally, a limitation of weak ion exchangers is their pH dependence. When working outside of their optimal pH range, these resins rapidly lose capacity, and more importantly, resolution.
Bio-Rad carries a wide range of anion and cation exchange resins in prepacked column form or as bulk resin. Some resins are more suited for analytical chromatography, whereas others are better suited for preparative and/or process chromatography. To select the best media for your intended application, visit the pages below for more detailed descriptions of Bio-Rad anion and cation exchange media:
This video presentation covers the basic principles of ion exchange chromatography including media choice, buffer selection, and factors that impact resolution.
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