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1. Spectrofluorimetry
or
fluorimetry

2. History
The term fluorescence comes from the mineral
fluorspar (calcium fluoride) when Sir George G.
Stokes observed in 1852 that fluorspar would
give off visible light (fluoresce) when exposed to
electromagnetic radiation in the ultraviolet
wavelength.

3. 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
LUMINESCENCE
FLUORESCENCE
PHOSPHORESCENCE
SPECTROSCOPY
CHEMILUMINESCENCE

4. LUMINESCENCE SPECTROSCOPY
Absorption first -
Followed by emission
in all directions, usually
at a lower frequency

5. LUMINESCENCE SPECTROSCOPY
• In favorable cases, luminescence
methods are amongst some of the most
sensitive and selective of analytical
methods available.
• Detection Limits are as a general rule at
ppm levels for absorption
spectrophotometry and ppb levels for
luminescence methods.

6. • 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 flourescent substances.

7. • 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.

8. • Chemiluminescence is based upon emission of light
from an excited species formed as a result of a
chemical reaction.
• Collectively, fluorescence and phosphorescence are
known as photoluminescence
• Fluorimetry is the most commonly used luminescence
method.
• Phosphorimetry usually requires at liquid nitrogen
temperatures (77K).
• The terms fluorimetry and fluorometry are used
interchangeably in the chemical literature.

9. Energy Level Diagram
s2
SINGLET STATES TRIPLET STATES
Ground
State
s1
T
T
1
2
INTERSYSTEM
CROSSING
VIBRATIONAL
RELAXATION
FLUORESCENCE PHOSPHORESCENCE
INTERNAL
CONVERSION CONVERSION
INTERNAL
   

11. • Following absorption of radiation, the molecule can lose the
absorbed energy by several pathways.
• One competing process is vibrational relaxation which involves
transfer of energy to neighbouring molecules which is very rapid
in solution (10-13 sec).
• In the gas phase, molecules suffer fewer collisions and it is more
common to see the emission of a photon equal in energy to that
absorbed in a process known as resonance fluorescence.
• In solution, the molecule rapidly relaxes to the lowest vibrational
energy level of the electronic state resulting due to internal
conversion which shifts the molecule from S2 to an excited
vibrational energy level in S1.
• Following internal conversion, the molecule loses further energy
by vibrational relaxation. Because of internal conversion and
vibrational relaxation, most molecules in solution will decay to
the lowest vibrational energy level of the lowest singlet electronic
state before any radiation is emitted.

12. • When the molecule has reached the lowest vibrational energy
level of the lowest singlet electronic energy level then a
number of events can take place.
• the molecule can lose energy by internal conversion without
loss of a photon of radiation (least likely).
• the molecule can emit a photon of radiation equal in energy to
the difference in energy between the singlet electronic level
and the ground-state, this is termed fluorescence.
• the molecule can undergo intersystem crossing which involves
an electron spin flip from the singlet state into a triplet state.
• Following this the molecule decays to the lowest vibrational
energy level of the triplet state by vibrational relaxation.
• the molecule can then emit a photon of radiation equal to the
energy difference between the lowest triplet energy level and
the ground-state in a process known as phosphorescence.

13. Fluorescence & Phosphorescence
• In fluorescence, the lifetime of the
molecule in the excited singlet state is
10-9 to 10-7 sec.
• In phosphorescence, the lifetime in the
excited singlet state is 10-6 to 10 sec
(because a transition from T1 to the
ground state is spin forbidden).

14. the principle of spectrofluorometer
• It is an analytical device depends on the fluorescence
phenomenon which is a short-lived type of
photoluminescence created by electromagnetic
excitation.
• That is, fluorescence is generated when a molecule
transmits from its ground state So to one of several
vibrational energy levels in the first excited electronic
state, S1, or the second electronic excited state, S2,
both of which are singlet states.
• Relaxation to the ground state from these excited
states occurs by emission of energy through heat
and/or photons.

16. • The difference between the excitation and
emission wavelengths is called the Stokes
shift.
• Stokes’ studies of fluorescent substances led
to the formulation of Stokes’ Law, which
states that the wavelength of fluorescent light
is always greater than that of the exciting
radiation. Thus, for any fluorescent molecule,
the wavelength of emission is always longer
than the wavelength of absorption.

17. Classification
• 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’s fluorescence: wavelength of emitted
radiation is shorter than absorbed radiation.
– Resonance fluorescence: wavelength of emitted
radiation is equal to that of absorbed radiation.

