Chapter 6 Molecular Fluorescence and Phosphorescence

Home , Doublet state, Intersystem crossing, Phosphorescence

Chapter 6

Molecular Fluorescence and Phosphorescence Sources of Luminescence

Luminescence can be classifieds according to the source of excitation into:

1. Photoluminescence: deactivation takes place after excitation with photons 2. Radioluminescence: ground state molecules are excited by collisions with high energy particles 3. Chemluminescence: ground state molecules are excitted by certain chemical reactions Characteristics of Photoluminescence

Fluorescence is short-lived with luminescence ending almost immediately.

Phosphorescence involves change in electron spin and may endure for several seconds.

In most cases, photoluminescent radiation tends to be at longer wavelengths than excitation radiation.

Chemiluminescence is based on an excited species formed by a chemical reaction. Types of Fluorescence/phosphorescence

• Resonance radiation (or fluorescence) – absorbed radiation is reemitted without alteration.

• More often, molecular fluorescence (phosphorescence) occurs as bands centered at wavelengths longer than resonance line. This shift to longer wavelengths is Stokes shift. Excitation and de-excitation process Molecular Multiplicity, M

M = 2S + 1 S = spin quantum number of the molecule =  net spin of the electrons in the molecule

•Most organic molecules have S = 0 because molecules have even number of electrons thus the ground state must have all electrons paired •M = 2 X (0) + 1 = 1; Molecules in the ground state

mostly have a singlet state, So. S1 and S2 for first and second excited states

• While molecules in the excited state, one e- may reverse its spin

• S = (+1/2) + (+1/2) = 1

M = 2(1) + 1 = 3 = Triplet State= T1

• A molecule with an even number of e- can not have a ground triplet state because the spins of all electrons are paired • Molecules with one unpaired electron are in doublet state (organic free radicals) Spin Orientations • The allowed absorption process will result in a singlet state.

• A change in electron spin is, technically, a "forbidden" process

•“Forbidden" process according to quantum mechanics means unlikely, not “ absolutely can’t happen” Electronic States

Singlet State: electron spins paired, no splitting of energy level. May be ground or excited state.

Doublet State: free radical (due to odd electron).

Triplet State: one electron excited to higher energy state, spin becomes unpaired (parallel). Difference between triplet and singlet states 1. Molecule is paramagnetic in the T excited state and diamagnetic in the S excited state

2. S T transitions (or reverse) are less probable than S S transitions Thus average lifetime of T excited state (10-4 s) is longer than the S excited state (10-5 - 10-8 s)

Also absorption peaks due to S-T transitions are less sensitive than S-S transitions

When an excited triplet state can be populated from an excited S state of certain molecules, a phosphorescence process will be the result

Energy of a Molecule (Jablonski energy-level diagram)

Energy Levels for Luminescence Transitions

+quenching Fluorescence in

the Jablonski energy-level diagram

absorption

internal internal

fluorescence conversion

S crossing

1 intersystem

+hν T0

-hν

internal internal radiationless transition conversion S 0 transition involving emission/absorption of photon Interpretation of the Energy Diagram

• Absorption : Ground state to Excited state • (10-15 sec) • Relaxation: Excited state to Ground state – Internal Conversion (IC) • nonradiative (thermal, collisional) relaxation of electrons through vibrational states (10-12 - 10-14 sec) – Emission • fluorescence (spontaneous emission: 10-10 - 10-8 sec) • phosophorescence (10-3 - 10-0 sec) – phosphorescence requires intersystem crossing (flip of electron spin) » Ground state singlet » Excited state singlet » Spin flip (now in Triplet state) » intersystem crossing » Need another Spin flip to be allowed to go back to Ground state singlet

– Once in the triplet state, de-excitation to the ground singlet state is “forbidden”. • Consequently, the molecule "hangs" in the triplet state for a considerably longer period of time than it would otherwise. When the emission finally comes, it is called phosphorescence.

Deactivation Processes

The molecule can rapidly dissipate excess vibrational energy as: 1. heat by collision with solvent molecules through vibrational relaxation process 2. EMR

-Internal Conversion IC -Inter System Crossing ISC - Quenching - Fluorescence - Phosphorescence

Rates of Absorption and Emission

• The rate at which a photon of radiation is absorbed is enormous, the process requiring on the order o f 10-14 to 10-15s. • Fluorescence emission, on the other hand, occurs at a significantly slower rate. – Here, the lifetime of the excited state is inversely related to the molar absorptivity of the absorption peak corresponding to the excitation process. • The favored route to the ground state is the one that minimizes the lifetime of the excited state. • Thus, if deactivation by fluorescence is rapid with respect to the radiationless processes, such emission is observed. • On the other hand, if a radiationless path has more favorable rate constant, fluorescence is either absent or less intense. Vibrational Relaxation

• This relaxation process is so efficient that the average lifetime of a vibrationally excited molecule is 10-12s or less, a period significantly shorter than the average lifetime of an electronically excited state. Internal Conversion

• The term internal conversion describes intermolecular processes by which a molecule passes to a lower energy electronic state without emission of radiation. • These processes are neither well defined nor well understood, but it is apparent that they are often highly efficient, because relatively few compounds exhibit fluorescence Predissociation

• As a result if internal conversion, electron may move from a higher electronic state to an upper vibrational level of a lower electronic state in which the vibrational energy is enough to cause rupture of a bond • In a large molecule there is an appreciable probability for the existance of bonds with sterngths less than the electronic excitation energy of the chromophores

Dissociation

• The absorbed radiation excites the electron of a chromophore directly to a sufficiently high vibrational level to cause rupture of the chromphoric bond. That is no internal conversion is involved. • Dissociation processes also competes with the fluorescent process

External Conversion • Deactivation of an excited electronic state may involve interaction and energy transfer between the excited molecule and the solvent or other solutes. • These processes are called collectively external conversion, or collisional quenching. • Evidence for external conversion includes the marked effect upon fluorescence intensity exerted by the solvent; furthermore, those conditions that tend to reduce the number of collisions between particles generally lead to enhanced fluorescence.

Intersystem crossing

• Intersystem crossing takes place from excited singlet to excited triplet state. • Transition occurs between the singlet ground state (electrons are anti-parallel & paired) to an excited state(electrons are parallel andunpaired) • Return to ground state is much slower process than fluorescence, or Phosphorescence. • Emitted radiation is of an even longer wavelength because the energy difference between the two is small.

Fluorescence De-excitation can occur via a radiative decay, i.e. by spontaneous emission of a photon. The radiative de-excitation process can be Electronic described as a monomolecular excited state process:

dnexc  kF nexc dt energy v=0 The vibrational relaxation of any Electronic electronic state is always much faster ground state than photon emission. Therefore, all observed fluorescence normally originates from the lowest vibrational v=0 level of the electronic excited state. Fluorescence

Most of the fluorescence spectrum is shifted to lower energies (longer wavelengths), compared to the absorption spectrum. Furthermore, the shape of the

Electronic emission spectrum is approximately excited state the mirror image of the absorption spectrum, providing that the ground

and excited state have similar

vibrational properties.

v=0 energy

Electronic ground state

v=0 Mirror Image Spectra

The above spectra are plotted as amplitude versus wave number. When plotted versus wavelength the mirror effect is not as pronounced. • The shortest wavelength in the fluorescence spectrum is the longest wavelength in the absorption spectrum Phosphorescence • Deactivation of electronic excited states may also involve phosphorescence. • After intersystem crossing to a triplet state, further deactivation can occur either by internal or external conversion or by phosphorescence. • External and internal conversions compete so successfully with phosphorescence that this kind of emission is ordinarily observed only at low temperatures, in highly viscous media or by molecules that are adsorbed on solid. Phosphorescence

Phosphorescence occurs when a “forbidden” spin exchange converts the electronic excited singlet state Electronic into a triplet state: excited state Intersystem Crossing   

The triplet state relaxes rapidly to the energy v=0 vibrational level, which has lower energy than the corresponding Electronic excited singlet state. The transition to ground state the electronic ground singlet state with the emission of a photon is spin- forbidden. Therefore the molecule gets trapped in the triplet state. Phosphorescence

In practice, the emission of a photon and the recovery of the ground state occurs, but with low efficiency. Electronic excited state Since the triplet state has generally lower energy than the excited singlet, phosphorescence occurs at longer

wavelengths (lower frequencies) and energy can easily be distinguished from fluorescence. The de-excitation of molecules due to phosphorescence is Electronic ground state described by: dn exc  k n dt IS exc Phosphorescence Being spin-forbidden, the transition from the excited triplet to the ground singlet occurs very slowly, with a radiative lifetime in the order of seconds, or longer. Phosphorescence can be observed only when other de-activating processes have been suppressed, typically in rigid glasses, at low temperature and in the absence of oxygen. In solution other de-excitation processes, such as quenching are much more efficient, and therefore phosphorescence is rarely observed. Quenching

•Energy gets transferred to the quencher, usually through collisions with a nearby residue or molecule

