Principles and Operations

of Fluorescence
Spectroscopy
Topics
• Theory on Molecular Fluorescence
• Fluorescence Instrumentation
• Applications of Fluorescence Methods
• Molecular Phosphorescence Spectroscopy
 The photograph is an immunofluorescent light
micrograph of HeLa cancer cells. The cell in the
center of the photo is in the prophase stage of
mitotic cell division.
The chromosomes have condensed before dividing to form two nuclei. The
cells are stained to reveal actin microfilaments and microtubules of the
cytoskeleton, which appear as the filamentary structures surrounding the cell
nuclei. The nuclei of the cells are visualized by exposing the cells to structure-
specific fluorescent antibodies, prepared by covalently attaching ordinary
antibodies to fluorescent molecules. The antibodies collect in the nuclei so
that when they are exposed to UV radiation, they glow as shown in the photo.
Introduction
Three related types of optical methods:
• molecular fluorescence,
• phosphorescence, and
• chemiluminescence.
Molecules of the analyte are excited to produce a species
whose emission spectrum provides information for qualitative
or quantitative analysis.
The methods are known collectively as molecular
luminescence procedures.
What is fluorescence?
photoluminescence process in which
atoms or molecules are excited by
absorption of electromagnetic radiation.
The excited species then relax to the
ground state, giving up their excess
energy as photons. One of the most
attractive features of molecular
fluorescence is its inherent sensitivity,
which is often one to three orders of
magnitude better than absorption
spectroscopy
Molecular Luminescence
Fluorescence and phosphorescence
• Both have excitation brought about by absorption of photons.
• The two phenomena are often referred to by the more general term
photoluminescence.
Fluorescence differs from phosphorescence in that the electronic energy
transitions responsible for fluorescence do not involve a change in electron spin.
Excited states involved in fluorescence are short-lived (<10-5s).
Phosphorescence accompanies a change in electron spin and the lifetimes of
the excited states are much longer often on the order of seconds or even
minutes.
Photoluminescence occurs at wavelengths longer than
that of the excitation radiation.
Molecular Luminescence
Chemiluminescence
• based on the emission of radiation by an excited species formed
during a chemical reaction.
• 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).
• The result is an emission spectrum characteristic of the oxidation
product of the analyte or the reagent rather than the analyte itself.
In other instances, the analyte is not directly involved in the
chemiluminescence reaction. Instead, the analyte inhibits or
has a catalytic effect on a chemiluminescence reaction.
Molecular Luminescence
Luminescence methods
• inherent sensitivity, with detection limits that are often one
to three orders of magnitude lower than those encountered
in absorption spectroscopy.
• selected species under controlled conditions, single
molecules have been detected by fluorescence
spectroscopy.
• has large linear concentration ranges, which also are often
significantly greater than those encountered in absorption
methods.
Theory of Molecular Fluorescence and Phosphorescence

photoluminescence emission is
measured at right angles to the incident
beam to avoid measuring the incident
radiation (recall Figure 23-1b). The short-
lived emission that occurs is called
fluorescence, while luminescence that is
much longer lasting is called
phosphorescence. The transitions giving
rise to fluorescence and
phosphorescence are radiative
transitions.
• Fluorescence occurs in simple as well as in complex gaseous,
liquid, and solid chemical systems.
• When the absorbed radiation is reemitted without a change
in frequency, it is termed resonance radiation or resonance
fluorescence.
• Many molecular species also exhibit resonance
fluorescence.
• Molecular fluorescence (or phosphorescence) hands center
at wavelengths longer than the resonance line. This shift
toward longer wavelengths is termed the Stokes shift.
Excited States Producing Fluorescence and Phosphorescence

