CHEM 362

Fluorescence spectrometry
What is luminescence ?
Luminescence is the emission of photons from electronically excited state.
Luminescence is divided into two types, depending upon the nature of the ground
and the excited states.
In a singlet excited state, the electron in the higher energy orbital has the opposite
spin orientation as the second electron in the lower orbital. These two electrons are
said to be paired. Return to the ground state from an excited singlet state does not
require an electron to change its spin orientation.
In a triplet state these electrons are unpaired, that is, their spins have the same
orientation. A change in spin orientation is needed for a triplet state to return to the
singlet ground state.

So diamagnetic S1 paramagnetic T1
Types of luminescence
(classification according to the means by which energy is supplied to excite the
luminescent molecule)
1) Photoluminescence : Molecules are excited by interaction with photons of
radiation.
Fluorescence :
Prompt fluorescence : S1→ S0 + h
The release of electromagnetic energy is immediate or from the singlet state.
Delayed fluorescence : S1→ T1→ S1→ S0 + h
This results from two intersystem crossings, first from the singlet to the triplet,
then from the triplet to the singlet.
Phospholuminescence : T1→ S0 + h
A delayed release of electromagnetic energy from the triplet state.
2) Chemiluminescence : The excitation energy is obtained from the chemical
energy of
reaction.
3) Bioluminescence : Chemiluminescence from a biological system: firefly, sea
pansy, jellyfish, bacteria, protozoa, crustacea.
 Fluorescence process
A: So + h → S1 or S2 Radiation process
Molecular fluorescence spectrometry is
based on the emission of light by molecules
that have become electronically excited
subsequent to the absorption of
visible(400~700nm), UV(200~400nm), or
NIR (700 ~ 1100nm) radiation. Excitation
process to the excited state from the ground
state is very fast, on the order of 10–15 s.
VR: vibrational relaxation,
non-radiational process, 10–11 s ~10–10 s.
IC : internal conversion, S2→ S1 S1→ S0
non-radiative process, 10–12 s.
ST : intersystem crossing, S1→ T1
F : fluorescence, S1→ S0 + h 10–10~10–6 s.
Jablonski diagram. P : phosphorescence, T1→ S0 + h
10–4 s ~104 s.
Example showing that phosphorescence comes at lower energy than
fluorescence from the same molecule. The phosphorescence signal is ~10
times weaker than the fluorescence signal and is only observed when the
sample is cooled.
 2
E21 = h21 = hc/21
Luminescence 1
L E2 = h2 = hc/2
E1 = h1 = hc/1
0
(b)
Sample
Incident Transmitted
radiation radiation
0  L

(a) 
2 1 21
(c)

Photoluminescence methods. Absorption of incident radiation from an external source

(a) causes excitation of the analyte to state 1 or state 2 (b). Excited species can dissipate
the excess energy by emission of a photon [luminescence (L)] or by radiationless processes
(dashed lines) in (b). Emission is isotropic (a), and the frequencies emitted correspond to
the energy differences between levels (c).
 Emitted 2
radiation E21 = h21 = hc/21
E 1
E2 = h2 = hc/2
E1 = h1 = hc/1
0

Sample (b)

E

Thermal, 
electrical, 2 1 21
or chemical
(a) (c)
energy

Emission and chemiluminescence(bioluminescence) methods. In (a) the addition of

thermal, electrical or chemical energy causes nonradiational excitation of the analyte
and emission of radiation in all directions (isotropic emission). The energy changes that
occur during excitation (dashed lines) or emission (soled lines) are shown in (b). The
energies of states 1 and 2 are usually relative to the ground level and often abbreviated
E1 and E2, respectively. A typical spectrum is shown in (c).
Fluorescence efficiency ; quantum yield of fluorescence

The ratio of the fluorescence radiant power to the absorbed radiant power where the
radiant powers are expressed in photons per second.

 = (luminescene radiant power) / ( absorbed radiant power)

= (number of photons emitted) / (number of photons absorbed)

10

The higher the value of , the greater the fluorescence of a compound.

