# Chemical Sciences: A Manual for CSIR-UGC National Eligibility Test for Lectureship and JRF/Fluorescence

Fluorescence is the emission of electromagnetic radiation light by a substance that has absorbed radiation of a different wavelength. In most cases, absorption of light of a certain wavelength induces the emission of light with a longer wavelength (and lower energy). However, under conditions in which intense radiation is being absorbed, it is possible for one electron to absorb two photons (multiple photon absorption), which can lead to the emission of radiation having a smaller wavelength than the excitation source. The energy difference between the absorbed and emitted photons is due to thermal losses. Dissipation of vibrational energy occurs on a much greater time scale than fluorescent emission. The most striking examples of this phenomenon occur when the absorbed photon is in the ultraviolet region of the spectrum, and is thus invisible, and the emitted light is in the visible region. Practical applications of this effect are found in mineralogy, gemology, chemical sensors, fluorescent labelling, dyes, biological detectors etc.
The term 'fluorescence' was coined by George Gabriel Stokes in his 1852 paper; the name was suggested "to denote the general appearance of a solution of sulphate of quinine and similar media". (Phil. Trans. R. Soc. Lond. 1853 143, 385-396, quote from page 387). The name itself was derived from the mineral fluorite (calcium difluoride), some examples of which contain traces of divalent europium, which serves as the fluorescent activator to provide a blue fluorescent emission. The fluorite which provoked the observation originally, and which remains one of the most outstanding examples of the phenomenon, originated from the Weardale region, of northern England.

## Equations
### Photochemistry

Fluorescence occurs when an orbital electron of a molecule, atom or nanostructure relaxes to its ground state by emitting a photon of light after being excited to a higher quantum state by some type of energy:
Excitation: $S_{0}+h\nu _{ex}\to S_{1}$
Fluorescence (emission): $S_{1}\to S_{0}+h\nu _{em}+heat$
here $h\nu$ is a generic term for photon energy with h = Planck's constant and $\nu$ = frequency of light. (The specific frequencies of exciting and emitted light are dependent on the particular system.)
State S$_0$ is called the ground state of the fluorophore (fluorescent molecule) and S$_1$ is its first (electronically) excited state.
A molecule in its excited state, S$_1$, can relax by various competing pathways. It can undergo 'non-radiative relaxation' in which the excitation energy is dissipated as heat (vibrations) to the solvent. Excited organic molecules can also relax via conversion to a triplet state which may subsequently relax via phosphorescence or by a secondary non-radiative relaxation step.
Relaxation of an S$_1$ state can also occur through interaction with a second molecule through fluorescence quenching. Molecular oxygen (O$_2$) is an extremely efficient quencher of fluorescence just because of its unusual triplet ground state.
Molecules that are excited through light absorption or via a different process (e.g. as the product of a reaction) can transfer energy to a second 'sensitized' molecule, which is converted to its excited state and can then fluoresce. This process is used in lightsticks to produce different colors.

### Quantum yield

The fluorescence quantum yield gives the efficiency of the fluorescence process. It is defined as the ratio of the number of photons emitted to the number of photons absorbed.
    $\Phi ={\frac {\textrm {Number\ of\ photons\ emitted}}{\textrm {Number\ of\ photons\ absorbed}}}$
The maximum fluorescence quantum yield is 1.0 (100%); every photon absorbed results in a photon emitted. Compounds with quantum yields of 0.10 are still considered quite fluorescent. Another way to define the quantum yield of fluorescence, is by the rates excited state decay:
    ${\frac {{k}_{f}}{\sum _{i}{k}_{i}}}$
where ${k}_{f}$ is the rate of spontaneous emission of radiation and
    $\sum _{i}{k}_{i}$
is the sum of all rates of excited state decay. Other rates of excited state decay are caused by mechanisms other than photon emission and are therefore often called "non-radiative rates", which can include: dynamic collisional quenching, near-field dipole-dipole interaction (or resonance energy transfer), internal conversion and intersystem crossing. Thus, if the rate of any pathway changes, this will affect both the excited state lifetime and the fluorescence quantum yield.
Fluorescence quantum yield are measured by comparison to a standard with known quantology; the quinine salt, quinine sulfate, in a sulfuric acid solution is a common fluorescence standard.

### Lifetime

The fluorescence lifetime refers to the average time the molecule stays in its excited state before emitting a photon. Fluorescence typically follows first-order kinetics:
    $\left[S1\right]=\left[S1\right]_{0}e^{-\Gamma t}$
where $\left[S1\right]$ is the concentration of excited state molecules at time $t$, $\left[S1\right]_{0}$ is the initial concentration and $\Gamma$ is the decay rate or the inverse of the fluorescence lifetime. This is an instance of exponential decay. Various radiative and non-radiative processes can de-populate the excited state. In such case the total decay rate is the sum over all rates:
    $\Gamma _{tot}=\Gamma _{rad}+\Gamma _{nrad}$
where $\Gamma _{tot}$ is the total decay rate, $\Gamma _{rad}$ the radiative decay rate and $\Gamma _{nrad}$ the non-radiative decay rate. It is similar to a first-order chemical reaction in which the first-order rate constant is the sum of all of the rates (a parallel kinetic model). If the rate of spontaneous emission, or any of the other rates are fast, the lifetime is short. For commonly used fluorescent compounds typical excited state decay times for fluorescent compounds that emit photons with energies from the UV to near infrared are within the range of 0.5 to 20 nanoseconds. The fluorescence lifetime is an important parameter for practical applications of fluorescence such as fluorescence resonance energy transfer.

