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Chromatography is a physico‐chemical method of separation of components within mixtures, liquid or gaseous, in the same vein as distillation, crystallization or fractionated extraction. The applications of this process are therefore potentially numerous, since many heterogeneous mixtures, or those in solid form, can be dissolved by a suitable solvent (which becomes, of course, a supplementary component of the mixture).

A basic chromatographic process may be described as (Figure 1.1):

1 A vertical, hollow glass tube (the column) is filled with a suitable finely powdered solid, the stationary phase.

2 At the top of this column is placed a small volume of the sample to be separated into individual components.

3 The sample is then forced through the column from inlet to outlet by continuous addition of the mobile phase, carrying the various constituents of the sample along with it. If the components migrate at different velocities, they will become separated from each other and can be recovered, each in solution with the mobile phase.

While this use of chromatography has continued since its origins, this process became a method of analysis with the idea of measuring the retention time of compounds through the column in order to identify them. To do so, it became essential to control certain parameters (flow rate, temperature, etc.) and a detector had to be placed at the column’s outlet to identify compositional changes in the mobile phase. This form of chromatography, whose goal is not simply to recover the components but to measure their retention time, has developed slowly.

The identification of a compound by chromatography is achieved by comparison. To identify a compound, which may be either A or B, using chromatography, we compare its retention time with those for the two reference compounds A and B previously recorded using the same apparatus and the same experimental conditions.

Figure 1.1 A basic experiment in chromatography. (a) The necessary ingredients (C, column; SP, stationary phase; MP, mobile phase and sample); (b) introduction of the sample; (c) start of elution; (d) recovery of the products following separation.

In this experiment, there was no true separation (A and B were pure products), only a comparison of the products’ retention times. However, this method does have three weaknesses: the procedure is fairly slow; absolute identification is unattainable; and the physical contact between the sample and the stationary phase could modify the sample’s properties, in particular the retention times.

This specific method of separation, in its modern form, was first undertaken at the beginning of the twentieth century by the botanist Mikhail Tswett (or Tsvet), who is credited with inventing the terms chromatography and chromatogram.

The technique has improved considerably since its beginnings. Nowadays, chromatographs are piloted by software programs that run highly efficient miniature columns able to separate nano‐quantities of sample. These instruments comprise a complete range of accessories designed to ensure repeatability of successive experiments by the perfect control of the different parameters of separation. Thus it is possible to obtain, during successive analyses of the same sample conducted several hours apart, recordings that are reproducible to within a second (Figure 1.2).

The specific recording that is obtained for each separation is called a chromatogram. It corresponds to a two‐dimensional diagram that reveals the variations of composition of the eluting mobile phase as it exits the column. To obtain this read‐out, a sensor, or detector, of which there exists a great variety, needs to be placed at the outlet of the column.

Figure 1.2 The principle of analysis by chromatography. The chromatogram, the essential graph of every chromatographic analysis, is obtained from variations, as a function of time, of an electrical signal emitted by the detector. It is either produced in real time or reconstructed at a later time from values that have been digitized and stored. The chromatography software recalculates these values and puts them in the desired format. This chromatogram illustrates the separation of a mixture of three principal components. Note that the order of appearance of the compounds corresponds to the relative position of each constituent on the column.

The identification of a molecular compound from the chromatogram can sometimes be risky. A better method consists in associating two different complementary methods, for example, a chromatograph coupled with a second instrument, such as a mass spectrometer or an infrared spectrometer. These coupled (or two‐dimensional) techniques provide two independent types of information (retention times and the spectrum). Therefore, it is possible to determine without ambiguity the composition of complex mixtures or the concentration of certain compounds on the nanogram level (confirmation analyses).