Electron Spin – Pauli exclusion principle

• The properties of a molecule in the excited triplet state differ significantly from
those of the excited singlet state.
– a molecule is paramagnetic in the triplet state and diamagnetic in
the singlet.
– a singlet-to-triplet transition (or the reverse), which also involves a change in
electronic state, is a significantly less probable event than the corresponding
singlet-to-singlet transition.
– the average lifetime of an excited triplet state may range from
10-4s to several seconds, compared with an average lifetime of ~10-8s for an
excited singlet state.
• An excited triplet state can be populated from an excited singlet state of certain
molecules. Phosphorescence emission is often a result of such a process.
Energy-Level Diagrams for Photoluminescent Molecules
• Next slide is a partial energy-level diagram, called a Jablonski diagram for a typical
photoluminescent molecule.
• The lowest heavy horizontal line represents the ground-state energy of the
molecule. which is normally a singlet state, and is labeled S0
• At room temperature. this state represents the energies of most of the molecules
in a solution.
• The upper heavy lines are energy levels for the ground vibrational states of three
excited electronic states.
• The two lines on the left represent the first (S1) and second (S2) electronic singlet
states.
• The one on the right (T1) represents the energy of the first electronic triplet state.
• The energy of the first excited triplet state is lower than the energy of the
corresponding singlet state.
Jablonski Diagram
Deactivation Process
• An excited molecule can return to its ground state by a
combination of several mechanistic steps.
• As shown by the straight, downward pointing, vertical
arrows in the figure, two of these steps, fluorescence and
phosphorescence involve the emission of a photon of
radiation.
• The other deactivation steps, indicated by wavy arrows, are
radiationless processes.
– Vibrational relaxation
– Internal conversion
Absorption Very Fast 10
-14
– 10
-15
sec .
Vibrational Relaxation
• Excited molecule rapidly
transfers excess vibrational
energy to the solvent/medium
through collisions.
• Molecule quickly relaxes into
the ground vibrational level in
the excited electronic level.
• Non-radiative process
• 10-11 – 10-10 sec.
Internal Conversion
•Transfers into a lower energy
electronic state of the same multiplicity
without emission of a photon.
• Favored when there is an overlap of
the electronic states’ potential energy
curves.
• Non-radiative process (minimal
energy change)
• ~10-12 s between excited electronic
states.
Fluorescence
• Radiative transition between
electronic states with the same
multiplicity.
• Almost always a progression
from the ground vibrational level
of the 1st excited electronic state.
• 10-10 – 10-5 sec.
• Occurs at a lower energy
than excitation.
 Intersystem Crossing
• Similar to internal conversion
(non radiative) except that it
occurs between electronic states
with different multiplicities.
• Slower than internal conversion.
• More likely in molecules
containing heavy nuclei.
• More likely in the presence of
paramagnetic compounds.
Variables Affecting Fluorescence and
Phosphorescence
Both molecular structure and chemical environment influence whether a substance
will or will not luminesce.
These factors also determine the intensity of emission when luminescence does occur.
• Quantum yield
• Transition types in Fluorescence
• Quantum Efficiency and Transition Type
• Fluorescence and Structure
• Effect of Structural Rigidity
• Effect of pH on Fluorescence
• Effect of Concentration on Fluorescence Intensity
• Dynamic Quenching
• Other Types of Quenching
Quantum Yield

the fluorescence quantum yield f for a compound is determined

by the relative rate constants kx for the processes by which the
lowest excited singlet state is deactivated. These processes are
fluorescence (kf), intersystem crossing (ki), external conversion
(kec), internal conversion (kic), predissociation (kpd), and dissociation
(kd). We can express these relationships by using equation 15-1:
Transition Types in Fluorescence
Quantum Efficiency and Transition Type
Fluorescent Species
• All absorbing molecules have the potential to fluoresce, but most
compounds do not because their structure allows radiationless pathways
for relaxation to occur at a greater rate than fluorescence emission.
• The quantum yield of molecular fluorescence is simply the ratio of the
number of molecules that fluoresce to the total number of excited
molecules, or the ratio of photons emitted to photons absorbed.
– Highly fluorescent molecules, such as fluorescein, have quantum
efficiencies that approach unity under some conditions.
– Species that do not fluoresce or that show very weak fluorescence
have quantum efficiencies that are essentially zero
Quantum Efficiency
Fluorescence and Structure
• Aromatic rings give the most intense and most useful
molecular fluorescence emission.
• Aliphatic and alicyclic carbonyl compounds as well as
highly conjugate double-bonded structures also fluoresce,
there are very few of these compared to the number of
fluorescent compounds containing aromatic systems.
• Unsubstituted aromatic hydrocarbons fluoresce in
solution, with the quantum efficiency increasing with the
number of rings and their degree of condensation.
The simplest heterocyclics, such as pyridine, furan,
thiophene, and pyrrole, do not exhibit molecular
fluorescence (see Figure 25-3), but fused-ring structures
containing these rings often do (see Figure 25-4).
Substitution on an aromatic ring causes shifts in the
wavelength of absorption maxima and corresponding
changes in the fluorescence bands. In addition, substitution
frequently affects the fluorescence efficiency. These effects
are demonstrated by the data in the Table 25-1.
The Effect of Structural Rigidity
Fluorescence is particularly favored
in rigid molecules. For example,
under similar measurement
conditions, the quantum efficiency
of fluorene is nearly 1.0 while that of
biphenyl is about 0.2. This rigidity
lowers the rate of nonradiative
relaxation to the point where
relaxation by fluorescence has time
to occur.
The influence of rigidity has also
been invoked to account for the
increase in fluorescence of
certain organic chelating agents
when they are complexed with
a metal ion. For example, the
fluorescence intensity of 8-
hydroxyquinoline is much less
than that of its zinc complex:
Temperature and Solvent Effects
In most molecules, the quantum efficiency of
fluorescence decreases with increasing temperature
because the increased frequency of collision at elevated
temperatures increases the probability of collisional
relaxation. A decrease in solvent viscosity 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; carbon
tetrabromide and ethyl iodide are examples. The
effect is similar to that which occurs when heavy
atoms are substituted into fluorescing
compounds.
Effect of pH on Fluorescence
• analytical procedures based on fluorescence frequently require
close control of pH.
• 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.
Effect of Concentration on Fluorescence Intensity
Quenching on Molecular Fluorescence
• Quenching
– relates to a process that reduces the fluorescence intensity of a
supplied substance.
– usually refers to nonradiative energy transfer from an excited
species to other molecules.
• A variety of methods can succeed in quenching, such as excited state
reactions, energy transfer, complex-formation and collisional quenching.
• As an outcome, quenching is often profoundly dependent on pressure
and temperature.
• Some examples of common chemical quenchers are molecular oxygen,
iodide ions and acrylamide.
Dynamic or Collisional Quenching
• interaction of an excited state fluorophore with the quencher
results in radiationless deactivation of the fluorophore to the
ground state.
• requires contact between the excited species and the
quenching agent.
• occurs as rapidly as the collision partners can diffuse together.
• The rate is temperature and viscosity dependent.
• The quencher concentration must be high enough that there is
a high probability of a collision between the excited species
and the quencher during the lifetime of the excited state.
• occurs when the molecules form a complex in the ground
state, i.e. before excitation occurs.
• the fluorophore forms a nonfluorescent complex with a
quencher causing a decrease in fluorescence intensity.
• Another proof of the occurrence of static quenching is the
presence of high values of kq which are higher than the
maximum scatter collision quenching constant value of 2 x 10-
10
M-1 s -1 .
• A decrease in Ksv with increasing temperature indicates that
the quenching occurs in a static process.
Difference between Static quenching and Dynamic quenching
Stern Volmer
The fluorescence quantum efficiency can be writtens as:
Emission and Excitation Spectra
Because the first step in generating
fluorescence emission is absorption of
radiation to create excited states, an
excitation spectrum is essentially
identical to an absorption spectrum
taken under the same conditions.
Fluorescence and phosphorescence
spectra (F and P, respectively), on the
other hand, involve excitation at a fixed
wavelength while recording the
emission intensity as a function of
wavelength
Instruments for Measuring Fluorescence and Phosphorescence

• The components of instruments for measuring

photoluminescence are similar to those found in
ultraviolet-visible photometers or spectrophotometers.
• Typical configuration for these components in
fluorometers and spectrofluorometers.
• Nearly all fluorescence instruments employ double-
beam optics as shown to compensate for fluctuations
in radiant power.
Components of Fluorometers and Spectrofluorometers

