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Molecular Luminescence Spectroscopy. Molecular Luminescence Spectroscopy.

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molecular luminescence spectroscopy1
Molecular Luminescence Spectroscopy

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.

singlet and triplet states
Singlet and Triplet States

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

slide5
In a singlet state, the spins of the two electrons are paired and thus exhibit no magnetic field and called diamagnetic. Diamagnetic molecules, containing paired electron, are neither attracted nor repelled by a magnetic field. On the other hand, molecules in the triplet state have unpaired electrons and are thus paramagnetic which means that they are either repelled or attracted to magnetic fields. The terms singlet and triplet stems from the definition of multiplicity where:

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).

slide6
Multiplicity = (2 * 0) + 1 =1

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.

slide7
Energy Level Diagram for Photoluminescent Molecules

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:

absorption
Absorption

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.

slide10
The most frequently used transition is the p-p* transition for the following reasons:

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).

vibrational relaxation
Vibrational Relaxation

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.

fluorescence
Fluorescence

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 and external conversion
Internal and External Conversion

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.

dissociation and predissociation
Dissociation and predissociation

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.

slide15
External conversion (EC) is a process whereby excited molecules lose their energy due to collisions with other molecules or by transfer of their energy to solvent or other unexcited molecules. Therefore, external conversion is influenced by temperature, solvent viscosity, as well as solvent composition.
intersystem crossing
Intersystem Crossing

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.

slide17
Intersystem crossing is facilitated by presence of nonbonding electrons as well as heavy atoms. The presence of paramagnetic atoms or species also enhances intersystem crossing.

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.

phosphorescence
Phosphorescence

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.

quantum yield and efficiency
Quantum Yield and Efficiency

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

slide21
The transitions most important in luminescence spectroscopy are n-p* and p-p*. However, fluorescence is encountered more often in molecules having p-p* transitions since this transition has a higher quantum efficiency in terms of a higher molar absorptivity and a shorter lifetime (10-7 – 10-9s) than the n-p* transition which has a longer lifetime (10-5 – 10-7s). Once again, a longer lifetime means a lower luminescent probability due to increased possibility of radiationless deactivation.
variables that affect fluorescence and phosphorescence
Variables That Affect Fluorescence and Phosphorescence

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

slide23

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.

slide24
Substitution of halogens to the aromatic ring has important influence on the fluorescent signal where a decrease in fluorescence is observed with an increase in the atomic weight of the halogen and a subsequent increase in phosphorescence. This is referred to as the heavy atom effect where promotion of intersystem crossing takes place. In addition, substitution of a carbonyl or carboxylic acid groups decreased fluorescence due to enhancement of intersystem crossing.
effect of structural rigidity
Effect of Structural Rigidity

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

slide26
In addition, some metal complexes have higher fluorescence efficiency than do the ligands; also as a result of increased structural rigidity. For example, the fluorescence intensity of 8-hydroxyquinoline is increased to a large extent in presence of zinc ions, due to more rigid complex formation.
slide27
Effect of Solvent Nature

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.

slide28
Effect of Temperature

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.

effect of dissolved oxygen
Effect of Dissolved Oxygen

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.

effect of concentration on fluorescence
Effect of Concentration on Fluorescence

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

Substitution gives:

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! + ….)

slide31
Only the first term is important at the very low concentrations used. The relation simplifies to:

F = kPo (2.303 A)

F = KPoebc

Where K = 2.303k

slide32
The relation F = KP0ebc implies the following

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.

deviation from linearity between fluorescence and concentration
Deviation from Linearity between Fluorescence and Concentration

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.

fluorescence instruments
Fluorescence Instruments

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.

sources
Sources

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.

xenon arc lamp
Xenon Arc Lamp

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.

lasers
Lasers

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.

1 fluorometers
1. Fluorometers

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:

slide43
Unlike the cell used in UV-Vis absorption studies, the cell used in luminescence studies has the two transparent faces perpendicular to each other, rather than parallel. This is important since luminescence is collected 90o to the incident beam. This configuration decreases the noise and fully excludes interferences from the incident beam.
2 spectrofluorometers
2. Spectrofluorometers

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:

excitation and emission spectra
Excitation and Emission Spectra

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.

slide49
After obtaining the excitation spectrum, the lex (resulting in highest absorbance) is determined. To obtain the emission spectrum, the excitation monochromator is adjusted at the determined lex and the emission monochromator is scanned so that the emission spectrum is recorded. lem is the wavelength with highest fluorescence signal. At this point we are ready to perform a fluorometric determination using the defined excitation and emission wavelengths.
slide50

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.

3 fiber optic fluorescence sensors
3. Fiber Optic Fluorescence Sensors

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.

4 phosphorimeters
4. Phosphorimeters

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.

slide55

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

applications of photoluminescence methods
Applications of Photoluminescence Methods

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.

slide58
In addition, too many precautions must be followed for a successful phosphorescent analysis preventing widespread use of these methods. Fluorescence methods are quantitative techniques that are usually highly sensitive. Either the analyte or a reaction product of the analyte must be fluorescent which makes the method highly applicable to many systems that can be made fluorescent.
chemiluminescence
Chemiluminescence

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).

slide60
This can be represented by the general reaction:

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:

determination of nitrous oxide no
Determination of Nitrous Oxide (NO)

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.

slide62
NO + O3 = NO2* + O2

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.

slide63
Determination of Sulfur Dioxide

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

luminol chemiluminescence
Luminol Chemiluminescence

One of the most common chemiluminescent reactions is that of luminal (5-aminophthalhydrazide) with hydrogen peroxide in basic medium.

slide65
Luminol + H2O2 + OH- = (3-aminophthalate)* + N2 + H2O

(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

slide66
In addition, this system can be extended for the analysis of many substrates that can indirectly be made to produce hydrogen peroxide such as cholesterol esters which can be hydrolyzed to cholesterol, using cholesterol estearase, followed by oxidation of generate cholesterol using cholesterol oxidase where hydrogen peroxide is produced.
instrumentation
Instrumentation

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: