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Chromatography literally means “color writing”. Chromatography was invented by the Russian botanist
Mikhail Tsvet in 1900. He used it to separate chlorophyll-containing extracts of plants.

Key idea is that molecules of interest interact differentially with the stationary phase and a mobile
phase, and thus can be separated.

Biomolecules are purified using chromatography techniques that separate them according to differences in
their specific properties. Ion exchange chromatography (IEX) separates biomolecules according to
differences in their net surface charge.




Gel filtration (GF), also called size exclusion




Chromatofocusing (CF)






Reversed phase chromatography (RPC)

Affinity chromatography (AC)


their net surface charge is highly pH dependent. offering high resolution and group separations with high loading capacity. This curve reflects how the overall net charge of the protein changes according to the pH of the surroundings.  The charged groups within a molecule that contribute to the net surface charge possess different pKa values depending on their structure and chemical microenvironment. . IEX is one of the most frequently used techniques for purification of proteins. Limitation: The technique is capable of separating molecular species that have only minor differences in their charge properties. their net surface charge will change gradually as the pH of the environment changes  Each protein has its own unique net charge versus pH relationship which can be visualized as a titration curve. for example two proteins differing by one charged amino acid. peptides. These features make IEX well suited for capture.  Since all molecules with ionizable groups can be titrated. intermediate purification or polishing steps in a purification protocol and the technique is used from microscale purification and analysis through to purification of kilograms of product.IEX for the separation of biomolecules was introduced in the 1960s and continues to play a major role in the separation and purification of biomolecules. which are built up of many different amino acids containing weak acidic and basic groups.  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. In the case of proteins. Today. IEC Principle and Theory Net surface charge and pH Ion exchange chromatography is a process that allows the separation of ions and polar molecules based on the charge properties of the molecules. nucleic acids and other charged biomolecules.

at a pH below its pI. Components of an IEC 1) Sample A sample is any biological material that is to be separated in a column. a protein will bind to a positively charged medium or anion exchanger and. showing how net surface charge varies with pH  IEX chromatography takes advantage of the fact that the relationship between net surface charge and pH is unique for a specific protein. works as transporter. In addition to the ion exchange interaction. other types of binding may occur. moves in a definite direction. In an IEX separation reversible interactions between charged molecules and oppositely charged IEX media are controlled in order to favor binding or elution of specific molecules and achieve separation. Theoretical protein titration curves. .  A protein that has no net charge at a pH equivalent to its isoelectric point (pI) will not interact with a charged medium. However. at a pH above its isoelectric point. but these effects are very small and mainly due to van der Waals forces and nonpolar interactions. It can be introduced either manually or with an autosampler. a protein will behind to a negatively charged medium or cation exchanger.Figure 2. The mobile phase in ion exchange chromatography is a buffered aqueous solution that carries the sample from the loop onto a column that contains some form of stationary phase material. into a sample loop of known volume. 2) Mobile Phase A mobile phase is a solution of gas or liquid components.

sulfate derivatives. and DEAE resin. and CM resins. DiEthylAminoEthane . Commonly used cation exchange resins are S-resin. Types of Ion Exchangers 1) Cation Exchanger Positively charged molecules are attracted to this negatively charged solid support. carboxylate derived ions 2) Anion Exchanger Negatively charged molecules are attracted to this positively charged solid support. Commonly used anion exchange resins are Q-resin. the positively charged analyte could be displaced by the addition of positively charged sodium ions. The stationary phase in ion exchange chromatography is a resin or gel matrix consisting of agarose or cellulose beads with covalently bonded charged functional groups. 4) Target analytes The target analytes. either the anions or cations. a Quaternary amine. Example: In cation exchange chromatography.3) Stationary Phase A stationary phase is a liquid or solid that absorbs or impedes different components of the solution to different degrees. are retained on the stationary phase but can be eluted by increasing the concentration of a similarly charged species that will displace the analyte ions from the stationary phase.

Depending on the conditions of the environment. a positively charged anion exchange resin is chosen to capture this protein. thus a negatively-charged cation exchange resin is chosen. When an ion exchange chromatography column is loaded with a sample at a particular pH. In a buffer with a pH greater than the pI of the protein of interest. if an anion exchange resin is chosen. the protein will carry a net negative charge. The choice of buffer pH then determines the net charge of the protein of interest. cationic (+) or zwitterion (no net charge) stage. it can exist in anionic (-).Selection of an ion exchanger Proteins are charged molecules. Note that isoelectric point is the pH where the net surface charge of a protein is zero. all proteins that are appropriately charged will bind to the resin. The pI value can be calculated based on the primary sequence of the molecule. all proteins that are negatively charged at the loading buffer pH will bind to the positively charged column resin. In a buffer with a pH lower than the pI of the protein of interest. For example. At a specific pH. therefore. A good rule of thumb for choosing a buffer pH is the following: . the protein will carry a positive net charge.

5 pH units less than the pI of the protein of interest IEC Set-up: .5–1.Anion exchanger — 0.5 pH units greater than the pI of the protein of interest Cation exchanger — 0.5–1.

depending on the total volume of sample being loaded. the proteins with the highest charge at a certain pH will be most strongly retained and will be eluted last. proteins are eluted by increasing the ionic strength (salt concentration) of the buffer or. when sample is loaded. 3. 2. The matrix is usually porous to give a high internal surface area.e. conditions are altered in order to elute the bound proteins. by changing the pH. An IEX medium comprises a matrix of spherical particles substituted with ionic groups that are negatively (cationic) or positively (anionic) charged. the UV signal has returned to baseline). The medium is packed into a column to form a packed bed. Similarly.Steps in an IEC Separation 1. 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 higher the . occasionally. The pH and ionic strength of the equilibration buffer are selected to ensure that. The bed is then equilibrated with buffer which fills the pores of the matrix and the space in between the particles. As ionic strength increases. The proteins which bind are effectively concentrated onto the column while proteins that do not have the correct surface charge pass through the column at the same speed as the flow of buffer. Most frequently. eluting during or just after sample application. proteins of interest bind to the medium and as many impurities as possible do not bind. The proteins with the lowest net charge at the selected pH will be the first ones eluted from the column as ionic strength increases. When all the sample has been loaded and the column washed so that all non-binding proteins have passed through the column (i.

the higher the ionic strength that is needed for elution. 4. By controlling changes in ionic strength using different forms of gradient. A wash step in very high ionic strength buffer removes most tightly bound proteins at the end of an elution. concentrated charge of the protein. The column is then re-equilibrated in start buffer before applying more sample in the next run. . proteins are eluted differentially in a purified.

A Closer look to how IEC works: .