Optics 101

An Introduction to Fluorescence Spectroscopy

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Spectroscopy is a broad technique, with many different types available to researchers. Each type of spectroscopy measures different bandwidths of radiation, and can be used to determine different chemical and material properties. In this article, we look at fluorescence spectroscopy.

Fluorescence spectroscopy is a method of measuring the fluorescence of a chemical sample to determine its concentration. Fluorescence spectroscopy is often used to measure compounds which are in solution and is often used for simple analyses.

It is often used for determining concentrations because it is a fast, simple and inexpensive method.

What is Fluorescence

At room temperature, most molecules occupy the lowest energy state. This is also known as the ground state. Within this ground state are vibrational levels, and before becoming excited, many molecules occupy the lowest vibrational level. Molecules that can exhibit fluorescence are called fluorophores.

When a molecule absorbs a certain wavelength of light, the adsorbed photon causes the molecule to adopt a higher vibrational energy state (usually the first excited singlet state). The molecules then collide with other molecules in the solution, causing the molecules to lose their vibrational energy and return to the lowest vibrational level of the excited state. At this point, the molecule can then return to any of the ground state vibrational levels.

When the molecule returns to the ground state, it emits a photon of light at a different wavelength to the wavelength that excited it. This is when the molecule exhibits a fluorescence. This fluorescence is measurable by fluorometers.

The fluorescence of an atom is very different. Atoms do not possess vibrational levels, so the expelled wavelength is the same as the radiation which excites the sample. This is called resonance fluorescence and is primarily a characteristic shown by atoms (although some molecules can exhibit this too).

How Does Fluorescence Spectroscopy Work?

A fluorescence spectrometer is used to excite fluorophore molecules and measure their emitted fluorescence. To do this, the spectrometer emits ultra-violet (UV) or visible light (180-800 nm wavelength) using an incident photon source – this could either be a laser, a xenon lamp, LEDs or mercury-vapour lamps. This light then passes through a monochromator which selects a specific wavelength. Monochromators in fluorescence spectrometers often use a diffraction grating and the light that exits comes out at a specific angle depending on its wavelength.

The monochromatic wavelength is then focused towards the sample and a wavelength is then emitted from the sample to the detector. The detector is usually set at a 90° angle to the light source to avoid any interference from the transmitted excitation light.

The emitted photon then hits a photo detector, and these detectors can either be single or multi channelled. Once detected, computer software generates a spectrum of both the emission and excitation of the molecule. The excitations spectrum shows which wavelengths are absorbed by the sample, and the emission spectrum shows which wavelengths are emitted by the sample.

Sensitivity can sometimes be an issue with fluorescence spectroscopy, especially if all the molecules in the sample do not fluoresce. The quantum efficiency describes the proportion of molecules which fluoresce when exposed to photons of a certain energy. If all the molecules fluoresce, then the quantum efficiency is categorized as 1 (the maximum), and if no molecules fluoresce the quantum efficiency is 0. Often, the quantum efficiency is closer to 0 than 1. Additionally, the presence of other molecules that absorb the intended wavelength and changes in the pH and temperature (quenching) can all affect the results.

An diagram illustrating a simple fluorescence spectrum. The absorption peak shows the wavelengths of light absorbed, the emission peak shows the wavelengths of radiation emitted. The shift in wavelength is due to energy lost via molecular vibrations. Source: Wikimedia.

Applications of Fluorescence Spectroscopy

Because fluorescence spectroscopy is concerned with determining the concentration of any solubilized molecules, its applications are widespread – and can feature in any application where the molecules can be dispersed in solution, can absorb either UV or visible light and can fluoresce.

It is not suitable for molecules that undergo a photochemical reaction at or above the wavelength of interest, opaque/non-clear samples and colloidal samples. For samples that do not naturally fluoresce (some biological materials), fluorescent dyes can be used to stain the molecules of interest.

As such, it is a widely used technique in the chemical, pharmaceutical and biomedical fields. Some specific applications include cancer diagnosis, studying marine petroleum pollutants, quantifying the levels of dissolved carbon in natural waterways and for determining the level of glucose, fungi, viruses and bacteria. But these are just a few examples.

Sources & Further Reading

https://www.chem.uci.edu/~dmitryf/manuals/Fundamentals/Fluorescence%20Spectroscopy.pdf

https://warwick.ac.uk/services/ris/impactinnovation/impact/analyticalguide/fluorescence/

http://www.oswego.edu/~kadima/CHE425/CHE425L/FLUORESCENCE_SPECTROSCOPY_08.pdf

http://www.chromedia.org/chromedia?waxtrapp=mkqjtbEsHiemBpdmBlIEcCArB&subNav=cczbdbEsHiemBpdmBlIEcCArBP

​“Emerging applications of fluorescence spectroscopy in medical microbiology field”- Shahzad A. et al, J. Transl. Med., 2009, DOI: 10.1186/1479-5876-7-99

“Applications of Fluorescence Spectroscopy”- Naresh K., Journal of Chemical and Pharmaceutical Sciences, 2014

“Applications of Fluorescence Spectroscopy for Predicting Percent Wastewater in an Urban Stream”- Goldman J. H., et al, Environ. Sci. Technol., 2012, DOI: 10.1021/es2041114

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