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Let us suppose that a sample contains two compounds 1 and 2 to be measured and that compound IS represents an additional compound for use as an internal standard (Figure 1.13).

In the first stage, a solution containing compound 1 at known concentration C₁, compound 2 at known concentration C₂, and the internal standard IS at known concentration C_IS is prepared and then injected onto the chromatograph. A₁, A₂, and A_IS are the areas of the elution peaks in the chromatogram due to the three compounds. If m₁, m₂, and m_IS represent the real quantities of these three substances introduced onto the column, then three equations of the type of Eq. (1.42) can be derived:

These ratios enable the calculation of the relative response factors of 1 and 2, with respect to IS. They are designated by K_1/IS and K_2/IS:

Figure 1.13 Method of analysis by internal standard.

Since the injected masses m_i are proportional to the corresponding mass concentrations C_i (m_i = C_i V), the above equations can be rewritten as: