MOLECULAR Fluorescence,

Phosphorescence
LUMINESCENCE and
SPECTROSCOPY Chemiluminescence
Pertemuan 6
PAK 325-KIMIA-KELAS A – Dr. Matlal F. Alif 16 Maret 2015
 Photoluminescence
What happens after a molecule has absorbed light ?

Exciting light Heat (80%)

Excitation More

Energy

Normal molecule Excited molecule Photobleaching

(try to avoid)

Emission of light (20%) (Photoluminescence)

 WHAT HAPPENS TO THE ABSORBED
EM ENERGY DETERMINES WHETHER
YOU HAVE…

 Absorbance
­ molecule returns to the ground or lower energy state via a non-radiative
transition such as vibration, collision with other molecules, etc. These give
off the energy absorbed rather than the emission of light.
 Fluorescence
­ Some energy is lost through various processes (e.g. non-radiative
transitions) and then light is given off.
 Phosphorescence
­ The molecule transitions from an excited triplet state to an lower energy
singlet state and gives off light. Non-radiative transitions intervene.
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 orient
ation. 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 + hν
The release of electromagnetic energy is immediate or from the singlet state.
Delayed fluorescence : S1→ T1→ S1→ S0 + hν
This results from two intersystem crossings, first from the singlet to the triplet,
then from the triplet to the singlet.
 Phospholuminescence : T1→ S0 + hν
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.
4) Triboluminescence : A release of energy when certain crystals, such as sugar, are broken.
5) Cathodoluminescence : A release of energy produced by exposure to cathode rays
6) Thermoluminescence : When a material existing in high vibrational energy levels emits energy at a t
emperature below red heat, after being exposed to small amounts of thermal energy.
A) Introduction

1.) Theory of Fluorescence and Phosphorescence:

For UV/Vis need to observe Po
10-8 – 10-9s and P difference, which limits
M* à M + heat detection

10-5 to 10-8 s fluorescence

10-4 to 10 s phosphorescence
10-14 to 10-15 s

- Excitation of e- by absorbance of hν.

- Re-emission of hv as e- goes to ground state.
-  Use hν2 for qualitative and quantitative analysis

Method Mass detection Concentration Advantages

limit (moles) detection limit
(molar)
UV-Vis 10-13 to 10-16 10-5 to 10-8 Universal

fluorescence 10-15 to 10-17 10-7 to 10-9 Sensitive

 2.) Fluorescence – ground state to single state and back.
Phosphorescence - ground state to triplet state and back.

Fluorescence Phosphorescence

10-5 to 10-8 s 10-4 to 10 s

Spins paired Spins unpaired

No net magnetic field net magnetic field

Example of
Phosphorescence

0 sec 1 sec
 Fluorescence process
A: So + hν → S1 or S2 Radiation process
Molecular fluorescence spectrometry is bas
ed on the emission of light by molecules that h
ave become electronically excited subsequent
to the absorption of visible(400~700nm), U
V(200~400nm), or NIR (700 ~ 1100nm) radi
ation. Excitation process to the excited state f
rom the ground state is very fast, on the orde
r 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
Jablonski diagram.
F : fluorescence, S1→ S0 + hν 10–10~10–6 s.
P : phosphorescence, T1→ S0 + hν
10–4 s ~104 s.
 Deactivation Processes:
a) vibrational relaxation: solvent collisions
- λ emission > λ excitation (Stokes shift)
- vibrational relaxation is efficient and goes to
lowest vibrational level of electronic state
within 10-12s or less.
- significantly shorter life-time then
electronically excited state
- fluorescence occurs from lowest vibrational
level of electronic excited state, but can go to
higher vibrational state of ground level.
b) internal conversion:
- crossing of e- to lower electronic state.
- S1 to S0 would also happen .
- efficient, therefore many compounds don’t
fluoresce (aliphatic)
- especially probable if vibrational levels of
two electronic states overlap, can lead to
predissociation or dissociation.
- dissociation: direct excitation
(absorption) to vibrational state with
enough energy to break a bond
- predissociation: relaxation to
vibrational state of a lower electronic
state with enough energy to break a bond
c) external conversion:

•  deactivation via collision with solvent (collisional quenching)

•  decrease collision à increase fluorescence or
phosphorescence
•  decrease temperature and/or increase viscosity
•  decrease concentration of quenching (Q) agent

d) intersystem crossing:
•  spin of electron is reversed
- change in multiplicity in molecule occurs (singlet to triplet)
- enhanced if vibrational levels overlap
- more common if molecule contains heavy atoms (I, Br)

e) Phosphorescence:
Deactivation from an ‘triplet” electronic
state to the ground state producing a
photon
PHOTOLUMINESCENCE
AND STRUCTURE
 The presence of the benzene ring and the nature of substituents on it
seem to favour the fluorescent behaviour of the molecule.
 The halogen substituents tend to decrease the fluorescence and shift
the fluorescence bands to longer wavelengths; the effects increase
with increase in the atomic mass of the substituted halogen.
PHOTOLUMINESCENCE
AND STRUCTURE
 Compounds with fused ring are found to be especially
fluorescent, and the extent of fluorescence is found to be
directly proportional to the number of rings in the
molecule
 The structural rigidity in a molecule favours fluorescence
Structural factors affecting fluorescence
1. Fluorescence is expected in molecules that are aromatic or multiple conjugated dou
ble 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, ofte
n 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 g
lassy state or viscous solution. Formation of chelates with metal ions also promotes flu
orescence. However, the introduction of paramagnetic metal ions gives rise to phosph
orescence but not fluorescence in metal complexes.
6. Changes in the system pH, if it affects the charge status of chromophore, may influ
ence fluorescence.
T  he fluorescence observed with rigid cyclic molecules
with pi-bonds is found to be enhanced by electron
donating groups e.g., −NH2, OR, – OH and OCH3,
 
T  he electron withdrawing groups such as COOH, NO2,
N=N and Br, I and CH2COOH tend to reduce it.

  n the other hand the nonrigid molecules do not

O
fluoresce much, as these rapidly lose the absorbed
energy through nonradiative means like, vibrational
relaxation or even degradation.
Typical aromatic molecules that do not Typical aromatic molecules that fluoresce.
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

Larry G. Hargis , Analytical Chemistry-principles and techniques, Prentice-Hall, 1988, p 435.

 Fluorescence of linear aromatics in a mixture of ethanol, isopropanol
and ether.

Compound Φ λex (nm) λ em (nm)

Benzene 0.11 205 278

Naphthalene 0.29 286 321
Anthracene 0.46 365 400
Naphthacene 0.60 390 480
•  Aliphatic and alicyclic carbonyl compounds or highly conjugated
double bond structures also show fluorescence.
 As regards phosphorescence, it has been observed that the
introduction of certain paramagnetic metal ions such as copper
and nickel give rise to phosphorescence. These ions do not induce
fluorescence, on the contrary Mg and Zn compounds show strong
fluorescence.
 Phosphorescence is affected by the molecular structure such as
unsubstituted cyclic and polycyclic hydrocarbons and those
containing –CH3, –NH2, –OH2, –COOH, –OCH3 substituents
which have lifetimes in the range of 5–10 seconds for benzene
derivatives and 1– 4 seconds for naphthalene derivatives.
 The introduction of a nitro group (NO2) in a structure diminishes
the intensity of phosphorescence, as does the introduction of
aldehyde and ketonic carbonyl groups.
 The emission life time (t) is in seconds in rigid media and is 102 –
100 seconds in fluid media.
FACTORS AFFECTING
FLUORESCENCE AND
PHOSPHORESCENCE
The common factors affecting the fluorescence are as follows.
• Temperature
• pH
• Dissolved oxygen
• Solvent
TEMPERATURE
 A rise in temperature is almost always accompanied by a
decrease in fluorescence.
 The change in temperature causes the viscosity of the
medium to change which in turn changes the number of
collisions of the molecules of the fluorophore with solvent
molecules.
 The increase in the number of collisions between molecules
in turn increases the probability for deactivation by
internal conversion and vibrational relaxation.
PH
 Relatively small changes in pH can sometimes cause substantial changes in
the fluorescence intensity and spectral characteristics of fluorescence.
­  For example, serotonin shows a shift in fluorescence emission maximum from 330 nm at
neutral pH to 550 nm in strong acid without any change in the absorption spectrum.

  In the molecules containing acidic or basic functional groups, the changes

in pH of the medium change the degree of ionisation of the functional
groups. This in turn may affect the extent of conjugation or the aromaticity
of the molecule which affects its fluorescence.
­  For example, aniline shows fluorescence while in acid solution it does not show fluorescence
due to the formation of anilinium ion.

  Therefore, pH control is essential while working with such molecules and

suitable buffers should be employed for the purpose.
DISSOLVED OXYGEN
 The paramagnetic substances like dissolved oxygen
and many transition metals with unpaired electrons
dramatically decrease fluorescence and cause
interference in fluorimetric determinations.
 Molecular oxygen is paramagnetic (has triplet ground
state), which promotes intersystem crossing from singl
et to triplet states in other molecules. The longer lifeti
mes of the triplet states increase the opportunity for r
adiationless deactivation to occur. Other paramagneti
c substances, including most transition metals, exhibit t
his same effect.
 P resence of dissolved oxygen influences
phosphorescence too and causes a large
decrease in the phosphorescence intensity.
 It is due to the fact that oxygen which is in triplet
state at the ground state gets the energy from an
electron in the triplet state and gets excited.
T  his is actually the oxygen emission and not the
phosphorescence. Therefore, it is advisable to
make phosphorescence measurement in the
absence of dissolved oxygen.
 SOLVENT
 The changes in the “polarity” or hydrogen bonding ability of the solvent
may also significantly affect the fluorescent behaviour of the analyte.
 The difference in the effect of solvent on the fluorescence is attributed to
the difference in their ability to stabilise the ground and excited states
of the fluorescent molecule.
 Besides solvent polarity, solvent viscosity and solvents with heavy atoms
also affect fluorescence and phosphorescence.
 Increased viscosity increases fluorescence as the deactivation due to
collisions is lowered.
 A higher fluorescence is observed when the solvents do not contain
heavy atoms while phosphorescence increases due to the presence of
heavy atoms in the solvent.
Temperature, Solvent & pH Effects:
- decrease temperature à increase fluorescence (deactivation)
- increase viscosity à increase fluorescence (less collisions)
- fluorescence is pH dependent for compounds with acidic/basic substituents.

more resonance forms stabilize excited state

H H H H H H
N N N

resonance forms of aniline

Effect of Dissolved O2:

- increase [O2] à decrease fluorescence
- oxidize fluorescing species
- paramagnetic property increase intersystem crossing (spin flipping)
Application of Fluorescence
- detecting inorganic species by adding a fluorophore

Ion Reagent Absorption (nm) Fluorescence (nm) Sensitivity (ppm) Interference

Be, Co, Cr, Cu,
Al3+ Alizarin garnet R 470 500 0.007 F-,NO3-, Ni,
PO4-3, Th, Zr

Al complex of Be, Co, Cr, Cu,

F- Alizarin garnet R 470 500 0.001 F-,Fe, Ni,PO4-3,
(quenching) Th, Zr

B4O72 Benzoin 370 450 0.04 Be, Sb

-

2-(0-Hydroxyphenyl)- NH3
Cd2+ 365 Blue 2
benzoxazole
Li+ 8-Hydroxyquinoline 370 580 0.2 Mg
Sn4+ Flavanol 400 470 0.1 F-, PO43-, Zr
B, Be, Sb, colored
Zn2+ Benzoin - green 10
ions

OH
OH HO
N O O OH
C C
HO N N
OH H

O SO3Na

8-Hydroxyquinoline flavanol alizarin garnet R benzoin

Instrumentation for fluorescence spectroscopy

Sample cell
Power Source Excitation monoc
hromator Slit
supply
Emission monochromator

Detector

Data processor

General layout of fluorescence spectrophotometer

INSTRUMENTATION
Basic design

•  components similar to UV/Vis

•  spectrofluorometers: observe
•  both excitation & emission spectra.

Extra features for phosphorescence

•  sample cell in cooled Dewar flask with liquid nitrogen

•  delay between excitation and emission
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
d. X-ray source for X-ray fluorescence
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 refelecting 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
Return beam vidicon
Intensified target vidicon

Stephen G. Schulman , (Alan Townshend Edt.), Encyclopedia of analytical science, Vol. 3, Academic Press, L
ondon, pp. 1358-1365.
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.
Stephen G. Schulman , (Alan Townshend Edt.), Encyclopedia of analytical science, Vol. 3, Academic Press, London,
p 1396.
Generation of fingerprint excitation-emission matrix. a) EEM of pure component, compound A, b) E
EM of pure component, compound B, c)fingerprint EEM of a mixture of compound A and B, d) isom
etric projection of fingerprint in c). Shelly et al., Clinical Chemistry 26, 1127-1132, 1980.
 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)
Fluorometers
- simple, rugged, low cost, compact
- source beam split into reference and sample beam
- reference beam attenuated ~ fluorescence intensity

A-1 filter fluorometer

Spectrofluorometer
- both excitation and emission spectra
- two grating monochromators
- quantitative analysis

