Instrumental Analysis

(Molecular Luminescence Spectroscopy)

Young Gyu Jeong

Department of Organic Materials Engineering
Chungnam National University

Chap 15.
Molecular Luminescence Spectrometry

 Theory of Fluorescence and Phosphorescence

 Instruments for Measuring Fluorescence and Phosphorescence

 Applications of Photoluminescence Methods

 Chemiluminescence

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  Spectroscopy
 the study of the interaction between matter and radiated energy

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Chap 15.
Molecular Luminescence Spectrometry

 Luminescence method
 Molecules of the analyte are excited to produce a species whose emission spectrum provides
information for qualitative or quantitative analysis
 Fluorescence, phosphorescence, and chemiluminescence
 One of the most attractive features is its inherent sensitivity, with detection limits that are often one to
three orders of magnitude lower than those encountered in absorption spectroscopy
 Generally, this method is less widely applicable for quantitative analyses than are absorption methods
because many more species absorb UV and visible radiation than exhibit photoluminescence
 Photoluminescence (fluorescence and phosphorescence)
 Fluorescence and phosphorescence are alike in that excitation is brought about by absorption of
photons
 In most instances, photoluminescence occurs at wavelengths longer than that of the excitation radiation
 The excited states involved in fluorescence are short-lived (<10-5 s)
 Chemiluminescence
 It is based on the emission of radiation by an excited species formed during a chemical reaction
 In some instances, the excited species is the product of a reaction between the analyte and a suitable
reagent (usually a strong oxidant such as ozone or hydrogen peroxide)
 In other instances, the analyte is not directly involved in the chemiluminescence. Instead, the analyte
inhibits or has a catalytic effect on a chemiluminescence reaction

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 Theory of Fluorescence and Phosphorescence

 Fluorescence
 Atomic fluorescence spectroscopy (AFS)  Chapter 9
 For vaporized sodium atoms:
 3s electrons can be exited to the 3p state by absorption of radiation of wavelengths 589.6
and 589.0 nm
 After about 10-8 s, the electrons return to the ground state and in so doing emit radiation of
the same two wavelengths in all directions
 Resonance radiation or resonance fluorescence
 Molecular fluorescence (or phosphorescence) bands center at wavelengths longer than the resonance
line. This shift toward longer wavelengths is termed the Stokes shift

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Theory of Fluorescence and Phosphorescence

Excited states producing fluorescence and phosphorescence
 The difference between fluorescence and phosphorescence phenomena is based on the electron spin
and the difference singlet and triplet excited state

 Electron spin
 The Pauli exclusion principle
 No two electrons in an atom can have the same set of four quantum numbers
 No more than two electrons occupy an orbital and furthermore the two have opposed spin states
 Under this circumstance, the spins are said to be paired
 Diamagnetic
 Because of spin pairing, most molecules exhibit no net magnetic field and thus they are neither
attracted nor repelled by static magnetic fields
 Paramagnetic
 Free radicals, which contain unpaired electrons, have a magnetic moment and consequently are
attracted by a magnetic field

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 Theory of Fluorescence and Phosphorescence

• 주 양자수 (principal quantum number)

• 방위 양자수 (azimuthal quantum number)
• 자기 양자수 (magnetic quantum number)
• 자기 스핀 양자수 (magnetic spin quantum number)

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Theory of Fluorescence and Phosphorescence

Excited states producing fluorescence and phosphorescence
 Singlet and triplet excited states
 Singlet state
 A molecular electronic state in which all electron spins are paired
 No splitting of electronic energy levels occurs when the molecule is exposed to a magnetic field
 Doublet state
 The ground state for a free radical is a doublet state because there are two possible orientations
for the odd electron in a magnetic field, and each imparts slightly different energies to the system
 Triplet state
 When one of a pair of electrons of a molecule is excited to a higher energy level, a singlet or a
triplet state is formed
Examples of atoms in singlet,
doublet, and triplet states.

Figure 15-1 Electronic spin states of molecules.