•This reduces photon emissions and decreases fluorescence intensity. Quenching •Two processes can diminish amount of light energy emitted from the sample: •Internal quenching due to intrinsic structural feature e.g. structural rearrangement. •External quenching interaction of the excited molecule with another molecule in the sample or absorption of exciting or emitted light by another chromophore in sample. •All forms of quenching result in non-radiative loss of energy. Quenching

De-excitation can result from collisions with other solute molecules (Q), capable of accepting the excess energy and therefore of quenching the excited states: exc  Q  ground  Q* [Q] [exc] dn exc  k n n  k' n dt quench exch quencher quench exch Usually Q is in large molar excess over the excited state and the observed kinetic is a pseudo-first order. Oxygen is an efficient quencher, with quenching rates limited basically by diffusion. At millimolar oxygen concentration this means

9 1 k'quench ~10 s Rate Constants and Quenching

• The rate constant for fluorescence is roughly proportional to the molar absorptivity

e 104 103 102 9 8 7 kf 10 10 10

• The rate constant for intersystem crossing depends upon the singlet-triplet gap, the smaller the gap the larger the rate constant • The rate constant for intersystem crossing is increased with Br and I substitution into the double bond structure • During the lifetime of the excited state a molecule can loose energy via collisions, this is called quenching k S** Q q S  Q  S  Q  heat 1 00

* kq SQTQ10       common quenchers are oxygen, molecules with heavy atoms, and molecules with unpaired spins Kinetics of Fluorescence and Phosphorescence

Intensity of absorbed light = I = Io - IT

Where I is known also as Rate of absorption That is exactly equal rate of deactivation

I = (kIC + kISC + kf + kQ [Q]) [S1]

kIC + kISC + kf + kQ are the first-order rate constants of the corresponding deactivation processes. kQ is the second-order quenching rate constant, [Q] is the quencher concentration

[S1] is the concentration if S1 molecules Vibrational relaxation has been included in kIC

Efficiency of fluorescence is measured in terms of the fluorescence quantum yield, f

f = # of photons emitted # of photons absorbed

Rate of fluorescence= If = I f = kf[S1] = f (kIC + kISC + kf + kQ [Q]) [S1]

f = kf / (kIC + kISC + kf + kQ [Q]) Fluorescence Quantum Yield • The higher the value of f the greater will be the observed fluorescence.  If the rate constants relative to other de-

excitation processes are small compared to kf

then the compound will have a value of f ~ 1.  So by definition a non-fluorescent compound has a value of f = 0, where all energy absorbed by the molecule is lost via nonradiative processes such as collisional deactivation. • The quantum yield of a compound is usually

determined relative to a standard for which f is already known. • The intensity of fluorescence of a fluorophore is referred to as brightness: the higher this is, the more

extinction coefficient (e) and the quantum yield (f ). • f allows a qualitative interpretation of many of the structural and environmental factors that affect fluorescent intensity

• The variables that lead to higher kf values and lower values to the other k terms will enhance fluorescence To obtain a large quantum yield:  find a molecule with a large molar absorptivity substitute a highly symmetric molecule with a group having a lone pair of electrons (-OH or –

NH2)  keep oxygen and free radicals out of the solution don't use molecules with heavy halogens ratio naphthalene 1-fluoro 1-chloro 1-bromo 1-iodo

p/f 0.093 0.068 5.2 16.4 >1000

The lifetime of the S1 state is given by:

= 1/ (kIC + kISC + kf + kQ [Q])

If all processes competing with fluorescence are absent, then

r (radiative lifetime) = 1 / kf

Thus,

f =  / r

’ For Phosphorescence p = 1/ (kp + k VR + kQP [Qp] and p / t = P / PR

Kp = First order decay const of T1 to S0 state ’ k VR = const. For vibrational relaxation of the T1 state kQP [Qp] = pseudo first-order rate const. For quenching of the triplet state by impurity quincher, Qp P and PR = lifetimes in, respectively, the presence and absence of the competitive radiationless processes

t = efficiency of formation of the triplet state

Effect of Concentration on Fluorescent Intensity

If = I f = f (Io – IT) ….(1) -ebc IT = Io X 10 …… (2) Where e is the molar absorptivity of the fluorescing molecule. Substituting Eq 2 in Eeq 1 -ebc If = fIo (1– 10 ) …. (3) The exponential term in Eq 3 can be expanded as a Maclaurin series to 2 3 If= fIo [2.303 ebc - (2.303 ebc ) /2! +(2.303 ebc ) /3!.. ) Provided 2.303 ebc < 0.05, all of the subsequent terms in the brackets become small with respect to the first. Thus, we may write