18. FLUORESCENCE AND
CHEMICAL STRUCTURE
Fluorescence is most commonly observed in
compounds containing aromatic functional
groups with low energy.
Most unsubstituted aromatic hydrocarbons
show fluorescence - quantum efficiency
increases with the no: of rings and degree of
condensation.

19. CONTD…
Simple heterocyclic do not exhibit
fluorescence.

20. Fusion of heterocyclic nucleus to benzene ring
increases fluorescence.

21. Substitution on the benzene ring shifts
wavelength of absorbance maxima and
corresponding changes in fluorescence
peaks
 Fluorescence decreases with
increasing atomic no: of the
halogen.
 Substitution of carboxylic acid or
carboxylic group on aromatic
ring inhibits fluorescence.

22.  Fluorescence is favored in molecules with
structural rigidity.
 organic chelating agents complexed with
metal ion increases fluorescence.

23. • Experimentally it is found that fluorescence is
favoured in rigid molecules, eg.,
phenolphthalein and fluorescein are
structurally similar as shown below. However,
fluorescein shows a far greater fluorescence
quantum efficiency because of its rigidity.
phenolphthalein

24. • It is thought that the extra rigidity
imparted by the bridging oxygen group in
Fluorescein reduces the rate of
nonradiative relaxation so that emission
by fluorescence has sufficient time to
occur.
Fluorescein

25. Fluorescence Spectra
• Photoluminescence spectra are recorded by
measuring the intensity of emitted radiation as a
function of either the excitation wavelength or
the emission wavelength.
• The excitation spectra is determined by
measuring the emission intensity at a fixed
wavelength , while varying the excitation
wavelength. It is useful for selecting the best
excitation wavelength for a quantitative or
qualitative analysis.
• The emission spectra is determined by
measuring the variation in emission intensity
wavelength for a fixed excitation wavelength.

27. What is The fluorescence quantum yield (Φf)?
• It is the quantitative expression of the
fluorescence efficiency, which is the
fraction of excited molecules returning to
the ground state by fluorescence.
• Quantum yields range from 1, when every
molecule in an excited state undergoes
fluorescence, to 0 when fluorescence does
not occur.

28. • A molecule’s fluorescence quantum yield is
influenced by external Variables such as:
• temperature
• viscosity of solvent
• pH
• Increasing temperature generally decreases Φf
because more frequent collisions between the
molecule and the solvent increases external
conversion.
• Decreasing the solvent’s viscosity decreases Φf
for similar reasons.
• For an analyte with acidic or basic functional
groups, a change in pH may change the analyte’s
structure and, therefore, its fluorescent
properties.

29. What can specrofluorometer do?
• It has been used for the direct or indirect
quantitative and qualitative analysis by
measuring the fluorescent intensity F.
• It is relatively inexpensive and sensitive (the
sensitivity of fluorescence is approximately
1,000 times greater than absorption
spectrophotometric methods).

30. • fluorescent intensity F is dependent on both
intrinsic properties of the compound
(fluorescence quantum yield Φf), and on readily
controlled experimental parameters including:
• intensity of the absorbed light I0
• molar absorption coefficient Ɛ
• path length of the cell b
• concentration of the fluorophor in solution c

31. • At low concentrations of fluorophore, the
fluorescence intensity of a sample is
essentially linearly proportional to
concentration.
• However, as the concentration increases, a
point is reached at which the intensity
increase is progressively less linear, and the
intensity eventually decreases as
concentration increases further.

32. fluorescence intensity
concentrations

33. • The most common reason for this is Inner
filter effect that, as the concentration of the
sample increases, the light intensity
experienced by some of the fluorescent
molecules is lower than that experienced by
others. When excitation intensity decreases,
so does fluorescence emission intensity.
• It is generally necessary to use
concentrations that result in absorbance
values of 0.1 or lower to observe
concentration dependent emission.

34. • As the concentration of molecules in a solution
increases, probability increases that excited
molecules will interact with each other and lose
energy through processes other than fluorescent
emission. Any process that reduces the probability of
fluorescent emission is known as quenching.
• Other parameters that can cause quenching include:
• presence of impurities
• increased temperature
• reduced viscosity of the solution media

35. Decrease in fluorescence intensity due to specific
effects of constituents of the solution.
Due to concentration, ph, pressure of chemical
substances, temperature, viscosity, etc.
Types of quenching
Self quenching
Chemical quenching
Static quenching
Collision quenching

36. Fluorescence
Concentration of
fluorescing species
Deviations at higher concentrations can be
attributed to self-quenching or self-absorption.
Fluorescence
Concentration of
fluorescing species
Calibration curve
(Low con)
calibration curve
(High con)

37. Here decrease in fluorescence intensity due to
the factors like change in pH, presence of oxygen,
halides &heavy metals.
 pH- aniline at pH 5-13 gives fluorescence but
at pH <5 &>13 it does not exhibit fluorescence.
 halides like chloride,bromide,iodide & electron
withdrawing groups like NO2,COOH etc. leads to
quenching.
 Heavy metals leads to quenching, because of
collisions of triplet ground state.

