After the peaks have been integrated and identified, the next step in the quantitative analysis is the calibration. The amount and response is rarely in direct proportion to the actual mass of the sample to be analyzed. This makes the calibration with reference materials necessary. Quantitation uses peak area or height to determine the amount of a compound in a sample.
A quantitative analysis involves many steps which are briefly summarized as follows:
Know the compound you are analyzing.
Establish a method for analyzing samples containing a known amount of this compound, which is called the calibration sample or standard.
Analyze the calibration sample to obtain the response due to that amount.
You may alternatively analyze a number of these standards with different amounts of the compounds of interest if your detector has a non-linear response. This process is referred to as multi-level calibration.
With the following calibration methods you can perform quantitation:
Compound specific calibration (ESTD, ISTD)
Indirect quantitation using calibration or response factor from another compound or group
Fixed response factor (Manual Factor)
The ESTD calibration curves and calculations are based on measured responses (area or height) of given amounts. The ISTD calibration curves and calculations are based on relative responses and relative amounts (see Relative responses with ISTD).