Perkin-Elmer 204
TOTAL FLUORESCENCE
INSTRUMENT
 Use array detector (CCD) to collect total fluorescence spectrum
TOTAL FLUORESCENCE
SPECTRUM
INSTRUMENTATION FOR
PHOSPHORESCENCE
MEASUREMENT
T  he basic difference between fluorescence and
phosphorescence is that the phosphorescence
emission occurs at a different time frame and can
be measured only if the sample is solid or is at
liquid nitrogen temperatures.
 The basic instrumentation for phosphorescence is
similar to that of fluorescence; however, two
aspects of the measurement need to be modified.
The first is the sampling technique and second
being the recording procedure.
SAMPLING
S  ince most of the measurements in
phosphorescence are carried out in rigid media at
cryogenic temperatures of liquid nitrogen we
need to use solvents that have certain special
characteristics.
  It is the most important requirement are
­  good solubility of the analyte.
­  The solvent must form a clear rigid glass at 77 K i.e., the
temperature of measurement.
­  In addition, it should be highly pure so that there is practically nil
background phosphorescence
 Ethanol is an excellent solvent for polar molecules though it
may require addition of small quantities of acid or base
to produce a clear solid.
 On the other hand a mixture of diethyl ether, isopentane
and ethanol in the ratio of 5: 5: 2 respectively, commonly
called EPA is an excellent choice for non-polar
compounds.
PHOSPHORIMETRY
 Spectrophophorimeter is similar to a Spectrofluorimeter except that
the former instrument must be fitted with
1) A Rotating-shutter device commonly called a phosphoroscope and
2) a sample system which is maintained at liquid nitrogen
temperature.
PHOSPHOROSCOPE
PHOSPHOROSCOPE
1)  The Rotating-Can Phosphoroscope:
•  it consists of hollow cylinder having one or more slit which are equally
spaced in the circumference.
•  This is rotated by a variable-speed motor.
•  when the rotating-can is rotated by a motor the sample is first illuminated
and then darkened.
•  Whenever, there is a dark, phosphorescence radiation passes to the
monochromator and be measured
2)The Becquerel or rotating disc phosphoroscope:
 It has two discs which are mounted on a common axis turn by a
variable speed motor.
 Both the discs are having openings equally spaced in their
circumference.
 On moving becqueral disc the sample is first illuminated and
then darkened.
 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 tempera
ture.
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

D.A. Harris, C.L. Bashford , Spectrophotometry & spectroflurimetry- a practical approach, IRL Press, Oxford, U
K, 1987, p.18-20.
 Problems with photoluminescence
1) Self-quenching
Self-quenching results when luminescing molecule collide and lo
se their excitation energy by radiationless transfer. Serious offen
ders are impurities, dissolved oxygen, and heavy atoms or param
agnetic species (aromatic substances are prime offenders).
2) Absorption of radiant energy
Absorption either of the exciting or of the luminescent radiation
reduces the luminescent signal. Remedies involve (a) dilution the s
ample, (b) viewing the luminescence near the front surface of the
cell, and (c) using the method of standard additions for evaluatin
g samples.

John A. Dean, Analytical Chemistry Handbook, McGraw-Hill, 1995, New York, p.5.55
3) Self-absorption
Attenuation of the exciting radiation a sit passes through the c
ell can be caused by too concentrated an analyte. The remedy i
s to dilute the sample and note whether the luminescence increas
es or decreases. If the luminescence increases upon sample diluti
on, one is working on the high-concentration side of the luminesc
ence maximum. This region should be avoided.

4) Excimer formation
Formation of a complex between the excited-state molecule a
nd another molecule in th ground state, called an excimer, causes
a problem when it dissociates with the emission of luminescent r
adiation at longer wavelengths than the normal luminescence. Dil
ution helps lesson this effect.

John A. Dean, Analytical Chemistry Handbook, McGraw-Hill, 1995, New York, p.5.55
 CHEMILUMINESCENCE
- chemical reaction yields an electronically excited species that emits
light as it returns to ground state.
- relatively new, few examples

A + B à C* à C + hν



Examples of Chemical Systems giving off light:
Luminol (used to detect blood)
NH2 O NH2

C COO-
NH O2/OH-
+ hν + N2 + H2O
NH
C
COO-
O

- phenyl oxalate ester (glow sticks)

 BIOLOGICAL SYSTEMS
Luciferase (Firefly enzyme)
O O
R2
Luciferase Spontaneous
Luciferin + O2 O C C R2 CO2 + O C* Light
R1
1
R
N
S

S
HO
N
O
Luciferin (firefly)
HO

“Glowing” Plants
Luciferase gene cloned into plants
OTHER APPLICATIONS
Determination of nitrogen monoxide

NO + O3 → NO2* + O2

NO2* + → NO2 + hν (λ = 600 – 2800 nm)

Determination of sulfur

4H2 + 2SO2 → S2* + 4H2O

S2* → S2 + hν (λ = 384 and 394 nm)