A non-fluorescent molecule is one whose quantum efficiency is zero or so
close to zero that the fluorescence is not measurable. All energy absorbed by
such a molecule is rapidly lost by collisional deactivation.
Fluorescence lifetime
Another important property of fluorescing molecules is the lifetime () of the lowest
excited singlet state. The lifetime of excited state is defined by the average time the
molecule spends in the excited state prior to return to the ground state. Generally,
fluorescence lifetimes are near 10 nsec. The quantum yield of fluorescence and  are
related by
 = kf / (kf+ kd) = kf 
where kf is the rates of fluorescence, kd is the radiationless rate of deactivation.
Fluorescence lifetime measurement is a valuable technique in the analysis of
multicomponent samples containing analytes with overlapping fluorescence bands.
Fluorescence related to concentration
The fluorescence radiant power F is proportional to the absorbed radiant power.
F = (Po – P)
where  = fluorescence efficiency, Po = incident power, P = transmitted power
The relationship between the absorbed radiant power and concentration can be obtained from
Beer’s law.
P/ Po = 10–A = 10–bC P = Po 10–bC F =  Po (1–10–bC)
When expanded in a power series, this equation yields
F =  Po [(lnbC)1/ 1! – (– lnbC)2 / 2! – (– lnbC)3 / 3! – (lnbC)4 / 4! } – … – (–lnbC)n /n!]
If bC is 0.05 or less, only the first term in the series is significant and equation can be written
as F =  Po (lnbC) = kbC
where k is a constant equal to  Poln. Thus, when the concentrations are very dilute and not
over 2% of the incident radiation is absorbed, there is linear relationship between fluorescent
power and concentration.
When bC is greater than about 1.5, 10–bC is much less than 1 and fluorescence depends
directly on the incident radiation power.
F =  Po
  Po

Concentration of fluorescing species

Theoretical behavior of fluorescence as a function of concentration.

Structural factors affecting fluorescence
1. Fluorescence is expected in molecules that are aromatic or multiple conjugated double
bonds with a high degree of resonance stability.
2. Fluorescence is also expected in polycyclic aromatic systems.
3. Substituents such as –NH3, –OH, –F, – OCH3, – NHCH3, and – N(CH3)2 groups, often
enhance fluorescence.
4. On the other hand, these groups decrease or quench fluorescence completely :
–Cl, –Br, –I, –NHCOCH3, – NO2, – COOH.
5. Molecular rigidity enhances fluorescence. Substances fluoresce more brightly in a
glassy state or viscous solution. Formation of chelates with metal ions also promotes
fluorescence. However, the introduction of paramagnetic metal ions gives rise to
phosphorescence but not fluorescence in metal complexes.
6. Changes in the system pH, if it affects the charge status of chromophore, may
influence fluorescence.
Typical aromatic molecules that do Typical aromatic molecules that fluoresce.
not fluoresce.
 Effect of molecular rigidity on
quantum yield. The fluorene
molecule is held rigid by the central
ring, two benzene rings in biphenyl
can rotate to one onother.

Effect of rigidity on quantum yield in

complexes. Free 8-hydroxyquinoline
molecules in solution are easily
deactivated through collision with solvent
molecules and do not fluoresce.
The rigidity of the Zn 8-hydroxyquinoline
complex enhances fluorescence.
Substitution effects on the fluorescence of benzene.

Substituent Changes in wavelength Changes in intensity

of fluorescence of fluorescence
Alkyl None None
OH, CH3, OC2H5 Decrease Increase
COOH Decrease Large decrease
NH2, NHR, NR2 Decrease Increase
NO2, NO - Total quenching
CN None Increase
SH Decrease Decrease
F, Cl, Br, I Decrease (F→ I) Increase ( F → I )
SO3H None None
Fluorescence and environment
1. Temperature:
A rise in temperature almost always is accompanied by a decrease in fluorescence
because the greater frequency of collisions between molecules increases the probability
for deactivation by internal conversion and vibrational relaxation.
2. pH :
Changes in pH influence the degree of ionization, which, in turn, may affect the extent
of conjugation or the aromaticity of the compound.
3. Dissolved oxygen :
Dissolved oxygen often decreases fluorescence dramatically and is an interference in
many fluorometric methods. Molecular oxygen is paramagnetic (has triplet ground state),
which promotes intersystem crossing from singlet to triplet states in other molecules.
The longer lifetimes of the triplet states increase the opportunity for radiationless
deactivation to occur. Other paramagnetic substances, including most transition metals,
exhibit this same effect.
4. Solvents :
Solvents affect fluorescence through their ability to stabilize ground and excited states
differently, thereby changing the probability and the energy of both absorption and
emission.
Common problems of fluorescence measurements

