Chromatography

The chromatography is a technique of qualitative and quantitative analysis of the analytical Chimie in which the sample containing one or more species is pulled by a mobile current of phase (liquid, gas or supercritical fluid) along a stationary phase (paper, Gélatine, silica, polymer, silica grafted etc). Each species moves at a clean speed depending on its characteristics and those of the two phases.

The analytical chromatography is used to identify or proportion the chemical compounds of a mixture and to appreciate their concentration.

The préparative chromatography is used to purify enough product for other uses. Its goal is to obtain substance; this is why, on any scale, it implies to collect fractions.

History

The Russian botanist Mikhail Tswett (1872 - 1919) was, in 1906, the first to use the term chromatography .

As from 1903, Tswett used columns of adsorption to separate from the Pigment S of plants. One thus speculated the etymology of the word chromatography starting from the Greek khrôma- for color and thus pigment. However, Tswett never gave this explanation; but it will be noted that tswett is the Russian word for color .

In 1952, Martin and Synge accepted the Nobel Prize of chemistry for their invention of the Chromatographie by partition.

Principle

The chromatography rests on the drive of a sample dissolved by a mobile phase through a stationary phase. This one more or less strongly retains the substances contained in the sample diluted according to the intensity of the forces of interactions of weak energy (like the forces of van der Waals, the hydrogen bonds, etc) carried out between the various molecular species and the stationary phase.

The various components of the sample have generally a characteristic speed which makes it possible to separate them, to even identify them. This bursting speed is strongly dependant on the nature of the mobile phase and the stationary phase.

Often, the sample is analyzed by comparison with substances already known in the sample. These substances are used as references and make it possible to identify each species by comparison bursting speeds (and possibly of other information given by detection).

In other cases, one is satisfied to separate the fractions, to collect them to identify them by other techniques.

There exist many types of chromatography; one can in particular classify them according to the nature of the mobile phase:

One can also name them according to the interactions developed by the stationary phase:

or according to the support of the stationary phase:

  • chromatography on column (gathering in particular HPLC and CPG);
  • the planar chromatography (which recovers Thin layer chromatography and chromatography on paper);

Chromatography on paper

The chromatography on paper is a technique of Chromatography in liquid phase.
Pour to carry out such a separation, an minor amount solutions to be analyzed is deposited on the edge of a paper band of chromatography. This sample is adsorbed by paper; what means that the molecules interact with this last and that they will tend to remain at the same place.

(NB: Any substance which would react with paper cannot be analyzed using this technique)

Paper is then soaked in a solvent (éluant) like a mixture water ethanol and is placed in a closed container. While the solvent (éluant) goes up along paper by capillarity, it meets the sample and involves it.

The various substances constituting the sample migrate at various speeds according to whether they more or less strongly interact with paper.

The chromatography on paper takes a certain time (generally several hours). Once the finished operation, generally when the solvent face (éluant) almost arrived in top of paper, paper is withdrawn from the tank and one lets evaporate solvent. The result is called chromatogram .

The chromatogram is used for comparison with other analyzes carried out on substances known and taken under identical conditions, to identify the substances of the sample. The substances can be identified by calculating the value RF which can be compared with those being in the tables. This value is calculated in the following way: RF = (distance covered by the sample)/(distance covered by solvent)

There are several ways of identifying the places where the products thus separate are:

  1. the products are coloured, it does not have nothing special there to make.
  2. the products are fluorescent S, one can identify them under a lamp Ultraviolet you.
  3. if not, it is necessary to use revealing which will react chemically with the products (by destroying them) and whose result will be coloured.

To separate from the complex mixtures of similar substances, it can be useful to use the chromatography with two dimensions. This one is carried out in two stages between which one changes solvent and one turns the paper of 90°. The interactions developed by new solvent will be different, which will modify separation in this second dimension and will allow a better total separation.

The chromatography on paper can also constitute a technique microphone-préparative: to recover the products thus separate, the portions of paper where they are located are cut out and redissolved then. The recoverable quantities are about the milligram or less.

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