Chromatography

Chromatography (from Greek χρώμα: chroma, colour) is the collective term for a family of laboratory techniques for the separation of mixtures. It involves passing a mixture through a stationary phase, which separates the analyte to be measured from other molecules in the mixture and allows it to be isolated. • • •

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The analyte is the substance which is to be purified or isolated during chromatography Analytical chromatography is used to determine the identity and concentration of molecules in a mixture A chromatogram is the visual output of the chromatograph. Different peaks or patterns on the chromatogram correspond to different components of the separated mixture A chromatograph takes a chemical mixture carried by liquid or gas and separates it into its component parts as a result of differential distributions of the solutes as they flow around or over the stationary phase The mobile phase is the analyte and solvent mixture which travels through the stationary phase Preparative chromatography is used to nondestructively purify sufficient quantities of a substance for further use, rather than analysis. The retention time is the characteristic time it takes for a particular molecule to pass through the system under set conditions. The stationary phase is the substance which is fixed in place for the chromatography procedure and is the phase to which solvents and the analyte travels through or binds to. Examples include the silica layer in thin layer chromatography.

Chromatography is a separation method that exploits the differences in partitioning behavior between a mobile phase and a stationary phase to separate the components in a mixture. Components of a mixture may be interacting with the stationary phase based on charge, relative solubility or adsorption. There are two theories of chromatography, the plate and rate theories.

Retention The retention is a measure of the speed at which a substance moves in a chromatographic system. In continuous development systems like HPLC or GC, where the compounds are eluted with the eluent, the retention is usually measured as the retention time Rt or tR, the time between injection and detection. In interrupted development systems like TLC the retention is measured as the retention factor Rf, the run length of the compound divided by the run length of the eluent front:

The retention of a compound often differs considerably between experiments and laboratories due to variations of the eluent, the stationary phase, temperature, and the setup. It is therefore important to compare the retention of the test compound to that of one or more standard compounds under absolutely identical conditions.

Plate theory The plate theory of chromatography was developed by Archer John Porter Martin and Richard Laurence Millington Synge. The plate theory describes the chromatography system, the mobile and stationary phases, as being in equilibrium. The partition coefficient K is based on this equilibrium, and is defined by the following equation:

K is assumed to be independent of concentration, and can change if experimental conditions are changed, for example temperature is increased or decreased. As K increases, it takes longer for solutes to separate. For a column of fixed length and flow, the retention time (tR) and retention volume (Vr) can be measured and used to calculate K.

Paper Chromatography For more details on this topic, see Paper chromatography. This is an older technique which involves placing a small spot of sample solution onto a strip of chromatography paper. The paper is placed into a jar containing a shallow layer of solvent and sealed. As the solvent rises through the paper it meets the sample mixture which starts to travel up the paper with the solvent. Different compounds in the sample mixture travel different distances according to how strongly they interact with the paper. This allows the calculation of an Rf value and can be compared to standard compounds to aid in the identification of an unknown substance.

Thin layer chromatography For more details on this topic, see Thin layer chromatography. Thin layer chromatography (TLC) is a widely-employed laboratory technique and is similar to paper chromatography. However, instead of using a stationary phase of paper, it involves a stationary phase of a thin layer of adsorbent like silica gel, alumina, or cellulose on a flat, inert substrate. Compared to paper, it has the advantage of faster runs, better separations, and the choice between different adsorbents. Different compounds in the sample mixture travel different distances according to how strongly they interact with the adsorbent. This allows the calculation of an Rf value and can be compared to standard compounds to aid in the identification of an unknown substance.