Absorbance spectrophotometer:

A schematic diagram of a simple instrument that is used to measure the absorption of visible light is shown in Figure below.

When studying a compound in solution by spectrophotometry, you put it in a sample holder called a cuvette and place it in the spectrophotometer.  Light of a particular wavelength passes through the solution inside the cuvette and the amount of light transmitted (passed through the solution-Transmittance) or absorbed (Absorbance) by the solution is measured by a light meter. While a spectrophotometer can display measurements as either transmittance or absorbance, in biological applications we are usually interested in the absorbance of a given sample.

Because other compounds in a solution (or the solvent itself) may absorb the same wavelengths as the compound being analyzed, we compare the absorbance of our test solution to a reference blank.  Ideally, the reference blank should contain everything found in the sample solution except the substance you are trying to analyze or measure. For instance, in today's lab exercise you will be measuring the absorbance of a dye, bromphenol blue that was dissolved in water. The reference blank in this case would be water alone.

The amount of light transmitted through a solution is referred to as transmittance (T). The transmittance is defined as the ratio of the light energy transmitted through the sample (I) to the energy transmitted through the reference blank (I0). Since the compound being tested is not present in the reference blank, the transmittance of the reference blank is defined as 100%T.