Fluorescence spectroscopy

Isn’t it obvious that fluorescence spectroscopy is based on the fluorescence principle? But what is it and how is it useful for performing a spectroscopic analysis? This article in our spectroscopic series address all these questions and much more. Simply, fluorescence is defined as the ability of certain chemical substances to emit visible radiations by absorbing electromagnetic radiations from a different region. Let’s find out how is this chemical property exploited in fluorescence spectroscopy.

What is fluorescence spectroscopy

Fluorescence spectroscopy is based on electronic excitation followed by de-excitation, stimulated by electromagnetic radiations. The electrons present in the sample molecules absorb energy from the radiant source in the form of photons. They get excited to an energy level higher than their ground state. Electrons stay unstable in this excited state therefore they return back by emitting light. This light emission is called fluorescence. Each wavelength of light emitted is characteristic of a specific chemical compound. It can thus be used to identify the compound present and how much of it is present in the sample i.e., its concentration.

What is the basic principle of fluorescence spectroscopy

A fluorescence spectrometer also called a spectrofluorometer consists of the following main components:

• Irradiation source
• Monochromator
• Sample holder
• Detector

Let us discuss one by one how all these components perform their required tasks.

Step I: Irradiation

• The chemical compounds to be tested are held in the sample compartment and irradiated using a light source.
• Circular, square, or rectangularly shaped cuvettes of 10 mm path length are principally used for holding the sample. The sample is mostly filled in it in a solution form. 
• A tungsten-halogen lamp, a xenon-arc lamp, or a mercury lamp are most often used as light sources in fluorescence spectroscopy. These supply radiations in the near-infrared or ultraviolet-visible (UV-visible) region i.e., 180-800 nm.

Step II: Wavelength selection

• A specific wavelength is selected by the monochromator to pass through the sample at a time.
• The monochromator also performs the task of selecting a specific wavelength emitted by the sample. This dual-functioning monochromator is a special feature in fluorescence spectroscopic instrumentation that differentiates it from UV-Vis spectroscopy.
• Simple filters or a diffraction grating is often used as the monochromator.  

Step III: Electronic interaction with radiant energy

• Electrons absorb photons from radiant light and are raised to an excited energy state.
• The excited state undergoes a rapid thermal energy loss by electronic vibrations.
• A photon is consequently emitted from the excited state and electrons are de-excited to the lower energy level. This is called radiative relaxation.
• The photon emission is called fluorescence. It usually lies in the visible region of the electromagnetic spectrum, so it is also called fluorescent light. Different energy changes in a fluorescent molecule are represented in the diagram below.

• The fluorescent light is collected by the detector and the fluorescence spectrum is plotted.
• Photomultiplier tubes are most commonly used as detectors in fluorescence spectroscopy.

Step IV: Recording the fluorescence spectrum

• Two types of fluorescence spectra are often recorded in fluorescence spectroscopy namely the fluorescence emission spectrum and fluorescence absorbance or excitation spectrum.
• The fluorescence emission spectrum is plotted as a graph of fluorescence intensity versus emission wavelength. It is recorded by keeping the excitation wavelength fixed while the emission wavelength is scanned using a monochromator.
• The fluorescence excitation spectrum is plotted as a graph of fluorescence intensity against excitation wavelength. The excitation wavelength is the wavelength at which the sample molecules absorbed radiation so as to emit only a single wavelength. The fluorescence excitation spectrum is recorded by supplying different wavelengths as excitation sources while the emission wavelength is kept fixed.
• In both cases, the fluorescence spectrum reveals the identity of the chemical substance. The intensity of a peak is proportional to the concentration of each chemical constituent.
• The sample concentration (c) is related to fluorescence intensity (F) by the formula given below:

F=QI_a εcl

where Q is constant for a particular chemical substance, I_a= intensity of absorbed light, ε= molar extinction coefficient, l= pathlength

 I_a can be calculated using Beer Lambert’s law by finding the difference between incident (I₀) and emitted (I_t) light intensities.

What types of chemical compounds are fluorescent

Some chemical compounds are naturally fluorescent. For example, the plant pigment chlorophyll is intrinsically fluorescent. It absorbs photons from sunlight and gives off a green color to plant leaves. Amino acid residues such as tryptophan, tyrosine, and phenylalanine are other examples of naturally fluorescent compounds.π-π conjugation or aromatic rings present in these molecules help them absorb UV light.

Fluorescent species such as organic dyes can also be artificially introduced into chemical compounds. These dyes include fluorescein, rhodamine, etc. In this way, fluorescence spectroscopy can be performed on otherwise non-fluorescent materials. Fluorescence spectroscopy is compatible with all types of samples, solid, liquid, and gas inclusive.  

 What is fluorescence spectroscopy used for

• Biological molecules can be tagged with fluorescent dyes and analyzed via fluorescence spectroscopy.
• Fluorescence spectroscopy can be used to evaluate antioxidants, vitamins, and aflatoxins present in a food or beverage. These chemical compounds contain multiple hydroxyl and aromatic functional groups in their chemical structures which can help them absorb UV light and emit visibly.
• Environmental pH and temperature strongly affect the fluorescing ability of plant pigments. So, the physiological state of a plant, its nutrient profile, and/or any environmental stress present on it can be studied through fluorescence spectroscopy of the plant pigments.  
• Fluorescence spectroscopy can be used to detect contaminants such as dissolved organic matter (DOM) present in water. Trace impurities as low as 1 in 10¹⁰  can be readily analyzed via fluorescence spectroscopy with high sensitivity.  
• It can also be employed as a diagnostic tool in the biomedical sector.

What are the advantages of fluorescence spectroscopy

• Fluorescence spectroscopy is a fast and sensitive method for chemical compound detection and identification.
• It is extremely specific,  dedicated to the analysis of special chemical compounds that can fluoresce.
• It is relatively simple and does not require an extensive instrumental setup like that required for NMR spectroscopy or FTIR spectroscopy.
• Fluorescence spectroscopy is a cost-effective analytical technique.

What are the limitations of fluorescence spectroscopy

The only limitation of fluorescence spectroscopy is that the fluorescence of naturally occurring substances is susceptible to environmental temperature and pH conditions and also to its interaction with neighboring molecules. Thus, even some small interferences can strongly impact spectroscopic results.

Check out this video on fluorescence spectroscopy to revise all the concepts learned through this article.

As a last thought, we would like to tell you that fluorescence is a sub-type of luminescence. There are some other types of luminescence as well that we have discussed in luminescence spectroscopy.

References

1. Fleming, K. G. (2010). Fluorescence Theory. Encyclopedia of Spectroscopy and Spectrometry (Second Edition). J. C. Lindon. Oxford, Academic Press: 628-634.

2. Gómez-Hens, A. (2005). FLUORESCENCE | Food Applications. Encyclopedia of Analytical Science (Second Edition). P. Worsfold, A. Townshend, and C. Poole. Oxford, Elsevier: 186-194.

3. Karoui, R. (2018). Chapter 7 – Spectroscopic Technique: Fluorescence and Ultraviolet-Visible (UV-Vis) Spectroscopies. Modern Techniques for Food Authentication (Second Edition). D.-W. Sun, Academic Press: 219-252.

4. Lakowicz, J. R. (1999). Principles of fluorescence spectroscopy Springer New York, NY. Senesi, N. and V. D’Orazio (2005). FLUORESCENCE SPECTROSCOPY. Encyclopedia of Soils in the Environment. D. Hillel. Oxford, Elsevier: 35-52.