Internal Standard ("ISTD")

An internal standard is a substance of which a known amount is added both to standard and unknown samples. The internal standard should have a similar retention time behavior as the analyzed substances, but it should be easily separable from them. Internal standards are used especially in gas chromatography but also in HPLC and IC to eliminate possible sample preparation errors.

As a rule, the amount of injected internal standard is constant; that is, all samples receive precisely the required amount of internal standard so that identical amounts reach the column when injecting the same volumes. In the case of a Dilution Series, the internal standard must be pipetted after the dilution. Therefore, the dilution cannot be simulated by variation of the injection volume but must be actually performed.

 Note:

With an appropriate Autosampler, such as the Dionex ASI-100, pipetting can be performed automatically. The quantitative accuracy thus reached is one level above the External standard method. The methods with internal standard also compensate minor injection inaccuracies, as the internal standard is equally affected by a possible loss or excess.

Substances serving as an internal standard are labeled as such in the Standard column of the peak table. Depending on the type of internal calibration, they are labeled ISTD Int/Ext or ISTD Internal.

Internal/External Principle

All substances contained in a sample are calibrated as usual with known standard substances (including the internal standard). As the same amount of ISTD is contained in all samples of a sequence, a uniform value for the ISTD should result. If this is not the case, this is an indication for an error during sample processing (if the same amount of the internal standard is actually contained in all samples). The results of the remaining substances of this sample can be relativized afterward.

Internal Principle

Instead of absolute areas and amounts, calculation is based on area and amount ratios. Calculation of all substance amounts is relative to the internal standard. By forming the ratio, inaccuracies in sample processing (from adding the ISTD) can be eliminated (const. Internal standard).

Due to the high experimental expenses, this type of calibration is rarely used (the internal standard must be added with a pipette to each sample, in the case of a dilution series this must be after the dilution).

Exceptions (= Variable Internal Standard)

The method itself or the properties of the internal standard make it impossible to add the same amount of ISTD to all samples. To solve this problem, Chromeleon supports the Use sample amount as reference (Variable) option (see F8 box of the Standard column in the peak table).

In this case (irrelevant whether Internal/External or Internal), the amount of the internal standard is entered in the ISTD Amount column of the sample list, not in the Amount column of the peak table. In this way, the amount of internal standard can be entered separately for each sample.

Also, refer to Standard