New applications in quality control and the development of novel fluorescence probes, particularly those based on expressed proteins, have greatly increased the sensitivity requirements of fluorescence spectroscopy.



The fluorescence spectra for the blue plastic star is shown here. Source: Thermo Fisher Scientific

Photoluminescence is the emission of light from a molecule or atom that has absorbed electromagnetic energy. Photoluminescence includes both phosphorescence and fluorescence. A good example of photoluminescence are glow in the dark materials such as stars placed on the ceiling of children’s rooms and glow sticks. All glow in the dark products contain phosphors, a substance which radiates visible light after being energized. The most common places we see phosphors are in a TV screen or computer monitor and fluorescent lights. For TV screens, an electron beam strikes the phosphor to energize it and ultraviolet light energizes the phosphor in a fluorescent light. These materials can be analyzed using a fluorescence spectrometer.

Fluorescence is becoming increasingly more important in the characterization of materials that both phosphoresce and fluoresce. Carbon nanostructures, biological assays on surfaces and common paper and textile samples can be evaluated using luminescence measurements from solid material samples. Typically, most of the samples analyzed with a fluorescence spectrometer are liquids placed in a traditional 10-millimeter cuvette. However, a flexible research grade instrument is capable of doing much more. There has been a steady increase in solid materials across all disciplines within both industry and academia. This article will describe the analysis of glow-in-the-dark plastic star samples to illustrate the capabilities of the latest fluorescence spectrometer technology.

This illustrates the phosphorescence spectra from the green and blue samples. Source: Thermo Fisher Scientific

Fluorescence Excitation and Emission Spectra

In the first experiment, the fluorescence emission and excitation spectra were obtained using the wavelength scanning application of the software on a fluorescence spectrometer. By using pre-aligned, modular monochromators and high quality optical components, the device provides high resolution measurements and superior sensitivity. The application has the capability of acquiring the fluorescence, phosphorescence or chemiluminescence emission and excitation spectrum. An excitation wavelength of 366 nanometers and an emission wavelength of 460 nanomaters provided the best results for the blue colored sample. The emission and excitation slits were both set to 5 nanometers and a 20-millisecond exposure was acquired at 1 nanomater data point intervals. The fluorescence emission and excitation spectra from the green star proved to be quite different, consisting of a main excitation peak at 462 nanomaters and a strong emission peak at 507 nanomaters.

This software application can also be used to measure the phosphorescence emission spectrum of the samples. In phosphorent materials, excitation causes the molecule to enter an excited state, which subsequently undergoes intersystem crossing to a symmetry-disallowed triplet state. Despite this transmission being forbidden, the molecule can actually return to the ground state. This is often as a result of emitting a photon of light. For materials that glow in the dark, the triplet state can exist for a period of between minutes and hours. In this experiment, the sample is exposed to the excitation light with the shutter being closed and the phosphorescence intensity measured.

In this example, the excitation grating was set to the zero order position, which allowed broadband light from the xenon source to excite the sample for three seconds. The fluorescence spectra described earlier were quite different for the two samples, but the phosphorescence spectra are quite similar. This suggests that the same compound was actually used in both plastics in order to generate the phosphorescence affect.

Luminescence is a third data mode available with the spectrometer. This enables a simple method to measure the spectrum of light emitted by the sample after being left under bright room lights for several minutes.



The excitation and emission slits were set to 10 nanometer and a 342-nanometer excitation light was used. Source: Source: Thermo Fisher Scientific

Measuring Phosphorescence Decay

The difference in the intensity of the luminescence spectra indicates that the phosphorescence signal decreases at a rapid rate after being exposed to light. In order to further examine this effect, a software application was used. A function allows the intensity of luminescence to be measured as a function of time, providing key information about the kinetics of the sample. A time scan was used to measure the intensity of the phosphorescence at 527 nanomaters. Data was collected over 200 milliseconds for a 30-second total measurement times. The excitation and emission slits were set to 10 nanometer and 342 nanometer excitation light was used.

The phosphorescence decay is very similar for the two samples even though the fluorescence spectra are different. Once again, this indicates that the same compound was added to the plastic. The final experiment in the analysis was to measure the decay curves for different light exposure times. Figure 5 shows the decay curves when the initial light exposure is reduced from 10 seconds to 0.2 seconds.

There is a similar decay rate when the sample is exposed to the source excitation in the ten seconds and five seconds prior to the closing of the shutting and the measuring of the intensity decay. Even with an exposure time of 0.2 seconds, a significant number of molecules are excited before the phosphorescence decay is measured.

This study has shown that several features of the latest fluorescence spectrometer technology can be used to accurately characterize fluorescence and phosphorescence from solid samples. The luminescence capability of the instrument used demonstrated that the spectrum for room light excitation was similar to the spectrum acquired with one of the software’s applications. This series of experiments provides an excellent example of the hardware and software tools are that are causing breakthroughs in the fluorescence field.



Phosphorescence decay with light exposure time set to 10 seconds, 5 seconds, 1 second and 0.2 second. Source: Thermo Fisher Scientific

Tech Tips

  • Fluorescence is becoming increasingly more important in the characterization of materials that both phosphoresce and fluoresce.
  • Part-to-part variations can be easily seen by plotting the resonant frequencies of each part. The closer the frequencies match from part to part, the more consistent the process is running.
  • There has been a steady increase in solid materials across all disciplines within both industry and academia.