Column Chromatography for Molecular Separations
How Compounds and Molecules are Separated by Column Chromatography
Oct 8, 2009
Chromatography is a useful, effective procedure and "tool" of chemistry and biology. Chromatography enables separation of molecular mixtures of different and important compounds. Column chromatography is one variation of several types of chromatography for this scientific inquiry.
Chromatography Types Paper, Gas, Liquid, HPLC, Gel Filtration, Ion Exchange, Affinity
Paper chromatography and simple column chromatography were the earliest and simplest chromatography types used to analyze pigments from plants and animals.
Gas, or liquid, chromatography is a physical-chemical, tubed system that promotes the separation of gaseous or liquid mixtures of compounds such as: drugs, proteins, sugars, amino acids, lipids, among other compounds. The compounds in mixtures are carried by gasses, or liquids, as mobile phases moving within glass,metal or plastic column walls. Mixtures are separated and various compounds are collected as distinct, separate fractions at the exit port of the column.
Gas and Liquid Column Chromatography, Gel Filtration, Ion Exchange, and Affinity Types
Chemical mixtures physically may be a mixture of chemical compounds:
- immersed in a suitable gas (as in the case of volatilized mixtures, injected through a heated port), or
- immersed in a liquid (mobile phase), and subjected to force (e.g., HPLC = High Pressure Liquid Chromatography, or gravity);
In either case, the chemical mixtures flow across a stationary phase. Imagine this as a flowing river in a tube or tunnel filled with rocks or pebbles (stationary phase) of defined size. Imagine, also, the dissolved or suspended materials flowing over the rocks and pebbles. The molecules of the different compounds in a river of liquid or gas will move at different speeds based on:
- size (molecular sieve),
- charge (ion exchange) or
- capture by another molecular type (affinity).
Therefore, chemical mixture separations are made possible by:
- molecular sieve, or gel filtration, chromatography: molecules of different compounds are retarded to different degrees, depending on sizes — large molecules retarded more, smaller molecules less, and
- ion exchange chromatography: e.g., negatively-charged compounds and molecules bind to positively-charged stationary molecules, or vice versa, and
- affinity chromatography: antibodies, or antigens, may be used for capture, respectively, of antigens or antibodies.
Chromatography Retention Time Variations Enable Purification and Identification of Compounds
Properly-prepared columns cause defined and different retention times, for the various, dissolved biochemical molecules. Some compounds interact strongly, moderately, weakly or, perhaps, not at all. This creates a gradient of the molecular types and permits separations to occur over the length and breadth of the column. The molecules come across "the finish line" at the column's terminus in chemical groups. In summary, the different mobile phase and stationary phase interactions within the column give different compounds different transit or retention times. These times may be documented and compared to known control biochemicals.
Click on the photo below to see fast-moving molecules of the red compound separating from the slower-moving molecules of the green compound. If separation is based on size differences it is molecular sieve chromatography; if separation is based on charge, it is ion exchange chromatography. Affinity chromatography usually captures select molecules and binds them until a chemical is added to wash off (elute, release) them. The eluant is the solution used to remove the bound molecules, the eluate is the detached molecular type.
Mass Spectroscopy and Chromatography, Molecular Identifications
A mass spectroscope may be connected to the chromatograph to analyze the separated molecular species as they exit and are collected at the port. Typically, flame ionization, or electron capture, detectors signal the exit of molecular compounds. These compounds then are recorded as to type and and amounts of each. The molecules, then, are transited to mass spectroscopy where unique molecular-atomic signatures of the compounds enable identification.
Resources
Lodish, H. et al. 2000. Molecular Cell Biology. Fourth Ed., W. H. Freeman and Co., New York, N.Y.
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