38. This occurs due to complex formation.
e.g.. caffeine reduces the fluorescence of
riboflavin by complex formation.
COLLISIONAL QUENCHING
It reduces fluorescence by collision. where no. of
collisions increased hence quenching takes place.

39. Factors affecting fluorescence intensity
• Conjugation: molecule must have conjugation ( π electron) so
that uv/vis radiation can be absorbed
• Nature of substituent groups:
– e- donating groups like NH2, OH groups enhance fluorescence.
– e- withdrawing groups like NO2, COOH reduce fluorescence.
• Fluorescent intensity is directly proportional to concentration.
• Increase in viscosity leads to decreased collisions of molecules
there by increasing fluorescent intensity.
• More rigid the structure of molecule, more the intensity of
fluorescence.
• Increase in temp leads to increased collisions b/w molecules
decreasing fluorescent intensity.
• Presence of O2 decreases the fluorescence and so de-aerated
solutions must be used and compare result obtained from that
of O2 containing solution.

40. INSTRUMENTATION
SOURCE OF LIGHT
FILTERS AND MONOCHROMATORS
SAMPLE CELLS
DETECTORS
Components of
fluorimeters and spectrofluorimeters

41. Spectrophotometer V/S Spectroflorimeter
• The fluorescence is often viewed at 90° orientation (in order to
minimise interference from radiation used to excite the
fluorescence).
• Spectroflorimeter has two monochromators
• As fluorescence is maximum between 25-30˚C, the sample holder
has the device to maintain the temperature

42. MERCURY ARC LAMP.
XENON ARC LAMP.
TUNGSTEN LAMP.
TUNABLE DYE LASERS.
Sources of light

43. In fluorimeter 10 filter (absorb Vis. radiation and
transmit UV radiation) and 20 filter (absorb UV
radiation and transmit Vis. radiation) are
present.
In spectrofluorometers, excitation
monochromators (isolates only the radiation
which is absorbed by the molecule) and
emission monochromator (isolates only the
radiation which is emitted by the molecule) are
present.
Filters and Monochromators

44. Sample and sample holder
The majority of fluorescence assays are
carried out in solution.
Cylindrical or rectangular cells fabricated
of silica or glass used.
Path length is usually 10mm or 1cm.
All the surfaces of the sample holder are
polished in fluorimetry.

45. Detectors
PHOTOVOLTAIC CELL
PHOTO TUBE
PHOTOMULTIPLIER TUBES – Best and
accurate.

46. Read out devices
The output from the detector is amplified
and displayed on a readout device which
may be a meter or digital display.
Microprocessor electronics provide
outputs directly compatible with printer
systems and computers, eliminating any
possibility of operator error in
transferring data.

47. SINGLE BEAM FLUORIMETER
DOUBLE BEAM FLUORIMETER
SPECTROFLUORIMETER(DOUBLE
BEAM)

48.  Source of light.
 The primary filter absorbs visible radiation and transmits
UV radiation.
 Emitted radiation measured at 90o by secondary filter.
 Secondary filter absorbs UV radiation and transmits
visible radiation.
Advantages
• Simple in construction
• Easy to use.
• Economical
Disadvantages
• It is not possible to use reference solution & sample solution
at a time.
• Rapid scanning to obtain Exitation & emission spectrum of the
compound is not possible.

49. Lamp
Primary
filter
Secondary
Filter
Photo
Multiplier
tube
I0
Ie
It

50. Similar to single beam instrument.
Two incident beams from light source pass through
primary filters separately and fall on either sample
or reference solution.
The emitted radiation from sample or reference pass
separately through secondary filter.
Advantages
• Sample & reference solution can be analyzed
simultaneously.
Disadvantages
• Rapid scanning is not possible due to use of filters.