If the two wavelength selectors are both filters, the

instrument is called a fluorometer. If both wavelength
selectors are monochromators, the instrument is a
spectrofluorometer. Some instruments are hybrids and use an
excitation filter along with an emission monochromator.
Fluorescence instruments can incorporate a double-beam
design in order to compensate for changes in the source
radiant power with time and wavelength. Instruments that
correct for the source spectral distribution are called
corrected spectrofluorometers.
Sources
Lamps
• The most common source for filter fluorometers is a lowpressure
mercury vapor lamp equipped with a fused silica window.
• This source produces useful lines for exciting fluorescence at 254,
302, 313, 546, 578, 691, and 773 nm.
• Individual lines can be isolated with suitable absorption or
interference filters.
• Because fluorescence can be induced in most fluorescing
compounds by a variety of wavelengths, at least one of the
mercury lines ordinarily proves suitable.
Lasers
• In photoluminescence instruments, of particular interest are
tunable dye lasers pumped by a pulsed nitrogen laser or a
Nd- YAG laser.
• Fixed-wavelength lasers are also used, particularly in
detectors for chromatography and electrophoresis.
• Most commercial spectrofluorometers use lamp sources
because they are less expensive and more applicable to
determining multiple analytes with different excitation
wavelengths.
Filters and Monochromators
• Interference and absorption filters 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
• Luminescence emission signals are typically quite low in intensity,
thus sensitive transducers are required.
– Photomultiplier tubes are the most common transducers in
sensitive fluorescence instruments. Often, these are operated
in the photon-counting mode to give improved signal-tonoise
ratios.
• Transducers are sometimes cooled to improve signal-tonoise ratios.
– Charge-transfer devices such as charge-coupled devices (CCDs),
are also used for spectrofluorometry. This permits the rapid
recording of both excitation and emission spectra and is
particularly useful in chromatography and electrophoresis
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.
• Even more than in absorbance measurements, it is important to
avoid fingerprints on cells because skin oils often fluoresce.
• Low-volume micro cells are available for situations in which sample
volumes are limited.
• Several companies make flow cells for fluorescence detection in
chromatography and in continuous flow analysis
• Sample handling accessories include micro-plate readers, microscope
attachments, and fiber-optic probes.
• Low volume cells are often used for room-temperature phosphorescence
and for chemiluminescence. Special cells and sample handling are needed
for low-temperature phosphorescence measurements.
• Although the right-angle geometry is most common, two other cell
configurations are used.
– Front-surface geometry allows measurements on solutions with high
absorbances or on opaque solids.
– The 180° , or in-line geometry is rarely used because the weak
luminescence signal must be isolated from the intense excitation beam
by the emission wavelength selector.
Data Manipulation
• Common data-manipulation and display options include
– blank signal subtraction,
– production of corrected excitation and emission spectra,
– calculation and display of difference and derivative spectra,
– peak detection and processing,
– deconvolution,
– production of three-dimensional total luminescence plots,
– fitting of calibration data,
– calculation of statistical parameters, and
– smoothing of spectra by various methods.
• Specialized software is available for kinetics, for highperformance liquid
chromatography (HPLC) detection, for analysis of mixtures, and for time-resolved
measurements.
Instrument Designs
• Filter fluorometers provide a relatively simple, low cost way of
performing quantitative fluorescence analyses.
• Filters are used to limit the wavelengths of the excitation and
emitted radiation.
• Fluorometers are compact, rugged, and easy to use.
• Schematic of a typical tilter fluorometer that uses a mercury lamp
for fluorescence excitation and a pair of photomultiplier tubes as
transducers.
• Some are simpler single-beam instruments. Some are portable
instruments for field work. The cost of such fluorometers ranges
from several hundred dollars to more than $10,000
Spectrofluorometers
• Capable of providing both excitation and emission spectra.
• The optical design employs two grating monochromators
provides perfectly satisfactory spectra for quantitative
analysis.
• Radiation from the excitation monochromator is split. part
passing to a reference photomultiplier and part to the
sample. The resulting fluorescence radiation, after
dispersion by the emission monochromator, is detected by
a second photomultiplier.
Fluorescence Methods
• Fluorescence methods
– used to study chemical equilibria and kinetics in much the same way as
absorption spectrophotometry.
– it is possible to study chemical reactions at lower concentrations because
of the higher sensitivity of fluorescence methods.
• If fluorescence monitoring is not feasible, fluorescent probes or tags can be
bound covalently to specific sites in molecules such as proteins, thus making
them detectable via fluorescence.
• These tags can be used to provide information about energy transfer
processes, the polarity of the protein, and the distances between reactive sites
• Quantitative fluorescence methods have been developed for inorganic,
organic, and biochemical species.
Fluorescence Methods
• Inorganic fluorescence methods can be divided into two classes:
– (1) Direct methods are based on the reaction of the analyte with a
complexing agent to form a fluorescent complex.
– (2) Indirect methods depend on the decrease in fluorescence, also called
quenching, as a result of interaction of the analyte with a fluorescent reagent.
• Quenching methods are primarily used for the determination of anions and
dissolved oxygen.
• Some fluorescence reagents for cations are shown in Figure 15-15.
• Nonradiative relaxation of transition-metal chelates is so efficient that these
species seldom fluoresce.
– most transition metals absorb in the UV or visible region, while non-
transition-metal ions do not.
fluorometric chelating agents for
metal cations.
Applications of Fluorescence Methods
• For this reason, fluorescence is often considered complementary to absorption for
the determination of cations.
• The number of applications of fluorescence methods to organic and biochemical
problems is impressive.
• Among the compound types that can be determined by fluorescence are amino
acids, proteins, coenzymes, vitamins, nucleic acids, alkaloids, porphryins, steroids,
flavonoids, and many metabolites.
• Because of its sensitivity, fluorescence is widely used as a detection technique for
liquid chromatographic methods, for flow analysis methods, and for
electrophoresis.
• In addition to methods that are based on measurements of fluorescence intensity,
there are many methods involving measurements of fluorescence lifetimes.
• Several instruments have been developed that provide microscopic images of
Fluorometric Determination of Inorganic Species

