What is Fluorescence ?

Fluorescence is the property of some substances to re-emit (in most cases at longer wavelength so at lower energy) the received electromagnetic radiation, in particular they absorb the ultraviolet radiation and re-emit it in the visible.

Examples of this process are the materials that contain fluorescent pigments, such as the ink of a highlighter and fluorescent paints.

The fluorescent properties of an object often become evident with the use of a Wood’s lamp which produces radiation in the ultraviolet band, but depending on the materials, lower wavelengths may be necessary.

The mechanism of fluorescence is the following : an incident radiation (in the example of the Wood lamp is ultraviolet radiation) excites the atoms of the fluorescent substance, promoting an electron to an energy level (orbital) less bound, with more energy and therefore more “external”. Within a few tens of nanoseconds, the excited electron returns to the previous level in two or more steps, that is, passing for one or more excited states at intermediate energy.

All decays except one are, usually, non radiative, while the last emits light at a longer wavelength than the incident radiation (not necessarily in the visible spectrum): this light is called “fluorescence”.

We can expect the emission spectrum to overlap the absorption spectrum at the wavelength corresponding to the 0 – 0 transition and the rest of the emission spectrum to be of lower energy, or longer wavelength.
In practice, the 0-0 transitions in the absorption and emission spectra rarely coincide exactly, the difference representing a small loss of energy by interaction of the absorbing molecule with surrounding solvent molecules. This difference in wavelength is called stokes shift.

λem > λa

λa and λem are absorption and emission spectra peaks

Example of Stokes Shift

Absorption and emission spectra of acridine orange. From the pictures we can see that the difference between the maxima is rather small : stokes shift = 537 – 525 = 12nm

The absorption of energy to produce the first excited state does not perturb the shape of the molecule greatly and this means that the distribution of vibrational levels is very similar in both the ground and first excited states. The energy differences between the bands in the emission spectrum will be similar to those in the absorption spectrum and frequently the emission spectrum will be approximate to a mirror image of the absorption spectrum.

Example of Mirror Spectra

Absorption and emission spectra of hematoporphyrin. There is coincidence of the first peaks of emission and absorption spectra. The shape of the two spectra are in a good agreement to the mirror-image rule

Since the emission of fluorescence always takes place from the lowest vibrational level of the first excited state, the shape of the emission spectrum is always the same, despite changing the wavelength of exciting light. This is also known as the Kasha rule

Example of Emission Spectra Excited by different wavelength

Emission spectra of fluorescein. There is coincidence of the maximum and shape of emission spectrum despite different exciting wavelength. In the second spectrum are shown both the anti-stokes (negative shift) emission and the stokes emission (positive shift)

Fluorescence is also influenced by the structure of the molecule. For example the rigid molecules that present systems of conjugated double bonds , are well suited to the fluorescence : in particular molecules where there are aromatic structures, in which the resonance phenomenon of the double bonds are scattered throughout the structure, if excited give rise to π → π * transitions, and thus facilitate the fluorescence.

 The Fluorescence spectrometer

For the study of the fluorescence we used the grating spectrometer already described in one of the previous posts, supplemented by a cell sample holder and by a source of excitation. The spectra has been acquired with the software Theremino Spectrometer.

Fluorescence Spectrometer Scheme

Excitation sources

Fluorescence Spectra

Chlorophyll

Chlorophyll is a green pigment found in chloroplasts of algae and plants. Chlorophyll is an extremely important biomolecule, critical in photosynthesis, which allows plants to absorb energy from light. Chlorophyll absorbs light most strongly in the blue portion of the electromagnetic spectrum, followed by the red portion. Conversely, it is a poor absorber of green and near-green portions of the spectrum, hence the green color of chlorophyll-containing tissues.
Measurement of the absorption of light is complicated by the solvent used to extract it from plant material, which affects the values obtained.
In diethyl ether, chlorophyll A has approximate absorbance maximum of 430 nm and 662 nm, while chlorophyll B has approximate maximum of 453 nm and 642nm. Chlorophyll A fluoresces at 673 nm (maximum) and 726 nm. The different absorption spectra of Chlorophyll A and B achieve a better absorption of the sun radiation in order to enhance the efficiency of the photosynthesis.
The alcoholic chlorophyll solution has been obtained with spinach leaves grinded and macerated in ethanol 95%

Fluorescein

Fluorescein is a synthetic organic compound available as a dark orange/red powder slightly soluble in water and alcohol. It is widely used as a fluorescent tracer for many applications.
Fluorescein is a fluorophore commonly used in microscopy, in a type of dye laser as the gain medium, in forensics and serology to detect latent blood stains, and in dye tracing. Fluorescein has an absorption maximum at 494nm and emission maximum of 521nm (in water). The major derivatives are fluorescein isothiocyanate (FITC). The disodium salt form of fluorescein is known as uranine or D&C Yellow no. 8. The fluorescence of this molecule is very intense; peak excitation occurs at 494 nm and peak emission at 521 nm.

Olive Oil

Extra-virgin olive oil has a high chlorophyll content which is easily evidenced in the fluorescence spectrum.

Heated Olive Oil

Olive oil subjected to heating (eg during frying) is degraded. Chemical degradation involves the formation of peroxides and the destruction of chlorophyll, and this is easily evidenced by fluorescence spectrometry.

Riboflavin (Vitamin B2) and Pyridoxine (Vitamin B6)

Riboflavin (vitamin B2) is part of the vitamin B group. It is the central component of the cofactors FAD and FMN and as such required for a variety of flavoprotein enzyme reactions including activation of other vitamins.
Riboflavin is a yellow-orange solid substance with poor solubility in water. It is best known visually as it imparts the color to vitamin supplements and the yellow color to the urine of persons taking it. It shows a strong green fluorescence The name “riboflavin” comes from “ribose” (the sugar whose reduced form, ribitol, forms part of its structure) and “flavin”, the ring-moiety which imparts the yellow color to the oxidized molecule (from Latin flavus, “yellow”).
Pyridoxine is one form of vitamin B6. Its hydrochloride salt pyridoxine hydrochloride is used as vitamin B6 dietary supplement.

Quinine

Quinine is a natural white crystalline alkaloid having antipyretic (fever-reducing), antimalarial, analgesic (painkilling), and anti-inflammatory properties and a bitter taste. It is a stereoisomer of quinidine, which, unlike quinine, is an antiarrhythmic. Quinine contains two major fused-ring systems: the aromatic quinoline and the bicyclic quinuclidine. Quinine is highly fluorescent (quantum yield ~0.58) in 0.1 M sulfuric acid solution and it is widely used as a standard for fluorescence quantum yield measurement.

Coumarine

coumarin is an aromatic compound. At room temperature is in the form of colorless crystals, with characteristic odor.
Isolated for the first time from Dipteryx odorata, whose name was indeed coumarin, coumarin is present in more than 27 families of plants, and is responsible for sweet smell of freshly cut grass.
It is the first of a class of compounds – called coumarins – that share the coumarin chemical structure.
Even idrossicoumarine are present in many plants : umbelliferone , esculetin and scopoletin are the most common in nature. More complex coumarins as furanocoumarins are limited to a few families (Rutaceae and Apiaceae); typical example are the phototoxic psoralens which are present in the essential oil of Bergamot (bergaptene).
Coumarin is also used as a gain medium in some dye laser and as a sensitizer in photovoltaic technologies.
Coumarin absorbs wavelengths less than 400nm and gives strong fluorescence at about 460nm.

Biofluorescence

Fluorescence is a phenomenon present in many living organisms. Many of the fish, shellfish, algae and jellyfish synthesize optically active molecules that exhibit fluorescence. One of the most famous and important examples is the aequorea victoria jellyfish that produces the protein called green fluorescent protein (GFP), which is widely used in molecular biology.
Another example of biofluorescence is that of scorpions. This is a curious feature of these night arachnids which, when exposed to ultraviolet light, emit a bright green glow.
Technically, the fluorescence of scorpions is given by the hyaline layer present in their cuticle that contains substances that belong to the coumarine family, but its function is still a mystery, although many hypotheses have been proposed about it.
In the pictures below you see a scorpion beneath the wood lamp and its excited UV spectrum emission spectrum.

 

Pdf document with the description of the fluorescence experiments : Fluorescenza_ENG

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