The Pharmaceutics and Compounding Laboratory
Spectrophotometry as an
Analytical Tool

Quantitative Measurements

A primary use of absorption spectroscopy lies in its applicability to quantitative measurements. This is a function of how much light is absorbed, and how that relates to the amount of the absorber present in the sample. The following derivation presents the basics of this relationship.

When we shine a light beam of a certain wavelength (l) and initial intensity (I 0 ) through an absorbing sample contained in a spectrophotometer cell, the intensity of the light beam transmitted through the sample (I t ) is dependent on three factors (see Figure 2). The first factor is whether the sample will absorb light at that wavelength. The second is the amount of sample which the light must pass through or, the cell width (b). The third factor is the concentration of the absorbing species in the sample solution (C). You can see through a test tube containing micromolar concentrations of the same species. The fraction of light transmitted, or transmittance (T), is defined as the following:



       Percentage of light transmitted (% T) = T × 100 (2)   (2)


       I 0 = initial intensity of the light beam, and I t = transmitted intensity of the light beam

The transmittance of the sample varies logarithmically with the cell width and the concentration of the absorbing species in the following way:

       log (1/T) = -log T = (proportionally constant) b C (3)  (3)

The proportionality constant depends on the chemical nature of the individual absorber, the wavelength at which the measurements are being made, and the units of b and C. In visible absorption spectroscopy, b is normally measured in centimeters. If C is also measured in mol/L (molar concentration, M), the proportionality constant is defined as the molar absorptivity (ε), which has units of l/M-cm. If C is measured in any other units (e.g., g/l), the constant is simply called the absorptivity (a), whose units will depend on C. Under normal operating conditions, ε or a is determined experimentally by measuring a standard of known concentration.

Figure 2: Factors that Determine Transmitted Light Intensity