Introduction:

Chromatography:
From the Greek chroma + graphia, literally "colour writing"
A technique for analysing mixtures of gases, liquids, or solutes by exploiting differences in their distribution between a stationary and a mobile phase.

Chromatography is a fundamental technique for the separation, detection, identification and quantitation of chemical species. It comes in two basic formats: planar and column chromatography. Chromatographic techniques can also be classified according to whether they are being used for identification and measurement (analytical, for microscale to trace quantities of analyte) or as a purification step (preparative, for semimicro to macroscale quantities of analyte). Today, chromatographic techniques are essential tools in areas such as chemistry, biology, medicine, forensic science, manufacturing, and the environment, and are arguably the most widely used of any family of analytical methods.

Planar Chromatography:

A Flash animation or Quicktime movie depicting the separation of a two-component mixture by TLC, with identification by matching migration distance with known standards. Requires either Adobe’s Flash plugin (version 9 or higher) or Apple’s Quicktime plugin to be visible.

The origins of planar chromatography can be traced back to the practice amongst dyers of testing pigments by placing a drop on paper and observing the colours as the drop spreads. The scientific use of paper chromatography can be traced to the mid-19th century.[1] The important separation of amino acids and peptides by paper chromatography was developed in 1994 by Consden, Gordon, and Martin[2]. Thin layer chromatography (TLC), as shown on the right, was developed in 1938 by Izmailov and Shraiber[3], based on Mikhail Tswett’s earlier description of column chromatography.

In planar chromatography, samples and standards are placed along a line at one end of a piece of paper or plate coated with a porous solid layer – the stationary phase. This is placed in a closed container together with an amount of solvent – the mobile phase. As the solvent rises up the plate by capillary action, the compounds migrate at different rates based on their relative affinities for the stationary and mobile phases. Identification is based on the distance migrated by each spot.

Column Chromatography:

A Flash or QuickTime animation showing separation of a three-component mixture by column chromatography, with collection of the separated components. Requires either a Flash or QT plugin to be visible.

Russian botanist Mikhail Tsvet invented column chromatography in 1906 as a means of studying plant pigments, but it soon became clear that the technique provided a means for separating many complex homogeneous mixtures into their individual components. In conventional column chromatography (as shown on the right) the stationary phase – consisting of a finely divided solid – is retained in a vertically-mounted tube. The mobile phase – consisting of one or more solvents – fills the voids between the solid particles. A sample mixture is introduced to the top of the column, and additional mobile phase is added and allowed to flow down through the column. As was the case with TLC (above), the individual sample components migrate at different rates depending on their relative affinities for the stationary and mobile phases. Each sample is collected as it exits the column, and identification is based on the time (or volume of mobile phase) taken for this to occur.

Available Chromatography Resources:

Footnotes:

  1. A.-M. Siouffi, “From Paper to Planar: 60 Years of thin layer chromatogtaphy” Separation & Purification Reviews, 2005, 34:2, 155-180. DOI: 10.1080/15422110500322990
  2. R. Consden, A.H. Gordon, & A.J.P. Martin, Biochem. J., 1944, 38, 224-232.
  3. N.A. Izmailov & U.M.S. Shraiber, Farmatsya, 1938, 3, 1ff..