Chemical Analysis. Francis Rouessac
This type of separation relies on ion exchange equilibria characterized by ionic distribution coefficients.
Size exclusion chromatography (SEC)
The stationary phase here is a material containing pores whose dimensions are selected as a function of the size of the species to be separated. This technique is used to separate synthetic or natural macromolecules. It is known as gel filtration (e.g. separation of natural macromolecules such as proteins) when the mobile phase is aqueous, or gel permeation (e.g. separation of synthetic macromolecules) when the mobile phase is an organic solvent. Thus, it involves a sort of selective permeation at the molecular scale. For this technique, the distribution coefficient is called the diffusion coefficient.
Affinity chromatography
Halfway between chromatography and extraction, this technique, though not often employed for analytical purposes, is used as a purification method in biochemistry. It consists of preparing a stationary phase (carrier resin) on which a specific ligand for the molecule we wish to separate is grafted to a maximum number of sites. As it has affinity for the ligand, the molecule is retained on the column. After elution of the other compounds present with the binding liquid phase, the latter is transformed into an elution buffer to retrieve the molecule of interest. The yield of this operation is related to the number of sites available on the resin, and the selectivity of the separation depends on the choice of ligand, which must be pure.
1.12.2 Gas Chromatography (GC)
The mobile phase is an inert gas with regards to the stationary phase and to the solutes to be separated. Three gases are used in most applications: nitrogen (not very efficient, see the Van Deemter curve), helium (more expensive and more efficient), and hydrogen (more efficient but also more dangerous). Depending on the physical state of the stationary phase, we shall distinguish between gas/solid chromatography (GSC) and gas/liquid chromatography (GLC).
Gas/solid chromatography (GSC)
This is the oldest application of GC. As with LSC, the stationary phase is a solid (such as graphite, silica gel or alumina) and the separation occurs via selective adsorption on the stationary phase. This type of gas chromatography is very effective for the separation of volatile species (gas or liquids that have a low boiling point).
Gas/liquid chromatography (GLC)
It was Martin and Synge who suggested the replacement of the liquid mobile phase in LLC with a gas. As indicated above, the stationary phase is a liquid immobilized on a support (inert silica particles for packed columns, inner wall of the column for capillary molecules) generally by grafting. Most often used for molecules in solution, the sample must be brought to its vapour state beforehand or at the time of its introduction. Therefore, this technique can be used for compounds whose boiling points are not very high, as well as for compounds which have been subject to a prior pyrolysis stage.
1.12.3 Supercritical Fluid Chromatography (SFC)
Here the mobile phase is a fluid in its supercritical state, such as carbon dioxide at about 50°C and more than 150 bar (15 MPa). The stationary phase can be a liquid or a solid. This technique combines the advantages of gas chromatography and liquid chromatography. It has many applications, even though it requires more complex equipment.
Quantitative Analysis by Chromatography
The significant development of chromatography in quantitative analysis is essentially due to its reliability and its precision. The relationship between the injected mass of the analyte on the column and the area of the corresponding peak on the resultant chromatogram leads to a comparative method used in many standardized assay protocols. The reproducibility of separations, allied with data processing software, makes it possible to automate all the calculations associated with these analyses. Trace and ultratrace analyses by chromatography are used, particularly in EPA methods for environmental analysis, although their costs are rather high. The three most widely used methods are described here, along with their simplest configuration.
1.13 PRINCIPLE AND BASIC RELATIONSHIP
To calculate the composition of a sample or to assay an analyte responsible for a peak on a chromatogram, two basic conditions must be met. Firstly, an authentic sample of the analyte to be measured must be available, as a reference, to determine the detector’s sensitivity to this compound. Secondly, software calculating the areas (or failing that, the heights) of the different eluting peaks of interest is also required. All of the quantitative methods in chromatography are comparative methods (which is very often the case in quantitative analysis).
For a given tuning of the instrument, it is assumed that a linear relationship (linearization) exists for each peak of the chromatogram, over the entire concentration range, between its area (or failing that, its height) and the quantity of the analyte responsible for this peak in the injected sample. This relationship can be used for concentration ranges bounded by the detectable quantity limit for lower concentrations and the linearity limit for higher concentrations. This hypothesis is translated into the following equation:
where mi is the mass of compound i injected on the column, Ki is the absolute response factor for compound i, and Ai is the area of the eluting peak for compound i.
The absolute response factor Ki (not to be confused with the partition coefficient) is not an intrinsic parameter of the compound, as it depends upon the tuning of the chromatograph. To calculate the response factor Ki of an analyte, according to the above expression, it is essential that both the area Ai and the mass mi, of compound i injected on the column, are known. However, this injected mass is difficult to determine with precision. This is why most chromatographic methods used for quantitative analysis, preprogrammed into the software, do not make use of the absolute response factor, Ki.
1.14 CHROMATOGRAPHY SOFTWARE
Equation (1.42) highlights the need to determine peak areas. To do so, chromatography software has been developed to ensure control of the chromatogram, the acquisition of chromatograms and the determination of certain information related to the observed peaks, such as the FWHM δ, the width at the base ω, and the peak height or its area. These software programs help correct the baseline, select the start and end, if necessary, of each peak for integration, and, most importantly for quantitative analysis, select the quantification method. Lastly, by taking into account compositions of reference solutions (calibration solutions), they calculate sample compositions.
The three quantification methods used in chromatography (and sometimes in other techniques using proportionality between composition and response of a measuring instrument) are described here.
1.15 EXTERNAL STANDARD METHOD
This method allows the measurement of the concentration (or percentage by mass) of one or more components that appear as resolved peaks on the chromatogram, even in the presence of other compounds yielding unresolved peaks whose concentrations are not of interest to the experimenter.
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