In order to be able to determine the concentration of the analytes in the sample, a calibration curve is needed.
Figure 1. Calibration curve showing the sample concentrations in x axis and the area under the curve in y axis.
The calibration curve is prepared by measuring several concentrations creating a linear line. These concentrations have include concentration that is both lower and higher than the expected concentration of the analyte in the real sample. When you do not know the analyte concentration in the real sample, typically you will prepare a calibration curve using minimum 8 different sample concentrations. When you know the expected analyte concentration in the real sample, you can reduce the number of sample concentrations needed for the calibration curve.