Spectrophotometer absorbance refers to the amount of light absorbed by a solution, as measured by a laboratory instrument called an absorbance spectrophotometer. In chemistry and biology, spectrophotometers are used for a variety of purposes. They can help identify compounds, determine concentrations of solutions, or estimate the number of cells suspended in a liquid. Spectrophotometers work by directing a filtered set of certain wavelengths of light through a sample solution and onto a light meter. The amount of light transmitted or absorbed by the sample, as well as the wavelengths absorbed, reveal some of the sample’s properties.
The light that human beings perceive visually is a form of energy, electromagnetic radiation, and includes a range of wavelengths over a small portion of the electromagnetic spectrum. Gamma rays, X-rays, and other short wavelengths under 400 nanometers (nm) are not visible to the human eye, and neither are wavelengths longer than 700 nm, such as infrared light or radio waves. The colors that humans perceive range from shorter blue and violet waves around 400 nm through the rainbow to red, which is closer to 700 nm. Spectrophotometers measure in the visible range with some overlap into the ultraviolet and infrared sections of the spectrum.
When we see a color, for example a green leaf, we are seeing the light wavelengths that are being transmitted by that item. In the case of the green leaf, a compound in the plant’s cells called chlorophyll is absorbing blue and red wavelengths from the white light of the sun, but is not strongly absorbing the green. Instead, the green and near-green wavelengths are being transmitted, and the plant appears green.
In any given liquid solution, some wavelengths of light will be absorbed in greater amounts than others. Spectrophotometers direct a beam of white light through the sample solution being studied. The spectrophotometer absorbance is the amount of light that is absorbed by the compound under study. This light is absorbed in varying amounts across a range of wavelengths known as the absorption spectrum.
The absorption spectrum can help to identify the sample compound. For example, some plant pigments absorb different wavelengths than chlorophyll and can be differentiated from each other by their absorption plots — graphs where spectrophotometer absorbance is displayed as a function of wavelength. The wavelengths that are absorbed in the highest amount will appear as spikes on the graph, giving each compound's graph a characteristic shape.
The concentration of a solution can also be deduced from its spectrophotometer absorbance. This is done through the Lambert-Beer law, also known as Beer’s law, which is an equation relating level of spectrophotometer absorbance to concentration through two other factors: the extinction coefficient and the path length, or width of the sample tube. The extinction coefficient is a chemical factor that is different for each compound, but it can be determined by testing a sample of known concentration in the spectrophotometer. Beer’s law can then be used to solve for unknown concentrations of the same compound.