Fluorescence Spectrophotometers: An Introduction to Fluorometry
Fluorescence spectrophotometers, or fluorometers, are powerful analytical tools. They have similarities to UV-Vis spectrophotometers (which we covered briefly in a recent article) since both involve light being absorbed by specific molecules in a test sample. However in fluorescence spectrophotometers, the intensity of light that is emitted after absorption due to the phenomenon known as fluorescence is measured as a function of wavelengths to create a fluorescence spectrum.
Basic Operating Principles of Fluorescence Spectrophotometers
All molecules contain a number of discrete energy levels, specific to the particular molecule. In a molecule’s ‘ground state’, its electrons occupy the lowest energy levels available. However when they absorb the appropriate energy from light they can transition to higher levels to form an ‘excited state’. These are inherently unstable, so the electrons will spontaneously drop back into lower levels. The light emitted in this process is fluorescence.
The excited electrons can drop back into various lower levels or right back to the ground state, resulting in energy emissions that are lower or equal to the incident light energy. The fluorescence spectrum therefore always occurs at longer wavelengths than that of the incident light beam. Since the fluorescence emissions occur in all directions but have much lower intensity than the incident beam, the fluorescence detector is positioned at 90˚ to the path of the beam through the test sample. This prevents the detector being swamped by the incident beam.
Making the most of Fluorescence Spectrophotometry
Although the fluorescence intensity emissions are much weaker than the signals in a UV-Vis spectrophotometer, the fluorometer has lower detection levels. This is because the fluorescence signal is measured directly. This is separate from the incident beam and can easily be amplified with a low noise contribution. However detecting low concentrations in a UV-Vis spectrophotometer means measuring a very small difference between a large transmission signal and the original incident beam and this is limited by the noise present in both beams.
Fluorometers can provide a number of different measurement options:
- The high intensity light source, typically covering the 200 – 750 nm wavelength range, is equipped with a diffraction grating-based monochromator so the wavelength of the incident beam can be tuned to a particular value. This means that the concentrations of multiple components in the test sample can be made using different excitation energies.
- By measuring fluorescence intensity as a function of time, rates of reactions taking place in the sample cell can be calculated
- Recording a series of fluorescence spectra as the wavelength of the incident light is scanned, allows a 3 Dimensional scan, or Excitation Emission Matrix to be built up, where the intensity of the fluorescent light emitted is dependent on both the excitation and emission wavelengths. This generates a ‘fluorescence fingerprint’ of the sample which can be used to measure composition, concentration and environmental load, for checking purity, abnormalities in raw materials, mixture compositions, freshness due to organic degradation, or determining the place of origin and more.
Fluorescence Spectrophotometers from Lambda
Lambda is a leading supplier of analytical instruments for a wide range of applications, including some of the best-in-class fluorescence spectrophotometers on the market. If you would like more information, simply contact a member of the team today.