Technical data

Understanding Your Agilent ChemStation 123

Quantification

ISTD Calculation

The ISTD procedure eliminates the disadvantages of the ESTD method by

adding a known amount of a component which serves as a normalizing factor.

This component, the internal standard, is added to both calibration and

unknown samples.

The software takes the appropriate response factors obtained from a previous

calibration stored in the method. Using the internal standard concentration

and peak areas or heights from the run, the software calculates component

concentrations.

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.

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 if the

calibration curve of the compound is non-linear.

In the internal standard analysis, the amount of the component of interest is

related to the amount of the internal standard component by the ratio of the

responses of the two peaks.

In a two-run ISTD calibration, the calculation of the corrected amount ratio of

a particular compound in an unknown sample occurs in the following stages:

Table 1 2 ISTD Procedure

Advantages Disadvantages

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.