An internal standard is a known amount of a compound, different from analyte, that is added to the unknown. Signal from analyte is compared with signal from the internal standard to find out how much analyte is present.
Internal standards are especially useful for analyses in which the quantity of sample analysed or the instrument response varies slightly from run to run for reasons that are difficult to control. For example, gas or liquid flow rates that vary by a few percent in a chromatography experiment (Figure 0-4) could change the detector response. A calibration curve is only accurate for the one set of conditions under which it is obtained. However, the relative response of the detector to the analyse and standard is usually constant over a wide range of conditions. If signal from the standard increases by 8.4% because of a change in solvent flow rate, signal from the analyte usually increases by 8.4% also. As long as the concentration of standard is known, the correct concentration of analyte can be derived. Internal standards are widely used in chromatography because the small quantity of sample solution injected into the chromatograph is not very reproducible in some experiments.
Internal standards are also desirable when sample loss can occur during sample preparation steps prior to analysis. If a known quantity of standard is added to the unknown prior to any manipulations, the ratio of standard to analyte remains constant because the same fraction of each is lost in any operation.
Using an internal standard
To use an internal standard, a known mixture of standard and analyte is prepared to measure the relative response of the detector to the two species (see detector response ratio).
In an experiment, a solution containing0.0837 M analyte (X) and 0.0666 M standard (S) gave peak areas of Ax=423 and As=347 (areas in arbitrary units).
To determine an unknown concentration of the analyte (X), 10.0 mL of 0.0146 M standard (S) was added to 10.0 mL of the unknown solution then the mixture was diluted to 25.0 mL in a volumetric flask. Analysis of this mixture resulted in peak areas of Ax=553 and As=582.
Using the known response factor (F = 0.970), the unknown concentration can now be calculated:
As X was diluted from 10.0 mL to 25.0 mL when the mixture with S was prepared, the original concentration of X in the unknown is: