Molecular Luminescence Spectroscopy. Molecular Luminescence Spectroscopy.
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Luminescence spectroscopy is a technique which studies the fluorescence, phosphorescence, and chemiluminescence of chemical systems. The analyte or its reaction product needs to be luminescent. The relative luminescence intensity is related to analyte concentration as will be seen shortly.
Electrons in molecular orbitals are paired, according to Pauli exclusion principle. When an electron absorbs enough energy it will be excited to a higher energy state; but will keep the orientation of its spin. The molecular electronic state in which electrons are paired is called a singlet transition. On the other hand, the molecular electronic state in which the two electrons are unpaired is called a triplet state. The triplet state is achieved when an electron is transferred from a singlet energy level into a triplet energy level, by a process called intersystem crossing; accompanied by a flip in spin
Multiplicity = 2S + 1
Where, S is the total spin. The total spin for a singlet state is zero since electrons are paired which gives a multiplicity of one (the term singlet state).
In a triplet state, the total spin is one (the two electrons are unpaired) and the multiplicity is three:
Multiplicity = (2 * 1) + 1 = 3
It should also be indicated that the probability of a singlet to triplet transition is much lower than a singlet to singlet transition. Therefore, the intensity of the emission from a triplet state to a singlet state is much lower than emission intensities from a singlet to a singlet state.
The following diagram represents the main processes taking place in a photoluminescent molecule when it absorbs and emits energy.
The different processes will be discussed below:
The absorption of UV-Vis radiation is necessary to excite molecules from the ground state to one of the excited states. Absorption of radiation promotes electrons in chemical bonds to be excited. However, we have seen earlier that not all transitions have the same probability and while certain transitions are practically very important, others are seldom used and are of either no or marginal importance. Therefore, from information we have discussed in Chapter 14 we concluded that there are four different types of electronic transitions which can take place in molecules when they absorb UV-Vis radiation. A s-s* and a n-s* are not useful while the n-p* transition requires low energy but the molar absorptivity for this transition is low and transition energy will increase in presence of polar solvents.
a. The molar absorptivity for the p-p* transition is high allowing sensitive determinations.
b. The energy required is moderate, far less than dissociation energy.
c. In presence of the most convenient solvent (water), the energy required for a p-p* transition is usually smaller.
Therefore, best molecules that may show absorption are those with p bonds or preferably aromatic nature as discussed earlier. Absorption to higher excited singlet states requires a very short time (in the range of 10-14s).
Absorption of radiation will excite molecules to different vibrational levels of the excited state. This process is usually followed by successive vibrational relaxations (VR) as well as internal conversion to lower excited states. In cases where transitions occur to the first excited state, vibrational relaxation to the main excited electronic level will take place and/or an intersystem crossing (ISC) to the triplet state can occur.
After vibrational relaxation to first excited electronic level takes place, a molecule can return to the ground state by emission of a photon, called fluorescence (FL). The fluorescence lifetime is much greater than the absorption time and occurs in the range from 10-7 to 10-9s. As the lifetime in the excited state is increased, the probability of fluorescence will be decreased since radiationless deactivation processes may take place. However, not all excited molecules can show fluorescence by returning to ground state and most return to ground state by losing excitation energy as heat or through collisions with other molecules or solvent.
Internal conversion (IC) is a radiationless deactivation process whereby excited molecules return to the ground state without emission of a photon. This process lacks rigid understanding but seems to be the most efficient deactivation process in luminescence spectroscopy, since most molecules do not show fluorescence. However, molecules with close electronic energy levels, to the extent that their vibrational energy levels of ground and excited states are overlapped, are believed to cause efficient internal conversion.
Internal conversion can result in a phenomenon called predissociation (PD) where an electron relaxes from a higher electronic state to an upper vibrational energy of a lower electronic state. When the vibrational energy is large enough and is greater than the bond senergy, bond rupture occurs in a process called predissociation. Dissociation should be differentiated from predissociation where dissociation involves absorption of high energy so that the molecule is directly promoted to a high energy vibrational level where bond rupture directly occurs.