Inorganic fluorometric methods are of two types.11

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. The latter technique
has been most widely used for anion analysis.
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.12 The structures of four common
reagents are shown in Figure 15-15. Selected
fluorometric reagents and their applications are
presented in Table 15-2. As can be seen, LODs are quite
low by most of these methods.
Methods for Organic and Biochemical Species
• There are more than 200 entries under the heading “Fluorescence
Spectroscopy of Some Organic Compounds,” including such diverse
compounds as adenine, anthranilic acid, aromatic polycyclic hydrocarbons,
cysteine, guanine, isoniazid, naphthols, nerve gases sarin and tabun,
proteins, salicylic acid, skatole, trypotophan, uric acid, and warfarin
(Coumadin).
• Many medicinal agents that can be determined fluorometrically are listed,
including adrenaline, morphine, penicillin, phenobarbital, procaine,
reserpine, and lysergic acid diethylamide (LSD).
• The most important applications of fluorometry include the analysis of
food products, pharmaceuticals, clinical samples, and natural products.
• The sensitivity and selectivity of molecular fluorescence make it a
particularly valuable tool in these fields.
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.
Laser-excited fluorescence is particularly important for
these applications because the beam can be readily
focused to a size on the order of the column diameter.
Phosphorimetric Methods
Phosphorescence and fluorescence methods tend to be
complementary because strongly fluorescing compounds
exhibit weak phosphorescence and vice versa.
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. The method has not,
however, found as widespread use as fluorometry, perhaps
because of the need for low temperatures and the generally
poorer precision of phosphorescence measurements.
Lifetime Measurements
• Fluorescence lifetime measurements can give information
about collisional deactivation processes, about energy
transfer rates, and about excited-state reactions.
• Lifetime measurements can also be used analytically to
provide additional selectivity in the analysis of mixtures
containing luminescent species. 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
Fluorescence Imaging Methods
• In recent years, it has become possible to combine
fluorescence spectroscopy with optical microscopy to
produce localized images of fluorophores in complex
matrices such as single cells.
• In some cases the intrinsic (native) fluorescence of
biomolecules can be used in conjunction with
microscopy to monitor dynamics in cells.
• In the absence of a native fluorophore, fluorescent
indicators can be used to probe biological events.
• An example in neurobiology, the dye Fura-2 has been used to
monitor the free intracellular calcium concentration following
pharmacological or electrical stimulation.
– When the cell is loaded with the Fura-2 fluorescent indicator,
sharp changes in fluorescence can be measured that correspond
to individual calcium action potentials.
– The changes are correlated to specific sites in the cell by means
of fluorescence imaging techniques. Figure 15-16 shows an
example of a fluorescence image.
– The interpretation of these kinds of patterns can have important
implications in understanding the details of synaptic activity