## Rules

There are several rules that deal with fluorescence. The Kasha–Vavilov rule dictates that the quantum yield of luminescence is independent of the wavelength of exciting radiation.
This is not always true and is violated severely in many simple molecules. A somewhat more reliable statement, although still with exceptions, would be that the fluorescence spectrum shows very little dependence on the wavelength of exciting radiation.
The Jablonski diagram describes most of the relaxation mechanism for excited state molecules.

## Applications

There are many natural and synthetic compounds that exhibit fluorescence, and they have a number of applications. Some deep-sea animals, such as the Greeneye, use fluorescence.

### Lighting

    For more details on this topic, see Fluorescent_light.
The common fluorescent tube relies on fluorescence. Inside the glass tube is a partial vacuum and a small amount of mercury. An electric discharge in the tube causes the mercury atoms to emit ultraviolet light. The tube is lined with a coating of a fluorescent material, called the phosphor, which absorbs the ultraviolet and re-emits visible light. Fluorescent lighting is very energy-efficient compared to incandescent technology, but the uneven spectrum of traditional fluorescent lamps may cause certain colours to appear different than when illuminated by incandescent or day light. The mercury vapor emission spectrum is dominated by a short-wave UV line at 254 nm (which provides most of the energy to the phosphors), accompanied by visible light emission at 436 nm (blue), 546 nm (green) and 579 nm (yellow-orange). These three lines can be observed superimposed on the white continuum using a hand spectroscope, for light emitted by the usual white fluorescent tubes. These same visible lines, accompanied by the emission lines of trivalent europium and trivalent terbium, and further accompanied by the emission continuum of divalent europium in the blue region, comprise the more discontinuous light emission of the modern trichromatic phosphor systems used in many compact fluorescent lamp and traditional lamps where better color rendition is a goal.
Fluorescent lights were first available to the public at the 1939 World's Fair, and improvements since then have largely been better phosphors, longer life and more consistent internal discharge, and easier-to-use shapes such as compact fluorescent lamps. Some HID lamps coupled their even greater electrical efficiency with phosphor enhancement for better color rendition. White light-emitting diodes became available in the mid 1990s as LED lamps, in which blue light emitted from the semiconductor strikes phosphors deposited on the tiny chip. The combination of the blue light that continues through the phosphor and the green to red fluorescence from the phosphors produces a net emission of white light.
Glow sticks sometimes utilize fluorescent materials to absorb light from the chemiluminescent reaction and emit light of a different color.

### Analytical chemistry

Many analytical procedures involve the use of a fluorometer, usually with a single exciting wavelength and single detection wavelength. Because of the sensitivity that the method affords, fluorescent molecule concentrations as low as 1 part per trillion can be measured.
Fluorescence in several wavelengths can be detected by an array detector, to detect compounds from HPLC flow. Also, TLC plates can be visualized if the compounds or a coloring reagent is fluorescent. Fluorescence is most effective when there is a larger ratio of atoms at lower energy levels in a Boltzmann distribution. There is then a higher probability of lower energy atoms being excited and releasing photons, making analysis more efficient.

### Equipment

Usually the setup of a Fluorescence assay involves a Light source, which may emit an array different wavelengths of light. Generally, a single wavelegth is required for proper analysis, so in order to selectively filter the light, it is passed through an excitation monochromator, and then that chosen wavelength is passed through the sample cell. After absorption and re-emission of the energy, many wavelengths may emerge due to Stokes shift and various electron transitions. To separate and analyze them, the fluorescent radiation is passed through an Emission monochromator, and observed selectively by a detector

