The basic theory for quantitation of sample components involves the measurement of peak heights or peak areas. For peaks that are well resolved, both peak height and area are proportional to the concentration.
Three different calibration methods, can be used in quantitative analysis:
i) The external standard
ii) The internal standard
iii) The standard addition method
For gas chromatography, the most commonly used quantitative methods are the internal standard and standard addition method.
An internal standard is a standard whose identity is different from the analyte’s, that is added to all samples and standards (calibrants) containing the analyte.
Since the analyte and internal standard in any sample or standard receive the same treatment, the ratio of their signals will be unaffected by any lack of reproducibility in the procedure. With this method, an equal amount of an internal standard (IS) is added to both the sample and calibrator solutions. The IS selected should be chemically similar to the analyte and have a similar retention time and similar derivatization. It is also important to ensure that the IS is stable and does not interfere with any of the sample components.
The IS should be added before any preparation of the sample so that extraction efficiency can be evaluated.
Quantitation is achieved by using ratios of peak areas of the component to the internal standard:
Standard addition: If only a few samples are to be chromatographed, it is possible to employ the method of standard addition(s). The chromatogram of the unknown is recorded. Then a known amount of the analyte(s) is added, and the chromatogram is repeated using the same reagents, instrument parameters, and procedures. From the increase in the peak area (or peak height), the original concentration can be computed by interpolation. The detector response must be a linear function of analyte concentration and yield no signal (other than background) at zero concentration of the analyte. Sufficient time must elapse between addition of the standard and actual analysis to allow equilibrium of added standard with any matrix interferant.
If an instrumental reading (area or height), Rx, is obtained from a sample of unknown concen-tration x and a reading R1 is obtained from the sample to which a known concentration C of analyte has been added, then x can be calculated from the relation:
x /( x + C) = Rx / R1
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