51.  The primary filter in double beam fluorimeter is
replaced by excitation monochromaters.
 The secondary filter is replaced by emission
monochromaters.
 The incident beam is split into sample and
reference beam using a beam splitter.
 The detector is photomultiplier tube.
SPECTROFLUORIMETER
Advantages
• Rapid scanning to get Exitation & emission
spectrum.
• More sensitive and accuracy when compared to
filter fluorimeter.

53. • Advantages of fluorescence spectroscopy:
SENSITIVITY : It is more sensitive as concentration is low
as µg/ml or ng/ml.
PRECISION : Upto 1 % can be achieved.
SPECIFICITY : More specific than absorption method
where absorption maxima may be same for two
compounds.
RANGE OF APPLICATION : Even non fluorescent
compounds can also be converted to fluorescent
compounds by chemical compounds.
• Disadvantages:
Not useful for identification
Not all compounds fluorescence
Contamination can quench the fluorescence and hence
give false/no results

54. Environmental Significance:
• To detect environmental pollutants such as polycyclic
aromatic hydrocarbons:
• pyrene
• benzopyrene
• organothiophosphorous pesticides
• carbamate insecticides
• 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
Geology:
• Many types of calcite and amber will fluoresce under
shortwave UV. Rubies, emeralds, and the Diamond
exhibit red fluorescence under short-wave UV light;
diamonds also emit light under X ray radiation.
Applications of Spectrofluorometer:

55. Analytical chemistry:
• to detect compounds from HPLC flow
• TLC plates can be visualized if the compounds or a
coloring reagent is fluorescent
• Plant pigments, steroids, proteins, naphthols etc can
be determined at low concentrations
Biochemistry:
• used generally as a non-destructive way of tracking or
analysis of biological molecules (proteins)
• Possible direct or indirect analysis aromatic amino
acids (phenylalanine- tyrosine-tryptophan)
• Fingerprints can be visualized with fluorescent
compounds such as ninhydrin.

56.  Medicine
• Blood and other substances are sometimes detected by
fluorescent reagents, particularly where their location was not
previously known.
• There has also been a report of its use in differentiating malignant,
bashful skin tumors from benign.
 Pharmacy:
• Possible direct or indirect analysis drugs such as:
• vitamins (vitamin A -vitamin B2 -vitamin B6 -vitamin B12 -vitamin E
-folic acid)
• catecholamines (dopamine-norepinephrine)
• Other drugs (quinine-salicylic acid–morphine-barbiturates –lysergic
acid diethylamide (LSD))
• to measure the amount of impurities present in the sample.

57. Fluorescent indicators
• Intensity and colour 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.
• Eg: Eosin – pH 3.0-4.0 – colourless to green
Fluorescien – pH 4.0-6.0 – colourless to green
Quinine sulphate: blue-violet.
Acridine: green-violet

58. APPLICATIONS
EX 1. Determination of polyaromatic
hydrocarbons
– Benzo[a]pyrene is a product of incomplete
combustion and found in coal tar.

59. • Benzo[a]pyrene, is a 5-
ring polycyclic aromatic
hydrocarbon that is
mutagenic and highly
carcinogenic
• It is found in tobacco
smoke and tar
• The epoxide of this
molecule intercalates in
DNA, covalently bonding
to the guanine base
nucleotide

60. Excitation and
fluorescence spectra for
benzo(a)pyrene in H2SO4.
In the diagram the solid
line is the excitation
spectrum (the
fluorescence signal is
measured at 545 nm as
the exciting wavelength
is varied). The dashed
line is the fluorescence
spectrum (the exciting
wavelength is fixed at
520 nm while the
wavelength of collected
fluorescence is varied).
Benzo(a)pyrene

61. EX 2. Fluorimetric Drug
Analysis
• Many drugs possess high
quantum efficiency for
fluorescence. For example,
quinine can be detected at
levels below 1 ppb.
Quinine
• In addition to ethical
drugs such as quinine,
many drugs of abuse
fluoresce directly. For
example lysergic acid
diethylamide (LSD)
whose structure is:
LSD

62. • Because LSD is active in minute quantities, an extremely
sensitive methods of analysis is required. Fluorimetricaly LSD
is usually determined in urine from a sample of about 5mL in
volume. The sample is made alkaline and the LSD is
extracted into an organic phase consisting of n-heptane and
amyl alcohol. This is a "clean-up" procedure that removes
potential interferents and increases sensitivity. The LSD is
then back-extracted into an acid solution and measured
directly using and excitation wavelength of 335 nm and a
fluorescence wavelength of 435 nm. The limit of detection is
approximately 1 ppb.