Electrons present at the first excited electronic level can follow one of three choices including emission of a photon to give fluorescence, radiationless deactivation to ground state, or intersystem crossing (ISC). The process of intersystem crossing involves transfer of the electron from an excited singlet to a triplet state. This process can actually take place since the vibrational levels in the singlet and triplet states overlap. However, crossing of the singlet state to the triplet state involves a flip in electron spin in order to satisfy the triplet state.
An electron in the triplet state can also cross back to the singlet state and can result in a photon as fluorescence but at a much longer time than regular fluorescence. This process is termed delayed fluorescence and has the same characteristics as direct fluorescence except for the large increase in lifetime.
Electrons crossing the singlet state to the triplet state with a flipped spin can also follow one of three choices including returning to the singlet state (including a flip in spin), relax to ground state by internal or/and external conversion, or lose their energy as a photon (phosphorescence, Ph) and relax to ground state with a second flip in spin to satisfy the singlet ground state. As can be rationalized from the processes involved in collecting phosphorescence photons, this involves an intersystem crossing and two flips in spin. This, in fact, requires a much longer time than fluorescence (10-4s to up to few s). Therefore, the probability of phosphorescence, and hence the intensity of the phosphorescence spectrum, is very low due to high possibility of radiationless deactivation.
The quantum yield or efficiency of fluorescence is the ratio of the number of fluorescing molecules to the total number of excited molecules. For highly fluorescent molecules, a quantum efficiency approaching one can be obtained. The quantum efficiency can be represented by the relation:
F = kFL/(kFL + kISC + kIC + kEC + kPD + kdiss)
Phosphorescence quantum efficiency is defined in the same manner
Factors affecting fluorescence and phosphorescence include both environmental and structural factors. Some of the important factors are discussed below:
Fluorescence and Structure
As indicated earlier, best luminescence is observed for molecules with p bonds and preferably those having aromatic rings due to presence of low energy p-p*. However, some heterocyclic aromatic rings do not show fluorescence. These include pyridine, furan, pyrrole, and thiophene
The lack of fluorescence in such molecules is largely believed to be due to existence of a low lying n-p* transition that rapidly converts the excited molecule to the triplet state and prevent fluorescence. However, fusion of a phenyl ring to any of the above molecules increase the possibility of the p-p* transitions and thus increase the fluorescence quantum efficiency.
The nature of the chemical structure of a molecule in terms of flexibility and rigidity is of major influence on the fluorescence and phosphorescence signal. Molecules that have high degree of flexibility will tend to decrease fluorescence due to higher collisional probability. However, more rigid structures have lower probability of collisions and thus have more fluorescence potential. Biphenyl has very low fluorescence quantum efficiency (~ 0.2) while fluorine has a quantum efficiency close
Solvents characteristics have important effects on luminescent behavior of molecules. Three main effects can be recognized:
a. Solvent Polarity
A polar solvent is preferred as the energy required for the p-p* is lowered.
b. Solvent Viscosity
More viscous solvents are preferred since collisional deactivation will be lowered at higher viscosities.
c. Heavy Atoms Effect
If solvents contain heavy atoms, fluorescence quantum efficiency will decrease and phosphorescence will increase.
Higher temperatures result in larger collisional deactivation due to increased movement and velocity of molecules. Therefore, lower temperatures are preferred.
Effect of pH
The pH of the solution is a very important factor that influences luminescence. For example, aniline shows fluorescence while aniline in acid solution (anilinium ion) does not. Most compounds luminesce in basic or slightly basic solutions while some show fluorescence in acidic medium. It is therefore important to adjust the pH so that maximum luminescence intensity is obtained. The pH also affects the emission wavelength where usually a longer emission wavelength is observed at higher pH.
Dissolved oxygen largely limits fluorescence since it promotes intersystem crossing because it is paramagnetic. However, dissolved oxygen affects phosphorescence more than it does to fluorescence. Although one would think that as far as intersystem crossing is increased in presence of oxygen, phosphorescence is expected to increase. On the contrary, phosphorescence is completely eliminated and quenched in presence of dissolved oxygen. This may be explained on the basis that the ground state of oxygen is the triplet state and it is easier for an electron in the triplet state to transfer its energy to triplet oxygen rather than performing a flip in spin and relax to singlet state. Therefore, oxygen will be excited and what we really observe is oxygen emission rather than phosphorescence. It is for this reason that oxygen should be totally excluded to be able to detect phosphorescence.
It can be undoubtedly said that the fluorescence is directly proportional to the amount of absorbed radiation where:
F = k(Po-P)
P = Po 10-A
F = kP0 (1-10-A)
This relation can be expanded by Mc Lauren series giving:
F = kP0 (2.303 A – (2.303 A)2/2! + (2.303 A)3/3! - (2.303 A)4/4! + ….)
F = kPo (2.303 A)
F = KPoebc
Where K = 2.303k
a. The fluorescence signal can be increased if the radiant power of the incident beam is increased. Therefore, always use more intense sources.
b. The fluorescence signal is directly related to the molar absorptivity and thus molecules of higher molar absorptivities are better fluorescers.
c. Fluorescence signal is directly proportional to path length.
d. Fluorescence signal is directly proportional to concentration. This is different from relation between absorbance and concentration which is logarithmic.
e. The linear correlation between fluorescence and concentration is only true when the absorbance is less than 0.05.
Negative deviations from the linear relation between fluorescence and concentration may be observed in the following cases:
a. At absorbances higher than 0.05.
b. Self-quenching whereby excited molecules lose their energies by collision with other molecules or solvent
c. Self-absorption which occurs when an emission band overlaps with an excitation (absorption) band. In this case, emitted photons excite other molecules in the ground state which results in no net emission.
Like any emission instrument, a fluorescence or phosphorescence instrument consists of a source, wavelength selectors, a sample cell, a detector, as well as a signal processor. Two wavelength selectors are used, the first is the excitation filter or monochromator which excites the sample while the other is the emission filter or monochromator that separates the fluorescence or phosphorescence wavelength.
We have seen earlier that the fluorescence signal is proportional to the radiant power of the source. Therefore, it is very important to select a source of as high radiant power as possible. In most instruments, a xenon arc lamp is usually the source of choice. However, some instruments use lasers.
A quartz envelope hosting two electrodes and xenon at high pressure (5-10 atm). The discharge which takes place between the two electrodes excites xenon and produces a continuum in the range from 200-1000 nm. The wavelength maximum occurs at about 500 nm. The radiant power produced by a xenon arc lamp is very high which makes the lamp suitable for luminescence analysis. Also, the coverage of the whole UV-Vis range adds to the assets of the lamp. However, a very good regulated power supply is essential since any fluctuations of the radiant power will be directly reflected on the fluorescence signal.
We have seen examples of lasers early in this course. However, it should be indicated that since lasers may have very high intensities, some molecules, which are otherwise non fluorescents, show good fluorescence and can thus be determined by fluorescence spectroscopy. This is called laser induced fluorescence (LIF) and is a common technique in fluorescence spectroscopy. In addition, one should remember that as the radiant power is increased, photodissociation may become a problem especially at shorter excitation wavelengths.
