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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.

The process is based upon the comparison of two chromatograms obtained successively without changing the control settings of the chromatograph (Figure 1.12). The first chromatogram is acquired from a standard solution (reference solution) with a known concentration C_ref of the analyte in a solvent. A volume V of this solution is injected. On the resulting chromatogram, the area A_ref of the corresponding peak is measured. The second chromatogram results from the injection of the same volume V of the sample in solution, containing an unknown concentration of the compound to be measured (conc. C_unk). The area of the corresponding peak is A_unk. Since an identical volume of both samples has been injected, the ratio of the areas is proportional to the ratio of concentrations, which depend upon the masses injected (m_i = C_i · V). Applied to the two chromatograms, Eq. (1.42) leads to Eq. (1.43), which characterizes this method:

(1.43)

Figure 1.12 Analysis by the external standard method. The precision of this basic method is improved when several solutions of varying concentrations are used in order to create a calibration curve. For trace analyses by liquid chromatography, it is sometimes advisable to replace the areas of the peaks by their heights, as the latter are less sensitive to variations in the mobile phase flow rate.

The single‐point calibration method, as depicted in Figure 1.12, assumes that the calibration line goes through the origin. Precision will be improved if the concentrations of the reference solution and of the sample solution are similar. That means the device settings do not need to be changed between injections.

This technique, employing the absolute response factors, yields very reliable results with current high‐performance chromatographs equipped with an autosampler: a combination of a carousel sample holder and an automatic injector. This gives numerous measurements without human intervention. A single reference solution compensates for a potential drift of the instrument with scheduled control re‐injections.

Precision of the assay can obviously be improved if several injections of the sample and the reference solutions are made, always using equal volumes. The average peak area is then calculated; however, unless several measurements are made, it is preferable to conduct a multilevel calibration, in which case equal volumes of a series of standard solutions are injected. The analytical results are obtained directly from the calibration curve, A = f (C).

This method, the only one adapted to gas samples but also applicable in LC, has the added advantage of its ease of execution and its rapidity. However, it requires perfect reproducibility of injected volumes, which automatic injectors can do nowadays.