Fluorescence Spectroscopy, Chemistry tutorial


Fluorescence is a photoluminescence phenomenon in that atom or molecules are excited through absorption of electromagnetic radiation. The excited atoms then return to the ground state, while giving up their excess energy as photons. One of the most interesting characteristics of molecular fluorescence is its inherent sensitivity that is a benefit over the conventional absorption spectroscopy. The fluorescence technique as well has a larger linear concentration range than those encountered in absorption spectroscopy. Fluorescence methods are much less widely applicable than the absorption techniques since of the bounded number of molecules that illustrate appreciable fluorescence.


Fluorescence is a radiation generated through an atom or molecule that has been excited through photons to a single and excited state.

Basic Concept of Molecular Fluorescence

When a fluorescent material absorbs a photon or quantum of light, one of its orbital electrons is elevated to a remote orbit and consequently elevates the molecule to a transient high energy excited state. As the elevated electron drops back to its original orbit, the photon activated molecule regains its original low energy ground state via emitting part of the absorbed energy as specific light wavelengths. Such an emission is termed fluorescence if the emitted light has a longer wavelength than the absorbed light and its emission commences almost instantly on exposure to the exciting short wavelengths and stops inside 10-8 sec of termination of the source exposure.

The energy level diagram of fluorescence radiation is exposed below: Where E0 is the ground state, while E1 and E2 are the analogous excited states.

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Fig: Energy level diagram of fluorescence emission

Relationship between Excitation and Fluorescence Spectra

The excitation spectra or absorption spectrum about illustrate as a mirror image of fluorescence spectrum. This is since the energy differences between vibrational states are about the similar for both ground and excited states. This consequence is revealed through the spectra of anthracene, as fluorescent substance.

1058_Excitation spectra.jpg

Fig: (a) Excitation spectra, (b) Emission spectra  

There are many exceptions for this mirror image rule, most particularly whenever the excited and ground states have dissimilar molecular geometries or when different fluorescence bands originate from different parts of the body.

Fluorescence and Structure

Under normal condition, compounds enclosing aromatic rings of benzene give the most intense and most helpful molecular fluorescence emission. Indeed aliphatic and alicyclic carbonyl compounds in addition to the extremely conjugated double bond compounds as well fluoresce, but such are extremely few as compared to those of aromatic ring structure. Most of the unsubstituted aromatic hydrocarbons fluoresce in solution and the efficiency enhances through the number of rings and their degree of condensation. The easiest heterocyclic molecules these as pyridine, furan, thiophene and pyrole don't exhibit or possess molecular fluorescence properties. But fused ring compounds structures containing such rings frequently fluorescence. As well substitution on an aromatic ring reasons the wavelength of absorption to shift in addition to corresponding transforms in the fluorescence peaks. Structural rigidity as well enhances the ability of a compound to fluoresce additionally, substitution influences the fluorescence efficiency. Instances of fluorescent compounds comprise: 

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Fig: Typical aromatic compounds that fluoresce

Effects of Temperature and Solvent

Generally, quantum efficiency of fluorescence reduces through enhance in temperature. This is since the enhanced frequency of collision at elevated temperatures increases the probability of collisional relaxation. As well a decrease in solvent viscosity leads to alike consequence.

Effect of Concentration

The intensity of fluorescence radiation (F) is proportional to the radiant power of the exciting beam absorbed by the system.

For example   F = K (I0 - I)   (1)

Where I0 is the power of the incident beam on the solution and I is the power of the transmitted radiation, while K is a steady.

Therefore, to relate F to the concentration (C) of the fluorescent substance, Beer's law is applied as follows:

I/I0=10- εbc

Where ε is the molar absorptivity of the fluorescing molecule and εbc is the absorbance (A)

Usually, F α C or

  F = KC

Therefore, a plot of the emitted fluorescence intensity against the concentration of the solution provides a linear graph. This is since for dilute solution, Beer's law is obeyed.

294_A graph of emitted fluorescence intensity against the concentration of solution.jpg

Fig: A graph of emitted fluorescence intensity against the concentration of solution

Note: At high concentration, the relationship is not linear. This consequence is as a product of primary absorption in that the incident beam is absorbed so strongly that fluorescence is no longer proportional to concentration. At extremely high concentrations, F attains a maximum and might even start to reduce by increasing concentration since of secondary absorption. This procedure takes place due to absorption of the emitted radiation via other analyte molecules.


Different kinds of fluorescence instruments are obtainable. The optical diagram of the typical instrument for fluorescence measurement is revealed below:

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Fig: A typical Fluorescence Spectrometer

The source of radiation is a mercury lamp. The emission is computed at right angle to the mercury arc lamp source. Fluorescence radiation is emitted in all direction and the 90 degrees geometry avoids the detector viewing the source. The instrument utilizes 2 grating monochromators which allow the scanning of excitation spectra and emission spectra (emission wavelength scanned at a fixed excitation wavelength). It as well synchronizes the 2 spectra together.

Applications of Fluorescence Methods

Fluorescence methods are used to study chemical equilibrium and kinetics in the similar way as absorption spectrophotometry is utilized. It is frequently possible to learn chemical reactions at lower concentrations due to elevated sensitivity of fluorescence technique. Fluorescent probes or tags can be connected covalently to precise site in molecules these as proteins for easy detection and monitoring. Such tags can as well be utilized to provide information about energy transfer processes.

In addition, quantitative fluorescence techniques have been expanded for the analysis of inorganic, organic and biochemical molecules. The inorganic fluorescence techniques could be completed directly or indirectly. The direct methods are depend on the reaction of the analyte through a complexing agent to form a fluorescent complex, while the indirect process depends on reduce in fluorescence as a consequence of interaction of the analyte through a fluorescent agent. By respect to organic and biochemical applications, fluorescence technique is employed to determine amino acids, proteins, coenzymes, vitamins, nucleic acids, alkaloid, flavonoids, steroids and many other metabolite. As well, fluorescence is extensively utilized as a detection technique for liquid chromatographic process, for fluoro-analysis methods and electrophoresis.

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