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.
Spectrophotometric analysis involves the following steps and data analysis. Try these with the simulation.
- Set the wavelength to 600 nm. This determines the color of the photons emitted by the light source.
- Set the pathlength to 1.00 cm. The determines how much solution each photon must pass through.
- Place a blank solution in the cell. The blank is a solution that does not contain the molecule that absorbs the light. Produce a blank solution by setting the concentration to zero.
- Run the simulation. Observe how all of the photons emitted by the light source reach the detector. Measure the intensity, I0, of light reaching the detector.
- Next place a sample solution in the cell. Set the concentration to 4.00 μM. (μM represents micromolar or 10-6 mole/L) 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 emitted by the light source 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 were 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
The absorbance is a base 10 logarithmic scale. If A = 0, then 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.
- The absorbance is related to the concentration of the molecules absorbing the light by Beer's Law: A = ε b C
C is the molar concentration of the absorbing molecules. In this case, C = 4.00 x 10-6 mole/L.
b is the cell pathlength, which is the distance a photon must travel to pass through the solution. In this case, b = 1.00 cm.
ε is the molar absorptivity, which describes the inherent tendency of a molecule to absorb a photon. The units for ε are L mole-1 cm-1. The value of ε depends upon the identity of the molecule and on the wavelength of light.
After trying the above exercise, explore the following questions using the simulation.
- What is the effect of the cell pathlength (b) on the absorbance?
- What is the effect of the concentration (C) on the absorbance?
- What is the effect of changing the wavelength of the light?
Spectrophotometry.html version 3.0
© 2000-2020 David N. Blauch