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.