The ISTD procedure eliminates the disadvantages of the ESTD method by adding a known amount of a compound which serves as a normalizing factor. This compound, the internal standard, is added to both calibration and unknown samples.
The compound used as an internal standard should be similar to the calibrated compound, both chemically and in retention/migration time, but it must be chromatographically distinguishable.
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Advantages |
Disadvantages |
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Sample-size variation is not critical. Instrument drift is compensated by the internal standard. The effects of sample preparations are minimized if the chemical behavior of the ISTD and unknown are similar. | The internal standard must be added to every sample. |
If the ISTD procedure is used for calibrations with a non-linear characteristic, care must be taken that errors which result from the calculation principle do not cause systematic errors. In multi-level calibrations, the amount of the ISTD compound should be kept constant, i.e. the same for all levels.
In the internal standard analysis, the amount of the compound of interest is related to the amount of the internal standard component by the ratio of the responses of the two peaks.
OpenLab CDS allows up to 5 ISTD compounds.
For the ISTD calculation relative responses and relative amounts are used instead of the "raw" responses and amounts. They are calculated by dividing the response and amount of the peak of interest by the response and amount of the corresponding ISTD compound:
Relative Response = Response / Response ISTD
Relative Amount = Amount / AmountISTD
The response can be Area, Area%, Height, or Height% (see Response type and response factor).
In an ISTD calibration, the calculation of the corrected amount ratio of a particular compound in an unknown sample occurs in several stages. These stages are described in the following sections.