A spectrophotometer measures the amount of light that a sample absorbs. The instrument operates by passing a beam of light through a sample and measuring the intensity of light reaching a detector.
The beam of light consists of a stream of photons, represented in the simulation below by the little circles moving from left to right across the screen.
The solution contains molecules that can absorb light. When a photon encounters one of these molecules, there is a chance the molecule will absorb the photon. Absorption of a photon reduces the number of photons in the beam of light, thereby reducing the number of photons reaching the detector.
Visualize this process by observing the simulation below. Click on the Start button to start the simulation and the Stop button to stop the simulation.
Watch the motion of the photons and observe how some of the photons disappear (are absorbed) as they pass through the cell containing the sample solution. The intensity of the light reaching the detector is less than the intensity emitted by the light source.
Once photons begin reaching the detector, start the Data Acquisition. The intensity of light (photons per second) reaching the detector will be displayed. Note that the simulation employs more photons than are shown on the screen.
The monochromator for the spectrometer is set to 600 nm. This determines the color of the light used in the experiment.
The cell containing the solution is 1.00 cm wide. The cell is placed in the light path (as shown in the simulation) so light can pass through the sample solution. The length of the solution each photon passes through is called the cell pathlength, which in this case is 1.00 cm.
The sample solution is colored, indicating it contains one or more compounds that absorb light in the visible region. The blank solution is colorless and does not absorb photons in the visible region.Spectrophotometric analysis involves the following steps and data analysis. Try these with the simulation.
- Place a blank solution in the cell. The blank is a solution that does not contain the molecule that absorbs the light.
- Run the simulation. Measure the intensity, I0, of light reaching the detector when there are no analyte molecules to absorb light.
- Next place the sample solution in the cell. Run the simulation again and measure the intensity, I, of light reaching the detector.
- Use the intensities you have measured to determine the solution transmittance, T. T represents the fraction of the light passing through the cell that ultimately reaches the detector. That is, T is the fraction of the photons that are not absorbed by the sample solution. The fraction 1 - T is the fraction of photons that are absorbed. (Do not confuse the transmittance with the temperature. The same symbol is used for both properties.)
- For analytical purposes, the absorbance, A, is more useful than the transmittance.
A = - log10 T
Absorbance uses a base 10 logarithmic scale. If A = 0, no photons are absorbed. Each unit in absorbance corresponds with an order of magnitude in the fraction of light transmitted. For A = 1, 10% of the light is transmitted (T = 0.10) and 90% is absorbed by the sample. For A = 2, 1% of the light is transmitted and 99% is absorbed. For A = 3, 0.1% of the light is transmitted and 99.9% is absorbed.
Spectrophotometry.html version 3.0
© 2000, 2014, 2023 David N. Blauch