 A molecule in a triplet state: paramagnetic, a molecule in a singlet state: diamagnetic

 Transition possibility: singlet-to-triplet transition << singlet-to-singlet transition
 Average lifetime: 10-5 to several seconds of an excited triplet state and ~10-8 s for an excited singlet state
 Intensity of absorption bands: singlet-to-triplet transition << singlet-to-singlet transition
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 Theory of Fluorescence and Phosphorescence
Excited states producing fluorescence and phosphorescence
 Energy-level diagrams for photoluminescent molecules

Figure 15-2 Partial energy-level

diagram for a photoluminescent
system.
Resonance fluorescence
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Theory of Fluorescence and Phosphorescence

Rates of absorption and emission
 The rate of photon absorption: 10-14 ~ 10-15 s

 The rate of fluorescence emission: 10-5 ~ 10-10 s

 The rate of phosphorescence emission: 10-4 ~ 10 s or more

 The average rate of a triplet-to-singlet transition is less than that of a corresponding singlet-to-
singlet transition

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 Theory of Fluorescence and Phosphorescence
Deactivation processes
 An excited molecule can return to its ground state by a combination of several mechanistic steps
 The emission of a photon of radiation: fluorescence and phosphorescence
 Radiationless process: vibrational relaxation, internal conversion, external conversion, intersystem
crossing

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Theory of Fluorescence and Phosphorescence

Deactivation processes
 Vibrational relaxation
 Collisions between molecules of the excited species and those of the solvent lead to rapid energy
transfer with a minuscule increase in temperature of the solvent
 The average lifetime of a vibrationally excited molecules is 10-12 s or less
 Internal conversion
 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)
 A crossover between two states of the same multiplicity (singlet-singlet or triplet-triplet)
 Internal conversion through overlapping vibrational levels is usually more probable than the loss of
energy by fluorescent from a higher excited state
 Ex) quinine: regardless of which wavelength is used to excite the molecule, the wavelength of
maximum emission is 450 nm (Figure 15.3)
 Internal conversion may result in the phenomenon of predissociation :
 The electron moves from a higher electronic state to an upper vibrational level of a lower
electronic state in which the vibrational energy is great enough to cause rupture of a bond,
especially for large molecules
 Rupture of the bond can occur as a consequence of absorption by the chromophore followed by
internal conversion of the electronic energy to vibrational energy associated with the weak bond

 Ref: Dissociation (in this case, no internal conversion is involved)

 The absorbed radiation directly excites the electron of a chromophore to a sufficiently high
vibrational level to cause rupture of the chromophoric bond
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 Theory of Fluorescence and Phosphorescence
Deactivation processes

Quinine

Figure 15-3 Fluorescence excitation and emission spectra for a solution of quinine.

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Theory of Fluorescence and Phosphorescence

Deactivation processes
 External conversion (collisional quenching)
 Deactivation of an excited electronic state may involve interaction and energy transfer between the
excited molecule and the solvent or other solutes

 Intersystem crossing
 A process in which there is a crossover between electronic states of different multiplicity
 The most common process is from the singlet state to the triplet state (S1  T1)
 The probability of intersystem crossing is enhanced if the vibrational levels of the two states overlap
 This is most common in molecules that contain heavy atoms, such as iodine or bromine (Heavy-atom
effect)

 Phosphorescence
 After intersystem crossing to the triplet state, further deactivation can occur either by internal or external
conversion or by phosphorescence
 The average lifetime of the excited triplet state: 10-4 ~ 10 s or more
 External and internal conversions compete to successfully with phosphorescence that this kind of
emission is ordinarily observed only at low temperatures in highly viscous media

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 Theory of Fluorescence and Phosphorescence
Deactivation processes
 Excited electrons can undergo intersystem crossing to a degenerate state with a different spin
multiplicity.