### Biochemistry and medicine

Fluorescence in the life sciences is used generally as a non-destructive way of tracking or analysis biological molecules by means of the fluorescent emission at a specific frequency where there is no background from the excitation light, as relatively few cellular components are naturally fluorescent (called intrinsic or autofluorescence). In fact, a protein or other component can be "labelled" with a extrinsic fluorophore, a fluorescent dye which can be a small molecule, protein or quantum dot, finding a large use in many biological applications.
The quantification of a dye is done with a Spectrofluorometer and finds additional applications in:
* when scanning the fluorescent intensity across a plane one has Fluorescence microscopy of tissues, cells or subcellular structures which is accomplished by labeling an antibody with a fluorophore and allowing the antibody to find its target antigen within the sample. Labeling multiple antibodies with different fluorophores allows visualization of multiple targets within a single image (multiple channels). DNA microarrays are a variant of this.
* Automated sequencing of DNA by the chain termination method; each of four different chain terminating bases has its own specific fluorescent tag. As the labeled DNA molecules are separated, the fluorescent label is excited by a UV source, and the identity of the base terminating the molecule is identified by the wavelength of the emitted light.
. Ethidium bromide fluoresces orange when intercalating DNA and when exposed to UV light.
* FACS (fluorescent-activated cell sorting)
* DNA detection: the compound ethidium bromide, when free to change its conformation in solution, has very little fluorescence. Ethidium bromide's fluorescence is greatly enhanced when it binds to DNA, so this compound is very useful in visualising the location of DNA fragments in agarose gel electrophoresis. Ethidium bromide may be carcinogenic - an arguably safer alternative is the dye SYBR Green.
* Immunology: An antibody has a fluorescent chemical group attached, and the sites (e.g., on a microscopic specimen) where the antibody has bound can be seen, and even quantified, by the fluorescence.
Additionally Fluorescence resonance energy transfer used to study protein interactions, detect specific nucleic acid sequences and used as biosensors, while fluorescent lifetime can give an additional layer of information.

### Gemology, mineralogy, geology, and forensics

Fingerprints can be visualized with fluorescent compounds such as ninhydrin. Blood and other substances are sometimes detected by fluorescent reagents, particularly where their location was not previously known.
Gemstones, minerals, fibers, and many other materials which may be encountered in forensics or with a relationship to various collectibles may have a distinctive fluorescence or may fluoresce differently under short-wave ultraviolet, long-wave ultra violet, or X-rays.
Many types of calcite and amber will fluoresce under shortwave UV. Rubies, emeralds, and the Hope Diamond exhibit red fluorescence under short-wave UV light; diamonds also emit light under X ray radiation.
Fluorescence in minerals is caused by a wide range of activators. In some cases, the concentration of the activator must be restricted to below a certain level, to prevent quenching of the fluorescent emission. Furthermore, certain impurities such as iron or copper need to be absent, to prevent quenching of possible fluorescence. Divalent manganese, in concentrations of up to several percent, is responsible for the red or orange fluorescence of calcite, the green fluorescence of willemite, the yellow fluorescence of esperite, and the orange fluorescence of wollastonite and clinohedrite. Hexavalent uranium, in the form of the uranyl cation, fluoresces at all concentrations in a yellow green, and is the cause of fluorescence of minerals such as autunite or andersonite, and, at low concentration, is the cause of the fluorescence of such materials as some samples of hyalite opal. Trivalent chromium at low concentration is the source of the red fluorescence of ruby corundum. Divalent europium is the source of the blue fluorescence, when seen in the mineral fluorite. Trivalent lanthanoids such as terbium and dysprosium are the principal activators of the creamy yellow fluorescence exhibited by the yttrofluorite variety of the mineral fluorite, and contribute to the orange fluorescence of zircon. Powellite (calcium molybdate) and scheelite (calcium tungstate) fluoresce intrinsically in yellow and blue, respectively. When present together in solid solution, energy is transferred from the higher energy tungsten to the lower energy molybdenum, such that fairly low levels of molybdenum are sufficient to cause a yellow emission for scheelite, instead of blue. Low-iron sphalerite (zinc sulfide), fluoresces and phosphoresces in a range of colors, influenced by the presence of various trace impurities.
Crude oil (petroleum) fluoresces in a range of colors, from dull brown for heavy oils and tars through to bright yellowish and bluish white for very light oils and condensates. This phenomenon is used in oil exploration drilling to identify very small amounts of oil in drill cuttings and core samples.

### Organic liquids

Organic liquids such as mixtures of anthracene in benzene, toluene, or stilbene in the same solvents, fluoresce with ultraviolet or gamma ray irradiation. The decay times of this fluorescence is of the order of nanoseconds since the duration of the light depends on the lifetime of the excited states of the fluorescent material, in this case anthracene or stilbene.

## Further reading

* Lakowicz, J.R. 2006. Principles of Fluorescence Spectroscopy, Third Edition, Plenum Press, New York. ISBN 0-387-31278-1.
* Valeur, B. 2001. Molecular Fluorescence: Principles and Applications, Wiley-VCH. ISBN 352729919X .
* Guilbault, G.G. 1990. Practical Fluorescence, Second Edition, Marcel Dekker, Inc., New York. ISBN 0-8247-8350-6.

## References

* ↑ Stokes, G. G. (1852). "On the Change of Refrangibility of Light". Philosophical Transactions of the Royal Society of London. 142: 463–562.
* ↑ Harris, D. C. et al, Exploring Chemical Analysis 4th ed., New York, NY (c)2009 by W.H. Freeman and Company
* ↑ Lakowicz, J.R., Principles of fluorescence spectroscopy. 3rd ed. 2006, New York: Springer. xxvi, 954 p.
* ↑ Invitrogen.com