Fluorescence Spectroscopy
Overview
Fluoresce spectroscopy is one of the most useful techniques for studying the optical properties
Introduction
When molecules unwind from electronic excited states, photon emission processes called Fluorescence and phosphorescence take place. These optical Polyatomic fluorescent compounds undergo changes between their electronic and vibrational states. (fluorophores). The main component of fluorescence spectroscopy is fluorophores. Molecules' fluorophores are the elements that give them this property. Tyrosine, Tryptophan, Fluorescein, and other molecules with aromatic rings are the most common fluorophores. A material emitting light without being heated is said to be luminescent, making it a type of cold body radiation. Chemical reactions, electrical energy, subatomic movements, or stress on a crystal are all potential causes. The following two conditions must be met for luminescence: The luminous substance needs to be a semiconductor with a non-zero band gap. If there is no band gap in a metal, light is not produced and this substance needs to receive the energy before luminescence can occur.
Types of luminescence
a) By Mechanism:
i) Fluorescence
ii) Phosphorescence
b) By Excitation Source:
i) Chemiluminescence
ii) Cathodoluminescence
iii) Electroluminescence
iv) Photoluminescence
A photon source is used to excite sample molecules in the sensitive optical emission method known as fluorescence spectroscopy. By detecting the intensity of that emission, it is possible to identify the molecules that relax through radiant emission.
Principle of fluorescence spectroscopy
Fluorescence occurs in molecules as a result of a subsequent to a series of physical phenomena, normally beginning with the absorption of light. This phenomenon is derived from the electromagnetic nature of light, molecular electronic structure and the nature of the environment of the luminescent molecule. It is fairly obvious that an appreciation of this phenomenon is necessary for the understanding of the relationships between molecular structure and luminescence spectroscopy to chemical and biological problems.
When light impinges upon matter, two things can happen. It can pass through the matter with no absorption taking place, or it can be absorbed either entirely or in part. Later, the energy is transferred to the molecule in the absorption process. The quantaenergy relationship can be expressed by the equation,
E = hν = hc/ λ
where h, ν, c and λ are respectively, Planck’s constant, the frequency of radiation, the velocity of light and the wavelength.
When light from external source hit the molecules and the energy absorbed is sufficient, the molecules may be excited by absorption of a photon to produce a transition from the ground state to an excited state. This process is known as excitation. Under normal conditions, the surplus energy of an excited molecule is invariably lost, with an ultimate return to the lowest vibrational level of the ground state. Several mechanisms may be involved in this process.
From this point, the molecule may return to the ground state by emission of photon (fluorescence), or by generation of heat (internal conversion), or may change to an excited triplet state (intersystem crossing) and then return to the ground state by emission of photon (phosphorescence), or may undergo chemical change. The processes that occur between the absorption and emission of light are usually illustrated by the Jablonski85 diagram.
Fluorescence is almost always the result of a transition between the lowest energy level of the first excited state (S1) and some of the ground state (S0). The part of the molecule responsible for the fluorescence is known as the fluorophore. The lifetime of an excited singlet state, and therefore the decay time of fluorescence is in the range 10-9 to 10-8 seconds.
The quantum energy of the emitted photon is equal to the difference in energy between these two levels. It follows that the quantum energy and therefore wavelength of the emission are independent of the wavelength of the photon producing excitation. Examination of the Jablonski diagram reveals that the energy of the emission is typically less than that of absorption. Because of that, the wavelength of the emission is longer than that of excitation. This phenomenon was first observed by Sir G. G. Stokes in 1852 at the University of Cambridge.
From the Jablonski diagram, although it is rare for molecules to enter an excited triplet state directly from the ground state, in many molecules there is an efficient process whereby an excited singlet state may be converted to an excited triplet state. This process is called intersystem crossing. It is immediately followed by vibrational relaxation whereby the molecule falls and return to the ground state from the triplet state by emission of a photon. This is called as phosphorescence. Phosphorescence decay is very much longer, typically milliseconds to seconds, therefore the wavelength of phosphorescence is generally longer than fluorescence.
