Chromatography is a technique that is used for separation of mixtures. The name combines the Greek word chroma, or “color,” with graphein, “to write.” The technique was invented in 1900 by Mikhail Tsvet, who used it “to write the colors” of plant pigments, like chlorophyll carotenes and xanthophylls. He did this by dissolving the plant mixtures in a fluid, or mobile phase, and allowing it to flow through a solid material called a stationary phase.
The components of the mixture to be separated flow through the stationary phase material at different rates, which creates waves or bands. Originally, those bands were detected by visible color differences. Detection methods are now based on the chemical and physical properties of the molecules being separated, including, but not limited to color, UV absorbance, size, charge, and hydrophobicity.
In modern chromatographic separations, the stationary phase comes in the form of media, either pre-packed in a chromatography column or added by the user to the separation apparatus. The choice of media depends on the type of molecule being separated, the means of separation, the purpose and scale of separation, as well as the desired purity of the separated sample.
Generally, the material used in chromatography media is a particle or resin packed into a column. The sample is loaded into the top of the column and is flushed through with the mobile phase liquid, gravity or under pressure. Chromatography may be carried out on a small scale for analytic purposes or on a medium-to-large scale for preparation and industrial purposes.
Ion exchange chromatography
One of the most popular methods for separating proteins is ion exchange chromatography. It separates proteins in a sample based on charge. If a negatively charged resin is used to capture positively charged molecules, the method is referred to as cation exchange chromatography.
The opposite, in which the resin is positively charged and the target molecule is negatively charged, is called anion exchange chromatography. An ion exchange resin is made using positively or negatively charged functional groups on a solid matrix like cellulose, agarose or polyacrylamide. A protein sample is loaded onto the column in a buffer of low salt and then flushed through the column with a gradient of salt concentration or change in pH.
Size exclusion chromatography
A simple and surprisingly effective physical property that can be used for chromatographic separations is size. In size exclusion chromatography (SEC), the sample is filtered through a gel made of spherical beads with specifically-sized pores.
These pores either include or exclude proteins flowing through in the mobile phase. This method is commonly used for separation of proteins. Molecules too large to enter the pores will flow through quickly, and molecules that enter the pores will be slowed down and pass through the column more slowly. SEC is often used as an early step in protein purifications, such as before an ion exchange separation and to desalt a sample or exchange buffers.
Affinity chromatography is based on binding interactions between a protein and a ligand immobilized to a resin. These binding interactions are highly selective. An affinity separation is a very precise separation and will almost always capture only one protein in a mixture.
Affinity chromatography can be used as a one-step purification method, in some circumstances, achieving a greater than 1000-fold purification of a specific protein. The resin material for an affinity column can be solid or a porous gel material. A wide range of materials have been used as a solid matrix material for affinity chromatography, including agarose, cellulose, dextran, polyacrylamide, and many more.
Porous gel matrices for affinity columns are particularly attractive, because the micro-bead format allows them to be dispensed as a wet slurry and packed into columns of any size. Affinity media for binding general classes of proteins or proteins with common fusion tags is available commercially. More specialized ligands can be coupled to commercially available activated affinity matrix products.
While ion exchange, size exclusion and affinity chromatography media are some of the most popular and commonly used options, there are dozens of other methods. These include hydrophobic interaction chromatography, reversed phase chromatography, two-dimensional chromatography and many others that are used to explore and achieve the perfect separation.
- Mikhail Tswett (1906) "Physikalisch-Chemische Studien über das Chlorophyll. Die Adsorption." (Physical-chemical studies of chlorophyll. Adsorption.)Berichte der Deutschen botanischen Gesellschaft, vol. 24, pp. 316–326.
- Bench Tips: Tips on Selecting a Liquid Chromatography Resin, www.biocompare.com/.../
- Ion Exchange Chromatography, http://www.proteinchemist.com/tutorial/iec.html
- Overview of Affinity Purification, www.thermofisher.com/.../overview-affinity-purification.html