The working formula:
A = ε b c
is derived from Lambert-Beer’s law, where A is the absorbance, E is molar extinction coefficient, b is the path length in cm and c is molar concentration (mol l1). The absorbance values of the standard solutions of known concentrations are determined from the spectrophotometer and a calibration graph is constructed. Absorbance value of the test solution is determined from the instrument and the concentration of the test solution is determined from the calibration groups.