Fluorimetry

Page 1 of 64

Introduction
When the molecules of the analyte are excited to give a
species whose emission spectrum provides information for
qualitative or quantitative analysis. The methods are known
collectively as molecular luminescence procedures.

Three types of Luminescence methods are:

(i) molecular fluorescence
(ii) phosphorescence
Photoluminescence
(iii) chemiluminescence

Page 2 of 64

Luminescence is the emission of light by a substance. It

occurs when an electron returns to the electronic ground
state from an excited state and loses its excess energy as a
photon.
It is of 3 types.
Fluorescence spectroscopy.
Phosphorescence spectroscopy.
Chemiluminescence spectroscopy

Page 3 of 64

FLUORESCENCE
When a beam of light is incident on certain substances they emit visible
light or radiations. This is known as fluorescence.
Fluorescence starts immediately after the absorption of light and stops as
soon as the incident light is cut off.
The substances showing this phenomenon are known as fluorescent
substances.

Page 4 of 64

PHOSPHORESCENCE
When light radiation is incident on certain substances they emit light
continuously even after the incident light is cut off.
This type of delayed fluorescence is called phosphorescence.
Substances showing phosphorescence are phosphorescent substances.

Page 5 of 64

Chemiluminescence
Chemiluminescence is another phenomenon that falls
in the category of luminescence. This refers to the
emission of radiation during a chemical reaction.
However, in such cases the excited state is not a result of
absorption of electromagnetic radiation.
The oxidation of luminol (3-aminophthalhydrazide) in an
alkaline solution is an example of chemiluminescence

Page 6 of 64

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 : The release of electromagnetic energy is immediate
or from the singlet state.
Delayed fluorescence : This results from two intersystem crossings, first
from the singlet to the triplet,
then from the triplet to the singlet.
Phospholuminescence : A delayed release of electromagnetic energy from
the triplet state.
2) Chemiluminescence : The excitation energy is obtained from the chemical
energy of reaction.

Page 7 of 64

THEORY

Classification of fluorescence
Based on the wavelength of emitted radiation when compared
to absorbed radiation
Stokes fluorescence: wavelength of emitted radiation is
longer than absorbed radiation
Anti-stokes fluorescence: wavelength of emitted radiation
is shorter than absorbed radiation.
Resonance fluorescence: wavelength of emitted radiation is
equal to that of absorbed radiation.

Page 8 of 64

Types of fluorescence and emission processes

Stokes fluorescence : This is the reemission of less energetic photons, which have a
longer wavelength than the absorbed photons.
One common cause of Stokes shift is the rapid decay to the lowest vibrational level of S1.
Furthermore, fluorophores generally decay to excited vibrational levels of So, resulting
in further loss of vibrational energy.
In addition to these effects, fluorophores can display further Stokes shifts due to
solvent effects and excited state reactions. In gas phase, atoms and molecules do not
always show Stokes shifts.
Anti-Stokes fluorescence :
If thermal energy is added to an excited state or a compound has many highly
populated vibrational energy levels, emission at shorter wavelengths than those of
absorption occurs.
This is often observed in dilute gases at high temperature.

Page 9 of 64

Resonance fluorescence :
This is the reemission of photons possessing the same energy as the absorbed photons.
This type of fluorescence is never observed in solution because of solvent interactions,
but it does occur in gases and crystals. It is also the basis of atomic fluorescence.
Rayleigh scattering : The emitted light has the same wavelength as the exciting light
since the absorbed and emitted photons are of the same energy.
Raman scattering : This is a form of inelastic scattering which involve a change in the
frequency of the incident radiation. Raman scattering involves the gain or loss of
vibrational quantum of energy by molecules

Page 10 of 64

 Sensitized fluorescenceWhen elements like thalium, zinc, cadmium or an alkali metal are
added to mercury vapour these elements are sensitized and thus gives
fluorescence.
Direct line fluorescence
Even after the emission of radiation, the molecules retain in
metastable state and finally comes to the ground state after loss of energy
by vibrational transmit.
Stepwise fluorescence
This is conventional type of fluorescence where a part of energy
is lost by vibrational transition before the emission of fluorescent
radiation.
Thermally assisted fluorescence
Here excitation is partly by electromagnetic radiation and partly
by thermal energy.