1) Reference materials is as fluorescent as the sample

Contaminating substances
Raman scattering, Rayleigh scattering
2) Fluorescence reading is not stable
Fogging of the cuvet when the contents are much colder than the ambient temperature.
Drops of liquid on the external faces of the cuvet.
Light passing through the meniscus of the sample.
Bubbles forming in the solution as it warms.
Quenchers : molecular oxygen
3) Sensitivity is inadequate
Excitation spectrum and emission spectrum
The excitation spectrum is a measure of the ability of the impinging radiation to raise a
molecule to various excited states at different wavelengths. An excitation spectrum is
recording of fluorescence versus the wavelength of the exciting or incident radiation and it is
obtained by setting the emission monochromator to a wavelength where fluorescence occurs
and scanning the excitation monochromator. An excitation spectrum looks very much like an
absorption spectrum, because the greater the absorbance at the excitation wavelength, the
more molecules are promoted to the excited state and the more emission will be observed.
The emission (fluorescence) spectrum is a measure of the relative intensity of radiation
given off at various wavelength as the molecule returns from the excited states to the
ground state. The emission spectrum is recording of fluorescence versus the wavelength of
the fluorescence radiation, and it is obtained by setting the excitation monochromator to a
wavelength that the sample absorbs and scanning the emission monochromator.
Since some of the absorbed energy is usually lost as heat, the emission spectrum occurs at
longer wavelengths (lower energy) than does the corresponding excitation spectrum. If an
emission spectrum occurs at shorter wavelengths than the excitation spectrum, the presence
of a second fluorescing species is confirmed.
Energy level diagram showing why structure is seen in the absorption and emission
spectra, and why the spectra seem roughly mirror images of each other.
Excitation and emission spectra of anthracene,
illustrating the mirror-image relationship between
absorption (A) and fluorescence (F),
Absorption and fluorescence
emission spectra of perylene
and quinine.
Joseph R. Lakowicz , Principles of
Fluorescence Spectroscopy, Plenum
Press, New York, 1983, p 3.
Absorption (black line) and emission (colored line) spectra of N-methlcarbazole
in cyclohexane solution, illustrating the approximate mirror image relationship
between absorption and emission.
Instrumentation for fluorescence spectroscopy

Sample cell
Power Source Excitation
monochromator Slit
supply
Emission monochromator

Detector

Data processor

General layout of fluorescence spectrophotometer

Schematic diagram of a typical spectrofluorometer.
1) Light sources
a. Gas discharge lamps :
Xenon arc lamp
High pressure mercury vapor lamp
b. Incandescent lamps : Tungsten wire filament lamp
c. Laser : tunable dye laser

2) Wavelength selection devices

a. Filters :
Absorption filters ---tinted glass or gelatin containing dyes sandwiched between glass
Interference filters ---thin transparent layer of CF2 or MgF2 sandwiched two parallel,
partially reflecting metal films
b. Monochromators :
Gratings
Prism
Cross-sectional view of an interference filter
3) Sample compartment
Fluorescence cells ---- right angle design or small angle(37o) viewing system
Quarz or fused silica ----200 nm ~ 800 nm
Glass or plastic ---- 300 nm ~

4) Detectors
Photomultiplier
Photoconductive target vidicon
Schematic of a fibre optic based multichannel fluorometer.
IDA=512 element intensified linear photodiode array detector, L=lens, OF1 and OF2 =
the excitation and emission fibres.
Applications
1) Direct measurement --- metal cations as fluorescent chelates
2) Indirect measurement where the fluorescence of the substance being determined is
measured prior to and after quenching
3) Indirect measurement where the fluorescence of the determined substance is enhanced
by the addition of a reacting material.
4) Tracer techniques --- bioengineered anlysis.
FISH(fluorescence in situ hybridization)
5) SFS( spectral fluorescent signatures)
Examples of naturally fluorescent organic compounds
Compound Wavelength or Range of em(nm) Compound Wavelength or Range of em(nm)

Aromatic hydrocarbons Drugs

Naphthalene 300-365 Asprine 335
Anthracene 370-460 Codeine 350
Pyrene 370-400 Diethylstibestor 435
1,2-Benzopyrene 400-450 Estrogens 546
Heterocyclic compound Lysergic acid diethylamide(LSD) 365
Quinoline 385-490 Phenobarbital 440
Quinine sulfate 410-500 Procaine 345
7-Hydroxycoumarine 450 Steroid
3-Hydroxyindol 380-460 Aldosterone 400-450
Dyes Cholesterol 600
Fluorescein 510-590 Cortisone 580
Rhodamine B 550-700 Prednisolone 570
Methylene Blue 650-700 Testosterone 580
Naphthol 516 Vitamines
Coenzymes, nucleic acids, pyrimidines Ribofravin(B 2) 565
Adenine 380 Cyanocobalamin(B 12) 305
Adenosine triphosphate(ATP) 390 Tocopherol(E) 340
Nicotinamide adenine
dinucleotide(NADH) 460
Purine 370
Thymine 380
Linear calibration curve for fluorescence
of anthracene measured at the wavelength
of maximum fluorescence.
Calibration curve for the
spectrofluorometric determination of
tryptophane in soluble proteins from
the lens of a mammalian eye.