When the wavelength selectors are filters, the instrument is called a fluorometer. It is a simple instrument that is usually used for quantitative analysis. The use of fluorometers implies that the excitation and emission wavelengths are predetermined by other means or are known from literature. The detector is usually a sensitive photomultiplier tube. A schematic of a simple fluorometer is shown below:
In this case, the excitation and emission wavelengths are selected using dispersive elements like gratings or prisms. The same instrumental configuration as fluorometers is usually used. The following schematic represents a basic configuration of a spectrofluorometer:
The first step in a successful fluorescence measurement is to determine the excitation and emission wavelengths. The excitation spectrum is determined by adjusting the emission monochromator at an arbitrary, but well selected, wavelength which is thought to be longer than the anticipated wavelength. Keeping the emission monochromator at the preset wavelength, the excitation spectrum is recorded by scanning the excitation monochromator in a range less than the preset wavelength of the emission monochromator.
The shape of the emission spectrum is expected to be a mirror image of the excitation spectrum since they originate from opposite processes However, instrumental artifacts result in excitation and emission spectra that are not exactly mirror images.
These are the same as conventional fluorescence instruments but the beam from the excitation monochromators is guided through a bifurcated optical fiber to the sample container where excitation takes place. The fluorescence at the emission wavelength is then measured and related to concentration of analyte in the sample.
Instruments that can measure phosphorescence are called phosphorimeters. They are very similar to fluorescence instruments but make use of the fact that phosphorescence has a much longer lifetime than fluorescence and thus can be time resolved from the fluorescence signal. This can be achieved by placing a rotating chamber with a hole directing the beam to the sample. When the hole is aligned so that the incident beam excites the sample, the sample gives both fluorescence and phosphorescence. However, as the chamber rotates the incident beam becomes blocked and the fluorescence ceases. Phosphorescence will continue since it has a much longer lifetime and as the hole faces the detector only phosphorescence will be measured.
As the chamber rotates, phosphorescence will be detected as the hole in the chamber becomes aligned with the detector slit. No fluorescence interfere as the fluorescence lifetime is much shorter than the time required by the rotating chamber to align its hole with the slit of the detector
Fluorescence is the most widely used luminescent technique for determination of many metal ions that react with organic ligands to form fluorescent molecules. On the other hand, although phosphorescence methods were used for analysis of a variety of analytes, they are still rarely used because of lower sensitivity and precision. Furthermore, few chemical systems really show good phosphorescence.
This luminescence technique emerges in systems where a chemical reaction produces enough energy to excite an analyte or a reaction product of the analyte. Upon returning to ground state, the excited molecule emits a photon and chemiluminescence is observed. Several systems show the phenomenon of chemiluminescence where the chemiluminescence intensity is proportional to analyte concentration (in the nM to fM range).
A + B = C* + D
C* = C + hn
Analytical Applications of Chemiluminescence
Several reactions are known to produce chemiluminescence under certain conditions, some are described below:
Nitrous oxide reacts with electrogenerated ozone to form an excited nitrogen dioxide molecules followed by emission of the excitation energy as photons (chemiluminescence). The intensity of chemiluminescence is proportional to concentration of nitrous oxide.
NO2* = NO2 + hn
A NO concentration down to 1 ppb was determined using this method. On the other hand, higher nitrogen oxides (NOx) were also determined by this method; by first reducing the oxide to NO followed by reaction with ozone.
Sulfur dioxide reacts with hydrogen to form an excited sulfur dimer species. The excited sulfur dimer then relaxes to ground state by emission of photons.
4 H2 + 2 SO2 = S2* + 4 H2O
S2* = S2 + hn
One of the most common chemiluminescent reactions is that of luminal (5-aminophthalhydrazide) with hydrogen peroxide in basic medium.
(3-aminophthalate)* = 3-aminophthalate + hn
This reaction is most important for determination of many bioanalytical substrates which produce hydrogen peroxide. Examples include glucose, cholesterol, alcohol, amino acids, lactate, oxalate, etc… which, in presence of the respective oxidase enzyme, produce hydrogen peroxide. The intensity of chemiluminescence is proportional to substrate concentration
The instrumentation used in chemiluminescence is rather simple and can be composed of a photomultiplier tube and a readout. However, the PMT should be of very high sensitivity and a very low dark current. A schematic of the instrument can be shown below: