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What Goes On Inside a Spectrophotometer?

Matt Bostick
Product Development

May 2017

A spectrophotometer is an analytical instrument used to measure the amount of light absorbed (or transmitted) as it passes through a sample, such as a solution containing food dye. Spectrophotometers work on a few basic principles:

  • The intensity of color is a measure of the amount of dissolved material in solution. For instance, imagine 2 solutions containing red food dye, with the first solution observed as having a lighter color than the second. When observing the 2 solutions, many science students would infer that as the color of the solution deepens, the concentration of food dye increases. Applying this common-sense principle, a spectrophotometer can be used to determine concentrations of compounds in solution.
  • Each substance absorbs or transmits only certain wavelengths of radiant energy (the energy of electromagnetic waves). Light is a form of electromagnetic radiation. When light interacts with a substance, certain wavelengths may be absorbed by the substance, while other wavelengths may be transmitted or reflected. For instance, consider a green plant. The chlorophyll molecule in green plants absorbs red and violet wavelengths within the visible light spectrum. However, the same chlorophyll molecule transmits green wavelengths, a factor that allows these transmitted wavelengths to appear as green when we perceive a green plant. Applying this principle, a spectrophotometer may be used to distinguish compounds by analyzing wavelengths absorbed and/or transmitted by a given sample.

Spectrophotometer basics

Spectrophotometers used in the classroom have a variable wavelength selector that allows the instrument to transmit light within a narrow range of wavelengths (i.e., 340–950 nm). As mentioned before, different compounds absorb light at different wavelengths. As specific wavelengths of light pass through a sample of dissolved material (referred to as an analyte) within a spectrophotometer, the instrument indirectly measures the amount of light absorbed by that sample by comparing the initial intensity of light reaching the sample (I0) with the light detected by a photocell as it exits the sample (I). The ratio of the 2 readings is referred to as the transmittance of light through the sample (Fig. 1). Often, this value is multiplied by 100 and interpreted as the percent transmittance of light, using this formula:

%T = I/I0 x 100

Figure 1.

The complement of percent transmittance is absorbance (A), the amount of light absorbed by the analyte. It is a logarithmic value with no units. It can be read directly from the instrument in absorbance mode, or it can be calculated from the decimal equivalent of percent transmittance read from the instrument (the values of absorbance and transmittance are inversely proportional). This calculation is:

A = –log T

How a spectrophotometer works

To better understand what happens within a spectrophotometer, it helps to know the role of each component in the instrument.

  • Light source—The light source provides wavelengths of light at great intensity that span from near infrared to within the ultraviolet range, including the visible light spectrum.
  • Diffraction grating or prism—The diffraction grating separates the light source into specific portions of spectrum. When you adjust the variable wavelength selector, you change the position of the diffraction grating so different wavelengths of light are directed towards the sample compartment containing the analyte.
  • Variable wavelength selector—Found on the exterior of the instrument, a variable wavelength selector allows the instrument to essentially filter the light, transmitting light only at a specific wavelength or range of wavelengths of interest.
  • Sample compartment—The sample compartment houses the cuvette (a transparent tube designed to hold samples for spectroscopic experiments), which contains the analyte. Selected wavelengths of light pass through the analyte for detection by a photodetector.
  • Photodetector—Light passed through the analyte strikes a photodetector composed of a semiconducting material. Electrons in this material are excited proportionally to the intensity of light striking the photodetector, creating a current of variable strength that is received by a signal processor.
  • Display—Many spectrophotometers display both transmittance and absorbance of a sample.

Spectrophotometer applications in the life science classroom

While there are several spectrophotometer applications within the chemistry classroom, the instrument can be equally useful to educators in the life sciences. For instance, spectrophotometers may be used to estimate the number of cells suspended in a medium. Consider yeast cells in solution. As all particles in suspension scatter light, yeast cells in solution will also scatter light as they grow, causing the solution to appear cloudy. As the turbidity (cloudiness) of the solution increases, less light will reach the photodetector. Light scattering is measured with the spectrophotometer set to report absorbance. However, it must be noted that different principles are used to measure light scattering and absorbance. Because of this, the amount of light scattered by a solution is referred to as its optical density instead of its absorbance.

Related resources

Consider the following Carolina products and investigations that utilize spectrophotometry.

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