External standard quantitation

General information

External standard quantitation is based on the use of a standard prepared in a number of concentration levels. A run is performed for each concentration level and calibration curves are produced to show the relationship between amount and peak size for each component. The calibration curves are used to quantitate the components in the sample.

Note: The standard should contain known amounts of all the components that are to be quantitated in the sample.

How to improve quantitation

External standard quantitation can be based on the use of a single standard concentration level, but the calibration curve is then limited to a linear through-the-origin relationship. The use of a number of different concentration levels of the standard broadens the range of the calibration curve. It also allows the development of non-linear calibration curves and improves precision. Multiple runs at each level improve precision further.

The description in this section is based on the use of a standard

  • that contains two components,

  • which is run at three different concentration levels.

How to perform External standard quantitation

The table below describes briefly how External standard quantitation is performed (based on the use of a standard which contains two components and which is run at three different concentration levels).

Step

Action

1

Perform a run for each standard level.

2

Integrate the curves to produce a peak table for each run.

3

Use the peak tables from the standard runs to produce a calibration curve for each component. This curve shows the relationship between amount and peak size.

Below is a calibration curve for one of the components:

4

Perform a run with the sample and peak integrate the curve.

5

Identify the components of interest by the peak identification settings from the sample peak table. Use the peak size(s) to calculate the concentration and amount from the calibration curve(s).

Illustration - how to use the calibration curve

The illustration below describes how the calibration curve is used to determine the amount based on the sample peak area.

Reliability

External standard quantitation normally produces accurate results and is fairly simple. The following reliability factors are specific to the technique.

  • Precision is limited by changes that may take place between the runs, for example column degradation and mobile phase variations.

  • There is no compensation for losses of sample during the sample preparation process prior to analysis.

2005-06-15