Page 11 of 64

ELECTRON SPIN
Acc to pauli exclusion principle, no two electrons in an atom can have the
same set of four quantum numbers. Ie no more than two electrons occupy
an orbital and the two will have opposite spin.
Spins are paired. molecules exhibit no net magnetic field Diamagnetic
They are neither attracted nor repelled by static magnetic field.
Free radicals unpaired electrons, have a magnetic moment and attracted
by magnetic field - Paramagnetic

Free radical
Page 12 of 64

 Singlet
All electron spins paired, no splitting of electronic energy levels
when the molecule is exposed to a magnetic field. (diamagnetic)
Double
The ground state for free radical, ie there are two possible
orientations for odd electrons in magnetic field, and each impart
slightly different energies to the system.
Triplet
Excited state electron promoted with unpaired spin, the spin of
the exited electrons have become unpaired and thus parallel.
(paramagnetic)
Less energetic that excited singlet state
Change between singlet and triplet unlikely, and less intense than
analogous singlet to singlet absorption
Higher lifetime (10 -4 s to several seconds)

Page 13 of 64

 Singlet ground state: state in which electrons in a molecule are paired. [ ]

Singlet excited state: state in which electrons are unpaired but of opposite
spins. [ ]
Triplet state: state in which unpaired electrons of same spin are present. [ ]
Excitation process: absorption of energy or light followed by conversion from
ground state to excite state.
Relaxation process: process by which atom or molecule losses energy &
returns to ground state.
Most molecules have an even number of electrons which means that all of
their electrons in the ground state must be paired

Page 14 of 64

ENERGY LEVEL DIAGRAM :

Let ground , the first excited, second excited etc. electronic states be: S0, S1,
S2 and etc..for all of the possible singlet states.
Triplet states would then be T1, T2 and so on .
All electronic state has several vibrational and rotational states. Let ground
, the first excited, second excited etc. electronic states be: S0, S1, S2 and
etc..for all of the possible singlet states.

Page 15 of 64

Partial energy level diagram Jablonski Diagram

Page 16 of 64

Page 17 of 64

Page 18 of 64

TERMS FROM ENERGY-LEVEL DIAGRAM

Term: Absorption
Effect: Excite
Process: Analyte molecule absorbs photon (very fast ~ 10-14
10-15 s); electron is promoted to higher energy state.
Slightly different wavelength excitation into different
vibrational energy levels.

Term: Vibrational Relaxation

Effect: Deactivate,
Radiationless
Process: Collisions of excited state analyte molecules with
other molecules loss of excess vibrational energy and
relaxation to lower vibrational levels (within the excited
electronic state)
Page 19 of 64

Term: Internal conversion

Effect: Deactivate,
Radiation less
Process: Molecule passes to a lower energy state
vibrational energy levels of the two electronic states overlap
(see diagram) and molecules passes from one electronic state
to the other.

Term: Fluorescence

Effect: Deactivate,
Emission of h
Process: Emission of a photon via a singlet to singlet
transition (short lived excited state ~10-7 10-9 s).

Page 20 of 64

Term: Intersystem Crossing

Effect: Deactivate,
Radiationless

Process: Spin of electron is reversed leading to change

from singlet to triplet state. Occurs more readily if
vibrational levels of the two states overlap. Common in
molecules with heavy atoms (e.g., I or Br)

Page 21 of 64

Term: External Conversion

Effect: Deactivate,
Radiationless

Process: Collisions of excited state analyte molecules with

other molecules molecule relaxes to the ground state
without emission of a photon.
Term: Phosphorescence
Effect: Deactivate,
Emission of h
Process: Emission of a photon via a triplet to single transition
(longlived excited state ~ 10-4 101s). after intersystem
crossing further deactivation occur by internal or external
conversion or phosphorescence. Ordinarily observed only at
low temperatures in highly viscous media or by using special
techniques to protect the triplet state
Page 22 of 64

At room temperature molecules stable electronic state ground state

Absorption of radiation
Excited state - The excited molecules are
extremely shot lived and deactivation occurs - emission of radiation
Fluorescence molecules in singlet excited state (unstable) to ground state emits
radiation as UV or visible light
Phosphorescence excited molecules undergo intersystem crossing (no unpaired
electrons to 2 unpaired electrons) to triplet excited state (metastable state) then to
ground state by emission

Molecules at excited state emits radiation ie., vibrational deactivation followed by

quenching ( decrease in fluorescence intensity)
Excitation wavelength where absorption of radiation takes place
Emission wavelength molecules emits radiation

Specific for any

substance

Excitation absorption of radiation 10-15 sec

Fluorescence emission of radiation at higher wavelength 10-6 to 10-10 sec
Intersystem crossing 10-7 to 10-8 sec
Phosphorescence 10-6 to 10 sec
Page 23 of 64

Deactivation processes for an excited state:

Vibrational relaxation: fluorescence always involves a transition
from the lowest vibrational states of an excited electronic state;
electron can return to any one of the vibrational levels of the
ground state; 10 -12 s;
Internal conversion: intramolecular processes by which a
molecule passes to a lower-energy electronic state without
emission of radiation.
External conversion: interaction and energy transfer between
the excited molecule and the solvent or other molecules.
Intersystem crossing: the spin of an excited electron is reversed
and a change in multiplicity of the molecule results.
Phosphorescence: an excited triplet state to give radioactive
emission. emission: a photon is emitted.

Page 24 of 64

Comparison of Triplet and Singlet

magnetic effect
electron transition for
emission
radiation induced
excitation
luminescence
life time

Singlet
diamagnetic
more probable
more probable

Triplet
paramagnetic
less probable
(unlikely)
less probable

Fluorescence
short, < 10-5 s to
10-9 s

Phosphorescence
long, 10-5 s to several
seconds or longer

Fluorescence and Phosphorescence

Page 25 of 64

Comparison of Fluorescence and Phosphorescence

Fluorescence
life time
short, < 10-5s
electron spin
no
excited states
singlet
quantum yield
high
temperature most temperature

Phosphorescence
long, several seconds
yes
triplet
low
low temperature more likely

Page 26 of 64

Fluorescence occurs when molecules absorb UV or Visible

radiation.
All the molecules are not fluorescent
A fluorophore (or fluorochrome, similarly to a chromophore)
is a fluorescent chemical compound that can re-emit light
upon light excitation. Fluorophores typically contain several
combined aromatic groups, or plane or cyclic molecules with
several bonds.

The extent of fluorescence can be quantified

Its denoted by () quantum yield or quantum efficiency
Page 27 of 64

Beer lamberts law

A= Kct

log I0/I = Kct

I0/I = 10 Kct

I/I0= 10 Kct

I = I010 Kct
I0- intensity of incident light I Intensity of transmitted light
Fluorescence F (I0-I) or F Ia
quantum yield or quantum efficiency
Page 28 of 64

F (I0-I)
F = K (I0- I010 Kct)

F = K I0 (1-10 Kct)
F = K I0 (1-10 A)

A= Kct

F = K I0 (2.303A)
At fixed instrumental condition I0 , t fluorescence is proportional to concentration
FC
F=KxC
K = 2.303I0kt

Page 29 of 64

Factors affecting fluorescence

1. Conjugation
2. Nature of substituents
3. pH
4. Adsorption
5. Oxygen
6. Temperature and viscosity
7. Photodecomposition
8. Intensity of incident light
9. Rigidity of structure
10. Quenchers
11. Solvent

Page 30 of 64

1. Conjugation
All the molecules does not exhibit fluorescence
Molecules that absorb radiation emits radiation

absorbance - fluorescence
Molecules having conjugated double bonds or bonds fluorescent.
Lowenergy * (aromatic): most intense fluorescence.
Heterocycles do not fluoresce; heterocycles fused to other rings fluoresce.
2. Nature of substituents

electron donating groups increases fluorescence eg: NH2, OH

Electron withdrawing groups decreases fluorescence eg: COOH, -NO2, -N=N-,
halides
substituents like SO3H, -NH4, alkyl groups does not have any effect on
fluorescence

Page 31 of 64

3. pH
The changes in pH of the medium change the degree of ionization of the
acidic/basic 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.
Buffers can be used to adjust the pH.
4. Adsorption
Adsorption of sample on surface of container wall affect fluorescence
Strong stock solutions should be prepared from which dilutions are made as
required
Eg: Quinine adsorbs easily into walls of container

Page 32 of 64

5. Oxygen
The paramagnetic substances like dissolved oxygen and many transition metals
with unpaired electrons dramatically decrease fluorescence and cause interference
in fluorimetric determinations.
The paramagnetic nature of molecular oxygen promotes intersystem crossing from
singlet to triplet states in other molecules.
The longer lifetimes of the triplet states increases the opportunity for radiation
less deactivation to occur.
6. Temperature and viscosity

Temperature
- viscosity
molecules by collision

- fluorescence

- due to deactivation of excited

7. Photodecomposition

Some compounds undergo photodecomposition with the incident light

Decomposition can be avoided by reducing intensity of incident light or by using
narrow slit width or filets ( neutral density)

Page 33 of 64

8. Intensity of incident light

Intensity of incident light - fluorescent intensity
By increasing the width of excitation slit
9. Rigidity of structure
Rigid structure have high fluorescent intensity
Flexible structure low fluorescent intensity

Anthracene rigid

Biphenyl flexible

Page 34 of 64

10. Quenchers
Quenching reduction in fluorescent intensity by presence of substances
There are four types
1. Collisional quenching dispersing the absorbed energy as heat due to
collision with quenching species
- can be influenced by presence of halides, heavy metals, temperature
changes
eg: Quinine in 0.05M H2SO4 highly fluorescent
Quinine in 0.1M HCl non fluorescent
2. Static quenching quenching species forms complex with fluorescent
substances and alter the fluorescence

Eg: caffeine reduces the fluorescent intensity of riboflavin by forming

complex

Page 35 of 64

3. Self quenching or concentration quenching - with change in concentration the

fluorescent property changes
Eg: at low concentration of substance (g, ng) linearity is obtained
at high concentration of substance (mg) no linearity
with increase in concentration constant increase in fluorescent intensity does
not occur
At high concentration the radiation emitted before falls on detector gets
absorbed by adjacent molecule followed by reemission
4. Chemical quenching presence of heavy metals, electron withdrawing groups,
halides , Oxygen and pH alters the fluorescent intensity
These agents decreases the fluorescent intensity

Page 36 of 64

EXCITATION AND EMISSION SPECTRA

Excitation spectrum: Emission wavelength is fixed; excitation
wavelength is scanned
Monochromator or filters selected to allow only one of
fluorescent light to pass through to the detector.
Excitation wavelength is varied at each excitation
increment fluorescent photons at the fixed emission are
collected.
The emission intensity (i.e., the number of fluorescent
photons collected) at each increment varies as the
excitation comes closer to or goes further from the of
maximum absorption this is why an excitation spectrum
looks like an absorption spectrum.

Page 37 of 64

 Emission spectrum: Excitation wavelength is fixed;

emission wavelength is scanned
Monochromator or filter is selected to allow only one of
excitation light to pass onto the sample.

Emission is varied fluorescent photons are collected

at each incremental emission .
The emission intensity (i.e., the number of fluorescent
photons collected) at each increment varies as the
emission is changed.
Spectrum shows at what the fluorescence intensity is a
maximum for a given excitation .

Page 38 of 64

Page 39 of 64

The absorption and emission spectra will

have an approximate mirror image
relationship if the spacings between
vibrational levels are roughly equal and if
the transition probabilities are similar.

Page 40 of 64

INSTRUMENTATION

Page 41 of 64

Page 42 of 64

1)SOURCE OF LIGHT: Mercury vapour lamp: Mercury vapour at high pressure give intense
lines on continuous background above 350nm.low pressure mercury
vapour gives an additional line at 254nm.it is used in filter fluorimeter.

Xenon arc lamp: It give more intense radiation than mercury vapour
lamp. it is used in spectrofluorimeter.

Page 43 of 64

 Tungsten lamp:- If excitation has to be done in visible

region this can be used. It is used in low cost instruments.

Tunable dye lasers :- pulsed nitrogen laser as the primary source

- radiation in the range between 250-650nm is produced

Page 44 of 64

2) FILTERS AND MONOCHROMATORS: Filters: these are nothing but optical filters works on the
principle of absorption of unwanted light and transmitting the
required wavelength of light. In inexpensive instruments
fluorimeter primary filter and secondary filter are present.
Primary filter:absorbs visible radiation and transmit UV radiation.
Secondary filter:absorbs UV radiation and transmit
visible radiation.

Page 45 of 64

 Monochromators: they convert

polychromatic
light
into
monochromatic light. They can
isolate a specific
range of
wavelength or a particular
wavelength of radiation from a
source.
Excitation monochromators:provides suitable radiation for
excitation of molecule .
Emission monochromators:- isolate
only the radiation emitted by the
fluorescent molecules.

Page 46 of 64

3) Sample cells: These are ment for holding liquid samples.

These are made up of quartz and can have various shapes
ex: cylindrical or rectangular etc.

4) Detectors: Photometric detectors are used

they are

Barrier layer cell/Photo voltaic cells

Photomultiplier cells
Page 47 of 64

1. Barrier layer /photovoltaic cell:

it is employed in inexpensive instruments.
For ex: Filter Fluorimeter.
It consists of a copper plate coated with a thin layer of
cuprous oxide (Cu2o). A semi transparent film of silver is
laid on this plate to provide good contact.
When external light falls on the oxide layer, the electrons
emitted from the oxide layer move into the copper plate.
Then oxide layer becomes positive and copper plate
becomes negative.

Page 48 of 64

 Hence an emf develops between the oxide layer and copper plate and
behaves like a voltaic cell. So it is called photovoltaic cell..
A galvanometer is connected externally between silver film and copper
plate and the deflection in the galvanometer shows the current flow
through it. The amount of current is found to be proportional to the
intensity of incident light

Page 49 of 64

BARRIER LAYER CELL

Page 50 of 64

2. Photomultiplier tubes:
These are incorporated in expensive instruments like
spectrofluorimeter. Its sensitivity is high due to
measuring weak intensity of light.
The principle employed in this detector Is that,
multiplication of photoelectrons by secondary
emission of electrons.
This is achieved by using a photo cathode and a series
of anodes (Dyanodes). Up to 10 dyanodes are used.
Each dyanode is maintained at 75-100Vhigher than
the preceding one.

Page 51 of 64

 At each stage, the electron emission is multiplied by a

factor of 4 to 5 due to secondary emission of electrons and
hence an overall factor of 106 is achieved.
.

PMT can detect very weak signals, even 200 times weaker
than that could be done using photovoltaic cell. Hence it is
useful in fluorescence measurements.
PMT should be shielded from stray light in order to have
accurate results.

Page 52 of 64

FIGURE 10
Page 53 of 64

INSTRUMENTS

The most common types are: Single beam (filter) fluorimeter

Double beam (filter )fluorimeter
Spectrofluorimeter(double beam)

Page 54 of 64

Single beam filter fluorimeter

It contains tungsten lamp as a source of light and has an
optical system consists of primary filter.

The emitted radiations is measured at 900 by using a

secondary filter and detector. Primary filter absorbs visible
radiation and transmit uv radiation which excites the
molecule present in sample cell.
In stead of 90 if we use 180 geometry as in colorimetry
secondary filter has to be highly efficient other wise both
the unabsorbed uv radiation and fluorescent radiation will
produce detector response and give false result.

Page 55 of 64

 Single beam instruments are simple in construction

cheaper and easy to operate.

Page 56 of 64

In double beam fluorimeter :

it is similar to single beam except that the two
incident beams from a single light source pass
through primary filters separately and fall on
the another reference solution.
Then the emitted radiations from the sample or
reference sample pass separately through
secondary filter and produce response
combinly on a detector.

Page 57 of 64

SPLITTER

FIGURE
Page12
58 of 64

In spectrofluorimeter: In this primary filter in double beam

fluorimeter is replaced by excitation
monochromator and the secondary filter is
replaced by emission monochromator.
Incident beam is split into sample and
reference beam by using beam splitter.

Page 59 of 64

Page 60 of 64

Difference between fluorometry and

spectrophotometry
Difference

Fluorometry

Spectrophotometry

Nature

Measuring emission

Measuring absorption

Sensitivity

Nanogram scale (102 -104

times sensitive)

Microgram

Instrumentation

Single beam or double beam

Use 2 filter monochromatic

Single or double beam

Only one

Selectivity

more

Less selective

Lambda
maximum

Absorption and emission

Absorption only

Equations

F=2.3 QI. B.C

A = . B.C

Calibration

Quinine in dilute H2SO4

Potassium chromate in H2O

Page 61 of 64

Applications of fluorimetry
Determination of Organic substances
Plant pigments, steroids, proteins, etc. can be determined at
low concentrations.
Generally used to carry out qualitative as well as quantitative
analysis for a great aromatic compounds present in cigarette
smoking, air pollutant concentrates & automobile exhausts.
Determination of inorganic substances
Extensively used in the field of nuclear research for the
determination of uranium salts.
Determination of vitamin B1 (thiamine) in food samples like meat
cereals etc.

Page 62 of 64

 Determination of Vitamin B2 (riboflavin). This method is generally

used to measure the amount of impurities present in the sample.
Most important applications are found in the analyses of food
products, pharmaceuticals, clinical samples and natural products.
Fluorescent indicators:
Intensity and color of the fluorescence of many substances depend upon the
pH of solutions. These are called as fluorescent indicators and are generally
used in acid base titrations.
Eosin : pH 3.0-4.0 colorless to green
Fluorescein : pH 4.0-6.0 colorless to green

Page 63 of 64

Page 64 of 64