If= 2.303 fIo ebc Or If = kC. If VS C is straight line at low concentration

Factors responsible for non linearity

1. The concentration: When 2.303 ebc is more than 0.05, the linearity is lost 2. Self quenching: collisions between excited molecules 3. Self absorption: When the wave length of emission overlaps an absorption peak. Fluorescence is then decreased as the emitted beam traverse the solution Excitation and Emission Spectra

Fluorescing molecules are characterized by two types of spectra: 1. Excitation Spectrum: Fluorescence intensity is observed as a function of exciting  at some fixed emission  2. Emission (Fluorescence and phosphorescence) spectrum: Emission intensity is measured as a function of emitted  at fixed exciting  3. Emission spectrum is usually used for analytical applications 4. Excitation spectrum is run first to confirm the identity of the substance 5. Fluorescence Spectrum occurs at  longer than does the excitation (absorption) spectrum 6. Only the longer  band of absorption and the shorter  band of fluorescence will generally overlap

7. Since the vibrational spacing in the ground state So and the first excited singlet state S1 will often be similar for large molecules the fluorescence spectrum is often mirror image of the absorption spectrum

8. Because phosphorescence emission occurs from the triplet state there is no mirror relationship with the absorption band of the lowest excited singlet 9. Since emission almost always occurs from the first excited state, the emission spectrum is independent of  of excitation 10. Since the quantum yield of emission is generally independent of  of excitation thus the excitation spectrum is independent of the emission  monitored • In order to scan the two types of spectra, tow monochromators are used: Excitation monochromator and Emission monochromator

• Excitation spectrum is recorded when the emission monochrom.

is set at fixed  max (fluor. or phosph.) and the excitation monochromator is allowed to vary. It is used when the compound to be studied for the first time

• Emission spectrum is recorded when the excitation

monochromator is set at a fixed  (max of absorption) and the emission monochromator is allowed to vary (This is usually used for analytical purposes)

Fluorescent Excitation and Emission Spectra

Excitation Spectrum Observe Emission at single wavelength while scanning excitation wavelengths

Emission Spectrum Observe Emission spectrum while keeping excitation at a single wavelength Sample Spectra Excitation (left), measure luminescence at fixed wavelength while varying excitation wavelength. Fluorescence (middle) and phosphorescence (right), excitation is fixed and record emission as function of wavelength. Electronic Transition Types in Fluorescence

• Seldom to have fluorescence by absorbing Uv at < 250 nm At this range of  deactivation of excited state may take place by predissociation (Rupture of bonds after IC) or dissociation (bond rupture after absorption)

Thus, Fluorescence due to  * -  transition is seldom observed • Fluorescence is limited to the less energetic * -  and * - n transitions depending upon which is less energetic • Fluorescence most commonly arises from transition from the first excited state to one of the vibrational levels of the ground state.

Quantum Efficiency and Transition Type

• f (* - ) > f (* - n) transition e for * -  transition is 100 – 1000 fold greater and this is a measure for transition probability Thus, the lifetime of * -  is shorter than * - n

and kf is larger • The rate constant for ISC is smaller for * -  because the energy difference for singlet/triplet states is larger. That is more energy is required to unpair the electrons of the * excited state. Thus, overlap of the triplet vibrational levels with those of the singlet state is less and the probability of ISC is smaller •In Summary: Fluorescence is more commonly associated with * -  transition state because: 1. * -  transitions possess shorter average lifetime 2. Deactivation processes that compete with fluorescence are less likely to occur

• Fluorescence is favored when 1. Energetic difference between the excited singlet state and triplet state is relatively large 2. Energetic difference between the first excited state and the ground state is sufficiently large to prevent appreciable relaxation to the ground state by radiationless processes Variables that Affect Fluorescence

•Structure and structural Rigidity •Temperature – increased temperature, decreased quantum yield

•Solvent Viscosity – lower viscosity, lower quantum yield

•Fluorescence usually pH-dependent

•Dissolved oxygen reduces emission intensity

•Concentration: Self-quenching due to collisions of excited molecules. Self-absorbance when fluorescence emission and absorbance wavelengths overlap. Fluorescence And Structure

• The most intense and the most useful fluorescence is found in compounds containing aromatic functional groups with low-energy  to  * transition levels. • Compounds containing aliphatic and alicyclic carbonyl structures or highly conjugated double-bond structures may also exhibit fluorescence, • Most unsubstituted aromatic hydrocarbons fluoresce in solution; the quantum efficiency usually increases with the number of rings and their degree of condensation. • The simple heterocyclics, such as pyridine, furan, thiophene, and pyrrole do not exhibit fluorescence; on the other hand, fused ring structures ordinarily do. • With nitrogen heterocyclics, the lowest-energy electronic transition is believed to involve n to * system that rapidly converts to the triplet state and prevents fluorescence. • Fusion of benzene rings to a heterocyclic nucleus, however, results in an increase in the molar absorptivity of the absorption peak. The lifetime of an excited state is shorter in such structures; fluorescence is thus observed for compounds such as quinoline, isoquinoline, and indole.

• Substitution of a carboxylic acid or carbonyl group on an aromatic ring generally inhibits fluorescence. • In these compounds, the energy of the n to * transition is less than that of the  to * transition; as pointed out earlier, the fluorescence yield from the former type of system is ordinarily low

Heavy Atom Effect

• Halogens constituents cause a decrease in fluorescence and the decrease increases with atomic number of halogens • The decrease in fluorescence with increasing atomic number of the halogen is thought to be due in part to the heavy atom effect, which increases the probability for intersystem crossing to the triplet state. • Spin/orbital interactions become large in the presence of heavy atoms and a change in spin is thus more favorable • Predissociation is thought to play an important role in iodobenzene (for example) that has easily ruptured bonds that can absorb the excitation energy following internal conversion. • Substitution of a carboxylic acid or carbonyl group on an aromatic ring generally inhibits fluorescence. In these compounds, the energy of the n,* transition is less than that of the  , * transition. • The electromagnetic fields that are associated with relatively heavy atoms affect electron spins within a molecule more than the fields associated with lighter atoms. • The addition of a relatively heavy atom to a molecule causes excited singlet and triplet electrons to become more energetically similar. That reduces the energetic difference between the singlet and triplet states and increases the probability of intersystem crossing and of phosphorescence. The probability of fluorescence is simultaneously reduced. • The increased phosphorescence and decreased fluorescence with the addition of a heavy atom is the heavy-atom effect. • If the heavy atom is a substituent on the luminescent molecule, it is the internal heavy-atom effect. The external heavy-atom effect occurs when the heavy atom is part of the solution (usually the solvent) in which the luminescent compound is dissolved rather than directly attached to the luminescent molecule.

• The effect that the halides have upon a luminescent molecule is an example of the internal heavy-atom effect. Fluorescence and Structure

• If a heteroatom exists in a luminescent molecule, the transition from the ground state to the first excited singlet state can be an n to * transition. • Electron in a nonbonding orbital that is associated with the heteroatom is excited to a * orbital of the molecule. • Molar absorptivities associated with n to * transitions are usually relatively small (less than 1000) in comparison with absorptivities associated with  to * transitions because nonbonding n orbitals do not overlap with * orbitals as much as bonding  orbitals do. • Consequently, less fluorescence generally is observed following excitation by an n to * transition than is observed following excitation by a  to * transition

Factors That Affect Photoluminescence

• Photoluminescence is favored when the absorption is efficient (high absorptivities). • Fluorescence is favored when 1. the energetic difference between the excited singlet and triplet states is relatively large 2. the energetic difference between the first excited singlet state and the ground state is sufficiently large to prevent appreciable relaxation to the ground state by radiationless processes. • Phosphorescence is favored when 1. the energetic difference between the first excited singlet state and the first excited triplet state is relatively small 2. the probability of a radiationless transition from the triplet state to the ground state is low. • Any physical or chemical factor that can affect any of the transitions can affect the photoluminescence. • These factors include: structural rigidity, temp., solvent, pH, dissolved oxygen. Effects of structural rigidity • Photoluminescent compounds are those compounds in which the energetic levels within the compounds favor de-excitation by emission of uv-visible radiation rather than by loss of rotational or vibrational energy • Fluorescing and phosphorescing compounds usually have a rigid planar structure • the quantum efficiencies for fluorene and biphenyl are nearly

1.0 and 0.2, respectively, under similar conditions CH2 causes more rigidity

• The rigidity of the molecule prevents loss of energy through rotational and vibrational energetic level changes. • Any subsistent on a luminescent molecule that can cause increased vibration or rotation can quench the fluorescence. • The planar structure of fluorescent compounds allows delocalization of the -electrons in the molecule. That in turn increases the chance that luminescence can occur because the electrons can move to the proper location to relax into a lower energy localized orbital. • Organic compounds that contain only single bonds between the carbons do not luminesce owing to lack of absorption in the appropriate region and lack of a planar and rigid structure. • Organic compounds that do luminesce generally consist of rings with alternative single and double bonds between the atoms (conjugated double bonds) in the rings. • The sp2 bonds between the carbons in the rings cause the desired planar structure, and the alternating double bonds give rigidity and provide the -electrons electrons necessary for luminescence.

Temperature Effect

• The quantum efficiency of fluorescence in most molecules decreases with increasing temperature • Due to increased frequency of collisions at elevated temperatures the probability for deactivation by external conversion is improved. Solvent Effect

• A decrease in solvent viscosity also increases the likelihood of external conversion and leads to the decrease in quantum efficiency • The fluorescence of a molecule is decreased by solvents containing heavy atoms or other solutes with such atoms in their structure; carbon tetrabromide and ethyl iodide are examples. • The effect is similar to what occurs when heavy atoms are substituted into fluorescing compounds; orbital spin interactions result in an increase in the rate of triplet formation and a corresponding decrease in fluorescence. • Compounds containing heavy atoms are frequently incorporated into solvents when enhanced phosphorescence is desired. Effect of pH on Fluorescence • Fluorescence of an aromatic compound with acidic ring substituents is usually pH-dependent. • Both  and the emission intensity are likely to be different for the ionized and nonionized forms of the compound. • The data for phenol and aniline shown illustrate this effect. • The changes in emission of compounds of this type arise from the differing number of resonance species that are associated with the acidic and basic forms of the molecules. • The additional resonance forms lead to a more stable first excited state; fluorescence in the ultraviolet region is the consequence. • Thus, close control of pH is required for fluorescence studies

Effect Of Dissolved Oxygen

• The presence of dissolved oxygen often reduces the intensity of fluorescence in a solution.

• This effect may be the result of a photochemically induced oxidation of the fluorescing species. • More commonly, however, the quenching takes place as a consequence of the paramagnetic properties of molecular oxygen, which promotes intersystem crossing and conversion of excited molecules to the triplet state. • Other paramagnetic species also tend to quench fluorescence. Fluorescence and Phosphorescence Instruments

Design luminescence instruments • Filter fluorometers (fluorometers, flurimeters) and filter phosphorimeters

Work at fixed exc and fixed emi

• Spectrofluorometers & spectrophophorimetrs Capable of  scanning. Two monochromators are required Features of Fluorescence and Phosphorescence Instruments

• Almost same components as Uv-Vis instruments • Most of them are double beam configuration to allow compensation of power source fluctuations • Though fluorescence is propagated in all directions the most convenient one is that at right angles to the excitation beam. – At other angles scattering from solutions and cell walls may become appreciable • The use of attenuator helps reducing the power of the reference beam to approximately that of the fluorescent radiation beam Components of Fluorometers and Spectrofluorometers

Sources • A source that is more intense than the tungsten or deuterium lamps employed for Uv-Vis.

• The magnitude of the output signal, and thus the sensitivity, is directly proportional to the source power Po.

• A mercury or xenon arc lamp is commonly employed • The most common source for filter fluorometers is a low-pressure mercury-vapor lamp equipped with a fused silica window.

• This source produces intense lines at 254, 366, 405, 436, 546, 577, 691, and 773 nm. Individual lines can be isolated with suitable absorption or interference filters. • Various types of lasers were also used as excitation sources for photoluminescence measurements. • Tunable dye laser employing a pulsed nitrogen laser as the primary source. Monochromatic radiation between 360 and 650 nm is produced. Filters And Monochromators

• Both interference and absorption filters have been employed in fluorometers.

• Most spectrofluorometers are equipped with grating monochromators. DETECTORS (Transducers)

• Luminescence signals are of low intensity thus, large amplifier gains are required • Photomultiplier tubes • Diode-array detectors • Cooling of detector is used sometimes to improve S/N ration Cells and Cell Compartments

• Both cylindrical and rectangular cells fabricated of glass or silica are employed for fluorescence measurements. • Care must be taken in the design of the cell compartment to reduce the amount of scattered radiation reaching the detector. Baffles are often introduced into the compartment for this purpose. Instrument Designs: Fluorometers

• The source beam is split near the source into a reference beam and a sample beam. • The reference beam is attenuated by the aperture disk so that its intensity is roughly the same as the fluorescence intensity. • Both beams pass through the primary filter, with the reference beam then being reflected to the reference photomultiplier tube. • The sample beam is focused on the sample by a pair of lenses and causes emission of fluorescent radiation. • The emitted radiation passes through a second filter and then is focused on the second photomultiplier tube. • The electrical outputs from the two detectors are fed into a solid state comparator, which computes the ratio of the sample to reference intensities; this ratio serves as the analytical parameter. Nearly all fluorometers (spectrofluorometers) are double-beam systems. Spectrofluorometer Fluorometer or Spectrofluorometer Filter Fluorometer Spectrofluorometers

• spectrofluorometers are capable of providing both excitation and emision pectra. • The optical design of one of these, which utilizes two grating monochromators, is shown above • Radiation from the first monochromator is split, part passing to a reference photomultiplier and part to sample. • The resulting fluorescence radiation, after dispersion by the second monochromator, is detected by a second photomultiplier. • The emission spectra obtained will not necessarily compare well with spectra from other instruments, because the output depends not only upon the intensity of fluorescence but also upon the characteristics of the lamp, detector, and monochromators. • All of these instrument characteristics vary with wavelength and differ from instrument to instrument. • A number of methods have been developed for obtaining a corrected spectrum, which is the true fluorescence spectrum freed from instrumental effects; many of the newer and more sophisticated commercial instruments provide a means for obtaining corrected spectra directly Spectrofluorometer based on Array Transducers

Transducer is a two-dimensional device that sees the excitation and emission radiation in two planes

Observe Fluorescent Excitation and Emission Spectra Simultaneously Phosphorimeters & Spectrophosporimeters • Instruments that have been used for studying phosphorescence are similar in design to the fluorometers and spectrofluorometers just considered, except that two additional components are required 1. Excitation must be gated in time to observe phosphorescence in the absence of fluorescence emission – A device that will alternately irradiate the sample and, after a suitable time delay, measure the intensity of phosphorescence. – The time delay is required to differentiate between long-lived phosphorescence and short lived fluorescence that would originate from the same sample 2. Ordinarily, phosphorescence measurements are performed at liquid nitrogen temperature (-196oc) in order to prevent degradation of the output by collisional deactivation (quenching). • Quenching effects are usually competitive enough to prevent phosphorescence observation at room temperatur • Thus, as shown in the Figure, a Dewar flask with quartz windows is ordinarily a part of a phosphorimeter. • At the temperature used, the analyte exists as a solute in a glass of solid solvent (a common solvent is a mixture of diethylether, pentane, and ethanol). Phosphorimeters

Rotating can and Dewar flask are used. Dewar is placed inside the rotating can that has two slits. As the slit moves into line with excitation beam the sample is excited. The speed of rotation is such that short lived fluorescence is ceased before the slit moves into line with the emission detector so that only phosphoriscence is observed. Applications of Photoluminescence Methods

• Fluorescence and phosphorescence methods are applicable to lower concentration ranges and are among the most sensitive analytical techniques • The enhanced sensitivity arises from the fact that the concentration-related parameter for fluorometry and phosphorimetry can be measured independent of the power of the source Po. • The sensitivity of a fluorometric method can be improved by increasing Po or by further amplifying the fluorescence signal. In spectrophotometry, in conrast, an increase in Po results in a proportionate change in P and therefore fails to affect A. • The precision and accuracy of photoluminescence methods are usually poorer than those of spectrophotometric procedures by a factor of perhaps two to five. • Generally, phosphorescence methods are less precise than their fluorescence counterparts. Fluorometric Determination of Inorganic Species

• Inorganic fluorometric methods are of two types. 1. Direct methods involve the formation of a fluorescent chelate and the measurement of its emission. 2. A second group is based upon the diminution of fluorescence resulting from the quenching action of the substance being determined. • The latter technique has been most widely used for anion analysis. Cations that form Fluorescing Chelates

Two factors greatly limit the number of transition-metal ions that form fluorescing chelates. 1. Many of these ions are paramagnetic; this property increases the rate of intersystem crossing to the triplet state. In solution most T states lose all of their electronic energy by collisional deactivation or by rapid conversion to their So state without emitting a photon. Thus paramagnetic metal ions (Fe3+, Co2+, Ni2+ and Cu2+) quench the fluorescence of their chelates. 2. Transition-metal complexes are characterized by many closely spaced energy levels, which enhance the likelihood of deactivation by internal conversion. • Nontransition-metal ions are less susceptible to the foregoing deactivation processes; it is for these elements that the principal inorganic applications of fluorometry are to be found. • It is noteworthy that nontransition-metal cations are generally colorless and tend to form chelates that are also without color. Thus, fluorometry often complements spectrophotometry. FLUOROMETRIC REAGENTS

• The most successful fluorometric reagents for cation analyses have aromatic structures with two or more donor functional groups that permit chelate formation with the metal ion.

Fluorometric Determination of Organic Species

• They are used for a wide variety of organic compounds, enzymes and co•enzymes, medicinal agents, plant products, steroids and vitamins. • It is important for Food products, pharmaceuticals, clinical samples, and natural products. Applications of Phosphorimetric Methods

• Phosphorescence and fluorescence methods tend to be complementary, because strongly fluorescing compounds exhibit weak phosphorescence and vice versa. • " For example, among condensed-ring aromatic hydrocarbons, those containing heavier atoms such as halogens or sulfur often phosphoresce strongly; on the other hand, the same compounds in the absence of the heavy atom tend to exhibit fluorescence rather than phosphorescence. • Phosphorimetry has been used for determination of a variety of organic and biochemical species including such substances as nucleic acids, amino acids, pyrine and pyrimidine, enzymes, petroleum hydrocarbons, and pesticides. • However, perhaps because of the need for low temperatures and the generally poorer precision of phosphorescence measurements, the method has not found as widespread use as has fluorometry. • On the other hand, the potentially greater selectivity of phosphorescence procedures is attractive. • Development of phosphorimetric methods that can be carried out at room temperature took two directions. 1. The first based upon the enhanced phosphorescence that is observed for compounds adsorbed on solid surfaces, such as filter paper. In these applications, a solution of the analyte is dispersed on the solid, and the solvent is evaporated. The phosphorescence of the surface is then measured. Presumably the rigid matrix minimizes deactivation of the triplet state by external and internal conversions. • The second is based on room-temperature method that involves solubilizing the analyte in detergent micelles in the presence of heavy metal ions. Lifetime Measurements

• The measurement of luminescence lifetimes was initially restricted to phosphorescent systems, where decay times were long enough to permit the easy measurement of emitted intensity as a function of time. • For analytical work, lifetime measurements enhance the selectivity of luminescence methods, because they permit the analysis of mixtures containing two or more luminescent species with different decay rates.

CHEMILUMINESCENCE

• The number of chemical reactions that produce chemiluminescence is small, thus limiting the procedure to a relatively small number of species. • Nevertheless, some of the compounds that do react to give chemiluminescence are important components of the environment. • Chemiluminescence is produced when a chemical reaction yields an electronically excited species, which emits light as it returns to its ground state. • Chemiluminescence reactions are encountered in a number of biological systems, where the process is often termed bioluminescence. • Examples of species that exhibit bioluminescence include the firefly, the sea pansy and certain jellyfish, bacteria, protozoa, and crustacea. • Several relatively simple organic compounds also are capable of exhibiting chemiluminescence. The simplest type of reaction of such compounds to produce chemiluminescence can be formulated as where C* represents the excited state of the species C. Here, the luminescence spectrum is that of the reaction product C Measurement of Chemiluminescence

• The instrumentation may consist of only a suitable reaction vessel and a photomultiplier tube. • Generally, no wavelength-restricting device is necessary, because the only source of radiation is the chemical reaction between the analyte and reagent. • Several instrument manufacturers offer chemiluminescence photometers. • The typical signal from a chemiluminescence experiment as a function of time rises rapidly to a maximum as mixing of reagent and analyte is complete; then more or less exponential decay of signal follows. • Usually, the signal is integrated for a fixed period of time and compared with standards treated in an identical way. • Often a linear relationship between signal and ,concentration is observed over a concentration range of several orders of magnitude.

A good example of chemiluminescence is the determination of nitrogen monoxide:

NO + O3 NO2* + O2

NO2* NO2 + hv (= 600 to 2800 nm)

spectral distribution of radiation emitted by the above reaction

Analytical Applications of Chemiluminescence

• Chemiluminescence methods are generally highly sensitive, because low light levels are readily monitored in the absence of noise. • Furthermore, radiation attenuation by a filter or a monochromator is avoided. • Detection limits are usually determined not by detector sensitivity but rather by reagent purity. They are in the ranges of ppb levels. Analysis of Gases

Determination of nitrogen monoxide • Ozone from an electrogenerator and the atmospheric sample are drawn continuously into a reaction vessel • Luminescence radiation is monitored by a photomultiplier tube. • A linear response is reported for nitrogen monoxide concentrations of 1 ppb to 10,000 ppm. • Instrumentally, for determination of nitrogen in solid or liquid materials containing 0.1 to 30% nitrogen. The samples are pyrolyzed in an oxygen atmosphere under conditions whereby the nitrogen is converted quantitatively to nitrogen monoxide; the latter is then measured by the method just described. Analysis of Inorganic Species in the Liquid Phase

• Many of the analyses carried out in the liquid phase make use of organic chemiluminescing substances containing the functional group

• These reagents react with oxygen, hydrogen peroxide, and many other strong oxidizing agents to produce a chemiluminescing oxidation product. • Luminol is an example of these compounds. Its reaction with strong oxidants, such as oxygen, hydrogen peroxide, hypochlorite ion, and permanganate ion, in the presence of strong base is given below. • Often a catalyst is required for this reaction to proceed at a useful rate. • The emission produced matches the fluorescence spectrum of the product, 3-aminophthalate anion; the chemiluminescence appears blue and is centered around 425 nm.