Determination of Analyte Concentration:
One of the most common applications of spectrophotometry is to determine the concentration of an analyte in a solution. There is a relationship between concentration and absorbance. This relationship is expressed by the Lambert-Beer law, which is more commonly known as Beer's law. This law states that the absorbance of a light absorbing material is proportional to its concentration in solution.
A = Ebc
E = the extinction coefficient of the substance, has units of M-i * cm-i (unique for each substance)
b = the sample path length measured in centemeters (i.e. the width of the cuvette-almost always i cm)
c = the molar concentration of the solution (you must express concentration in terms of molarity)
It is because of this relationship that biologists measure absorption rather than transmission. The Lambert-Beer law can be used to calculate the concentration of a solution if its extinction coefficient is known. To determine the extinction coefficient, you measure the absorbance of a known concentration of solution and then rearrange the equation to solve for E.=
Table. Visible Spectrum and Complementary Colors