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Theory of Fluorescence and Phosphorescence

Variables affecting fluorescence and phosphorescence
 Molecular structure and chemical environment
 These factors influence whether a substance will or will not luminesce
 They also determine the intensity of emission when luminescence does occur
 Quantum yield or quantum efficiency for fluorescence or phosphorescence
 The ratio of the number of molecules that luminescence to the total number of excited molecules
 For a highly fluorescent molecule (such as fluorescein), the quantum efficiency ~ 1
kf

kf  ki  kec  kic  kpd  kd

fluorescence external conversion predissociation

intersystem crossing internal conversion dissociation

 Transition types in fluorescence

 Fluorescence seldom results from absorption of UV radiation of wavelengths shorter than 250 nm
(~140 kcal/mol) because such radiation is sufficiently energetic to cause deactivation of the excited
states by predissociation or dissociation
 Fluorescence is confined to the less energetic    and   n processes
 The most common fluorescence arises from a transition from the lowest vibrational level of the first
excited electronic ground state

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 Theory of Fluorescence and Phosphorescence
Variables affecting fluorescence and phosphorescence
 Quantum efficiency and transition type
 Fluorescence is more commonly found in compounds in which the lowest transition is of a    type
than in compounds in which the lowest energy transition is of the n   type
 The great quantum efficiency associated with the ,  state
 The molar absorptivity of a    transition is ordinarily 100- to 1000-fold greater than for an n
  process
 The inherent lifetime associated with the , states (10-7 to 10-9 s) is shorter compared with that
for the n, states (10-5 to 10-7 s)
 kf of the , states is larger than that of the n, states
 Fluorescence and structure
 The most intense and the most useful fluorescence is found in compounds containing aromatic
functional groups with low-energy    transitions
 Most unsubstituted aromatic hydrocarbons fluoresce in solution, the quantum efficiency usually
increasing with the number of rings and their degree of condensation

Simple heterocyclics  No fluorescence Fused ring structures  Fluorescence

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Theory of Fluorescence and Phosphorescence

Variables affecting fluorescence and phosphorescence
 Substitution on the benzene ring causes shifts in the wavelength of absorption maxima and
corresponding changes in the fluorescence emission

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 Theory of Fluorescence and Phosphorescence
Variables affecting fluorescence and phosphorescence
 Effect of structural rigidity
 Fluorescence is particularly favored in molecules with rigid structures:

Quantum efficiency: ~1.0 ~0.2

 Organic chelating agents complexed with a metal ion increase the intensity of fluorescence

8-hydroxyquinoline:

 Lack of rigidity in a molecule probably causes an enhanced internal conversion rate (kic) and a
consequent increase in the likelihood for radiationless deactivation

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Theory of Fluorescence and Phosphorescence

Variables affecting fluorescence and phosphorescence
 Temperature and solvent effects
 The quantum efficiency of fluorescence in most molecules decreases with increasing temperature
because the increased frequency of collisions at elevated temperatures improves the probability for
deactivation by external conversion
 A decrease in solvent viscosity also increases the likelihood of external conversion and leads to the
same result
 The fluorescence of a molecule is decreased by solvents containing heavy atoms or other solutes with
such atoms in their structure (ex: carbon tetrabromide and ethyl idodide)

 Effect of pH on fluorescence
 The fluorescence of an aromatic compound with acidic or basic ring substituents is usually pH
dependent  ex) phenol and aniline in Table 15-1

The additional resonance forms lead

to a more stable first excited state 
fluorescence in the UV region is the
consequence

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 Theory of Fluorescence and Phosphorescence
Variables affecting fluorescence and phosphorescence
 Effect of dissolved oxygen
 The presence of dissolved oxygen often reduces the intensity of fluorescence in a solution
 The effect may be the result of a photochemically induced oxidation of the fluorescing species
 Effect of concentration on fluorescence intensity
 The power of fluorescence emission F is proportional to the radiant power of the excitation beam that is
absorbed by the system

F  K   P0  P 
P
 10 bc
P0
F  K P0 1  10 bc 
  2.303 bc   2.303 bc  
2 3

F  K P0  2.303 bc    Maclaurin series

 2! 3! 
F  2.3K  bcP0
Provided that 2.303 bc  A  0.05
F  Kc
 Negative departures from linearity at high concentration
 Primary and secondary absorption: inner filter effect (absorption effect)

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Theory of Fluorescence and Phosphorescence

Emission and excitation spectra
 Three types of photoluminescence spectra of phenanthrene
Relative intensity

Wavelength, nm

Excitation spectrum Fluorescence spectrum Phosphorescence spectrum

(absorption)

Figure 15-5 Spectra of phenanthrene.

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 Theory of Fluorescence and Phosphorescence
Emission and excitation spectra
 The total luminescence spectrum

Scattered
light

Intensity
Intensity

Emission wavelength, nm Excitation wavelength, nm

Figure 15-6 Total luminescence spectra. (a) the total fluorescence

spectrum of a mixture of anthracene and ovalene is shown as a three-
dimensional plot. (b) the total fluorescence spectrum of 8-
hydroxybenzo[a]pyrene is shown as a contour plot.

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Theory of Fluorescence and Phosphorescence

Emission and excitation spectra
 The total luminescence spectrum

Excitation
Synchronous signal
Fluorescence signal

spectrum
Emission
spectrum

Wavelength, nm Wavelength, nm

Figure 15-7 Synchronous fluorescence spectra.

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Instruments for Measuring Fluorescence and Phosphorescence

 Fluorometers and spectrofluorometers

Figure 15-8 Components of a fluorometer or

superfluorometer.

Figure 15-9 Fluorescence spectra for 1 ppm

anthracence in alcohol: (a) excitation spectrum; (b)
emission spectrum.
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Instruments for Measuring Fluorescence and Phosphorescence

Components of fluorometers and spectrofluorometers
 Source
 More intense sources are used in luminescence methods than the tungsten or deuterium lamps used in
absorption measurements
 For filter fluorometers, the most common source is a low-pressure mercury vapor lamp (produces
useful lines for exciting fluorescence at 254, 302, 313, 546, 578, 691, and 773 nm) equipped with a
fused silica window
 For spectrofluorometers, a source of continuum radiation is required, a 75- to 450-W high-pressure
xenon arc lamp is commonly employed  a continuum radiation of 300~1300 nm wavelength

 Lasers
 Since the 1970s, various types of lasers have also been used as excitation sources for
photoluminescence measurements.
 Tunable dye lasers or a Nd-YAG laser
 Advantages of laser sources
 When samples are very small, as in micropore chromatography and capillary electrophoresis
where the amount of sample is a microliter or less
 In remote sensing, as in fluorometric detection of hydroxyl radicals in the atmosphere or of
chlorophyll in bodies of water, where the collimated nature of laser beams is vital
 When highly monochromatic excitation is needed to minimize the effects of fluorescing
interferences

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Instruments for Measuring Fluorescence and Phosphorescence
Components of fluorometers and spectrofluorometers
 Filters and monochromators
 Interference and absorption filers have been used in fluorometers for wavelength selection of both the
excitation beam and the resulting fluorescence radiation
 Spectrofluorometers are equipped with at least one and often two grating monochromators

 Transducers
 Photomultiplier tubes are the most common transducers in sensitive fluorescence instruments
 These are operated in the photon-counting mode to give improved signal-to-noise ratios
 Charge-transfer devices, such as charge-coupled devices (CCDs), are also used for spectrofluorometry

 Cells and cell compartments

 Both cylindrical and rectangular cells fabricated of glass or silica are employed
 Cell compartments should be designed to reduce the amount of scattered radiation reaching the
detector

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Instruments for Measuring Fluorescence and Phosphorescence

Instrument designs
 Fluorometers

Figure 15-10 A typical fluorometer.

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Instruments for Measuring Fluorescence and Phosphorescence
Instrument designs
 Spectrofluorometers

Figure 15-11 A spectrofluorometer.

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Instruments for Measuring Fluorescence and Phosphorescence

Instrument designs
 Spectrofluorometers based on array detectors
Intensity

Figure 15-12 Three-dimensional spectrofluorometer. (a) schematic of an optical system for obtaining total
luminescence spectra with a CCD detector. (b) excitation and emission spectra of a hypothetical
compound. (c) total luminescence spectrum of compound in (b).
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Instruments for Measuring Fluorescence and Phosphorescence
Instrument designs
 Fiber-optic fluorescence sensors
 Radiation from a laser source travels through an optical fiber
and excites fluorescence in sample solutions. Fluorescence
emission then travels back through the same fiber to a detector
for measurement
 Phosphorimeters
 Fluorometers or spectrofluorometers + two additional
components
 The first is a device that alternately irradiates the sample
and, after a suitable time delay, measures the intensity of
phosphorescence
 The second component is needed because
phosphorescence measurements are usually performed
at liquid nitrogen temperature in a rigid medium to
minimize collisional deactivation of the long-lived triplet
state

Figure 15-13 Dewar flask and cell for low-temperature

phosphorescence measurements. The optical path
traverses the unsilvered part of the flask.

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Instruments for Measuring Fluorescence and Phosphorescence

Correction and compensation schemes
 Source compensation
 The luminescence instruments (Figures 15-10 and 15-11) monitor the Rhodamine B
source intensity via a reference photomultiplier.
 Most commonly, the ratio of the sample luminescence signal to the signal
from the reference detector is continuously obtained. This can compensate
for source intensity fluctuations and drift
 Both double-beam-in-space and double-beam-in-time designs are
employed
 Corrected excitation spectra
 To correct the wavelength dependence of the source or the efficiencies of
the optical components and excitation wavelength selector, a quantum
counter has been used
 The quantum counter: a reference cell filled with a concentrated
fluorophore of high quantum efficiency, such as Rhodamine B
 3- to 8-g/L Rhodamine B in glycerol essentially absorbs all incident light
from 220 to 600 nm

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Instruments for Measuring Fluorescence and Phosphorescence
Instrument standardization
 Because of variation in source intensity, transducer sensitivity, and other instrumental variables, it is
impossible to obtain with a given fluorometer or spectrofluorometer exactly the same reading for a
solution of a set of solutions from day to day
 Standardization is carried out with a standard solution of a stable fluorophore.
 The most common reagent is a standard solution of quinine sulfate having a concentration of ~10-5 M
(excitation at 350 nm and emission at 450 nm).

Corrected
Uncorrected
Relative Intensity

Excitation Emission

Wavelength, nm
Figure 15-14 Corrected and uncorrected spectra for quinine sulfate in 0.2 M H2SO4.
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Applications of Photoluminescence Methods

 Fluorescence and phosphorescence methods

 Inherently lower limits of detection than absorption based spectrophotometric measurements
 The most sensitive analytical techniques
 The enhanced sensitivity arises from the concentration-related parameter for fluorometry and
phosophorimetry F being directly proportional to the source radiant power P0

P P0
F  Kc  10 bc A   log10 T  log   bc
P0 P

 The sensitivity of a fluorometric method can be improved by increasing P0 or by further amplifying the
fluorescence signal
 On the other hand, the precision and accuracy of photoluminescence methods is usually poorer than
spectrophotometric procedures by a factor of 2 to 5
 The precision of photoluminescence methods is often limited by source flicker noise and drift
 The accuracy is often limited by concomitants, or particles, in the sample that cause additional
fluorescence and scattering or that quench the analyte fluorescence

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 Applications of Photoluminescence Methods
Fluorometric determination of inorganic species
 Inorganic fluorometric methods
 Direct methods involve the formation of a fluorescing chelate and the measurement of its emission
 A second group of methods is based on the decrease in fluorescence emission because of the
quenching action of the substance being determined
 most widely used for anion analysis
 Cations forming fluorescing chelates
 Two factors greatly limit the number of transition-metal ions that form fluorescing chelates
 Many of these ions are paramagnetic, which increases the rate of intersystem crossing to the
triplet state  fluorescence is deactivated, but phosphorescence may be observed.
 Transition-metal complexes are characterized by many closely spaced energy levels, which
enhance the likelihood of deactivation by internal conversion
 Flurometric reagents
 Selected fluorometric reagents for cation analyses: Figure 15-15 and Table 15-2

Figure 15-15 Some fluorometric chelating agents for metal cations.

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Applications of Photoluminescence Methods

Fluorometric determination of inorganic species

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 Applications of Photoluminescence Methods
Methods for organic and biochemical species
 The number of applications of fluorometric methods to organic chemistry
 “Fluorescence Spectroscopy of Some Organic Compounds” by Dean
 More than 20 entries, including such diverse compounds as adenine, anthranilic acid, aromatic
polycyclic hydrocarbons, cysteine, guanine, isoniazid, naphthols, nerve gases sarin and tabun,
proteins, salicylic acid, etc.
 Many medicinal agents including adrenaline, morphine, penicillin, phenobarbital, procaine, reserpine,
and lysergic acid diethylamide (LSD)
 Food products
 Pharmaceuticals
 Clinical samples
 Natural products

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Applications of Photoluminescence Methods

Phosphorimetric methods
 Phosphorescence and fluorescence methods tend to be complementary because strongly fluorescing
compounds exhibit weak phosphorescence and vice versa
 Among condensed-ring aromatic hydrocarbons, those containing heavier atoms such as halogens or
sulfur often phosphoresce strongly
 It has been used for determination of a variety of organic and biochemical species including nucleic
acids, amino acids, pyrine and pyrimidine, enzymes, petroleum hydrocarbons, and pesticides
 This method has not found as widespread use as fluorometry, perhaps because of the need for low
temperatures and the generally poorer precision of phosphorescence measurements

Fluorescence detection in liquid chromatography

 Photoluminescence measurements provide an important method for detecting and determining

components of a sample as they elute from a chromatographic or capillary electrophoresis column

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 Applications of Photoluminescence Methods
Lifetime measurements

Intensity

Time, ns
Figure. Curves of fluorescence lifetime. A: excitation
pulse, B: decay curve during measurement, C: corrected
decay curve.

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Applications of Photoluminescence Methods

Fluorescence imaging methods

Figure 15-16. Calcium transients in cerebellar Purkinje cell.

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 Chemiluminescence
The chemiluminescence phenomenon
 Chemiluminescence
 Chemiluminescence of organic compounds:

A  B  C*  D
C *  C  hv

 Emission intensity of chemiluminescence:

dC dC
I CL  CL  EX EM
dt dt

Quantum efficiency
Emission quantum efficiency
0.01~0.2 (photons per excited state)

Excitation quantum efficiency

(excited states per molecule reacted)

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Chemiluminescence
Measurement of chemiluminescence

Figure 15-17. Chemiluminescence emission intensity as a function of

time after mixing reagents.

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 Chemiluminescence
Analytical applications of chemiluminescence
 Analysis of gases
 Highly sensitive methods for determining atmospheric pollutants such as ozone, oxides of nitrogen, and
sulfur compounds
 Determination of nitrogen oxide:
NO  O3  NO*2  O 2
NO*2  NO 2  hv (  600 ~ 2800 nm)
 Determination of atmospheric sulfur compounds such as H2S, SO2, and mercaptans:

4H 2  2SO 2  S*2 +4H 2O

S*2  S2  hv (Blue emission of 384 and 394 nm)

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Chemiluminescence
Analytical applications of chemiluminescence
 Analysis for inorganic species in the liquid phase
 Organic chemiluminescing substances containing the function group of

 These reagents react with oxygen, hydrogen peroxide, and many other strong oxidizing agents to
produce a chemiluminescing oxidation product

Luminol

3-aminophthalate anion

(Blue emission centered ~425 nm)

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 Chemiluminescence
Analytical applications of chemiluminescence
 Determination of organic species
 Glucose, cholesterol, choline, uric acid, amino acids, aldehyde, and lactate

 It is practice to precede a chemiluminescence step by an enzyme reaction for which the desired analyte
is the substance and one of the products is detected by chemiluminescence
 Biosensor designs

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