Standard addition quantitation

General information

Standard addition quantitation is a simple way to obtain measurements of amount in your sample (concentration is not calculated). It requires only a first sample run and a second sample run which has been spiked with a known amount of the component of interest. The technique is straight-forward and relatively fast when you are running only a few samples. Standard addition can be useful when you want to use the internal standard technique but do not have a suitable internal standard.

Disadvantages

The disadvantages of Standard addition quantitation are

  • its limited precision compared to the external and internal standard techniques

  • its lack of ability to measure more than one component.

How to perform Standard addition quantitation

The table below describes briefly how Standard addition quantitation is performed.

Step

Action

1

Perform a sample run.

2

Perform a second run with a sample that has been spiked with a known quantity of the component of interest prior to the sample preparation.

See illustration below:

3

Perform peak integration on both curves in the Evaluation module to produce a peak table for both the spiked and the unspiked sample.

Result: The difference in peak area between the spiked and the unspiked sample represents the peak area from the added amount.

4

With the assumption of a linear proportionality between the peak area and amount, and with the added amount known, the software calculates the amount of the component of interest in the sample:

Reliability

Standard addition is the least precise of the quantitation techniques since it is restricted to a single concentration level and the amount in the sample is calculated by extrapolation. Below are factors that determine if standard addition can be used with reliable results:

  • The component of interest must be completely resolved from all other components in the chromatogram. Overlapping peaks will produce unreliable results.

  • The peak integration parameters (baseline settings) must be correctly selected. The default settings will be satisfactory in many cases, but the integration results have to be checked for all chromatograms.

  • The standard addition technique assumes a linear through-the-origin relationship between the amount of component and peak size. This is a good approximation for small quantities under normal conditions.

  • Standard addition has no way of compensating for changes that are made between the runs. However, if losses during sample preparation are constant between the two runs, they may be accounted for by spiking the sample prior to the sample preparation.

  • A spike amount which is of the same order of magnitude as the sample must be used to maximize precision.

  • All the runs must be performed consecutively to reduce systematic errors and thereby maximize precision.

2005-06-15