Another process which has similar spectral but different temporal distribution86 is called delayed fluorescence. The delay is due to a double intersystem crossing, from singlet to triplet and back to singlet. It is a form of luminescence which takes place over a time scale similar to the phosphorescence but otherwise has the nature of fluorescence
Instrumentation of spectrofluorometer
Spectroflourometer mainly consists of:
A. Source of light
-Mercury vapour lamp
-Xenon arc lamp
-Tungsten film
B. Filters and monochromators
-Primary filters and secondary filters
-Excitation monochromators
-Emission monochromators
C. Sample cells, Detectors.
Fluorescence measurements fall into two categories, the measurement of the emission spectra, called fluorimetry, and measurement of the time dependence of the emission, called fluorometry. All fluorescence instruments consist of light source, wavelength selectors, sample compartment and detector system.
The light source produces light photons over a broad energy spectrum, typically ranging from 200 to 900 nm. Photons impinge on the excitation monochromator, which selectively transmits light about the specified excitation wavelength. The transmitted light passes through adjustable slits, then into the sample cell causing fluorescent emission by fluorophors within the sample. Emitted light enters the emission monochromator, which is positioned at a 90° angle from the excitation light path to eliminate background signal and minimise noise due to stray light. Finally, the emitted light entering the photomultiplier tube which the signal is amplified and creates a voltage that is proportional to the measured emitted intensity. In principle, the greatest sensitivity can be achieved by the use of filter or monochromator, together with the highest intensity source possible. In practice, to realise the full potential of the technique, only a small band of emitted wavelengths is examined and the incident light intensity is not made excessive, this is due to minimise the possible photodecomposition of the sample.
Factors affecting fluorescence
Conjugation
Molecule must have unsaturation i.e. it must have π electrons so that UV/vis radiation can be absorbed. If there is no absorption of radiation, there will not be fluorescence.
Rigidity of structures
Rigid structures will produce more fluorescence, while flexible structure will produce less fluorescence.
Nature of substituent groups
Electron donating groups like amino, hydroxyl groups enhance fluorescence activity. Electron withdrawing groups like Nitro, carboxylic group reduce fluorescence. Groups like SO3H or on NH4 + have no effect on fluorescence intensity.
Effect of temperature
Increase in temperature leads to increase in collisions of molecules and decrease in fluorescence intensity while decrease in temperature leads to decrease in collisions of molecules and increased fluorescence intensity.
Viscosity
Increase in viscosity leads to decreased collisions of molecules which will enhance fluorescence intensity while decrease in viscosity causes increased collisions of molecules which cause decreased fluorescence intensity.
Oxygen
Oxygen decreases the Fluorescence intensity in two ways: Oxidises fluorescence substances to non fluorescence substances. It quenches fluorescence because of paramagnetic properties.
Effect of pH
a. Aniline: Neutral or alkaline medium shows visible fluorescence while acidic conditions give fluorescence in UV region only.
b. Phenols : Acidic conditions do not give fluorescence while alkaline conditions gives good fluorescence.
Effect of concentration on fluorescence intensity
Fluorescence intensity = Q x Ia
Where, Q = Fluorescence efficiency
Ia = Intensity of absorbed light
Since emission is proportional to absorption, Ia has to be known
Where, Io = Intensity of incident light and It = Intensity of incident light
Advantages
- It’s one of the newer methods and its potentialities are still largely unexplored.
-It also affects precision. Up to 1% can be achieved easily in Flourimetric.
-The method is very sensitive and also possesses specificity because there is a choice of wavelength not only for the radiation emitted, but also for the light which excites it.
Limitations
-Careful buffering is necessary as fluorescence intensity may be strongly dependent
-Ultraviolet light used for excitation may cause photochemical changes or destruction of the fluorescent molecule .
-The presence of dissolved oxygen may cause increased photochemical destruction.
-Traces of iodide and nitrogen oxides are efficient quenchers and therefore interfere.
-The method is not suited for determination of major constituents of a sample, because the accuracy is very less for large amounts.
-The extent of applicability of this technique is limited, because of the fact that all elements and compounds are unable to exhibit fluorescence.