Spectrophotometers measure the amount of light absorbed by a solution to identify compounds, determine concentrations, and estimate cell numbers. The absorption spectrum can differentiate compounds based on their absorbance graphs, and the Lambert-Beer law can infer concentration.
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 properties of the sample.
The light that humans perceive visually is a form of energy, electromagnetic radiation, and comprises a range of wavelengths over a small portion of the electromagnetic spectrum. Gamma rays, X-rays, and other short wavelengths below 400 nanometers (nm) are invisible to the human eye, and neither are wavelengths above 700 nm, such as infrared light or radio waves. The colors that humans perceive range from the 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 in the ultraviolet and infrared sections of the spectrum.
When we see a color, such as a green leaf, we are seeing the wavelengths of light that are being transmitted by that item. In the case of the green leaf, a compound in the plant’s cells called chlorophyll absorbs the blue and red wavelengths from the sun’s white light, but doesn’t absorb green strongly. Instead, the green and near-green wavelengths are transmitted and the plant appears green.
In a given liquid solution, some wavelengths of light will be absorbed in greater quantities than others. Spectrophotometers direct a beam of white light through the sample solution under test. The absorbance of the spectrophotometer is the amount of light that is absorbed by the compound under test. This light is absorbed in varying amounts across a range of wavelengths known as the absorption spectrum.
The absorption spectrum can help identify the compound in the sample. For example, some plant pigments absorb different wavelengths than chlorophyll and can be differentiated from each other based on their absorption graphs, graphs in which spectrophotometer absorbance is displayed as a function of wavelength . The wavelengths that are absorbed in the greatest amount will appear as peaks on the graph, giving each compound’s graph a characteristic shape.
The concentration of a solution can also be inferred from its spectrophotometric absorbance. This is done through the Lambert-Beer law, also known as Beer’s law, which is an equation that relates the absorbance level of the spectrophotometer to the concentration through two other factors: the extinction coefficient and the path length , or sample tube width. The extinction coefficient is a different chemical factor for each compound, but 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.
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