Fluorescence spectroscopy and its theory, instrumentation

1. FLUORIMETRY
(MOLECULAR LUMINESCENCE
SPECTROSCOPY)
Mrs. Poonam Sunil Aher (M.Pharm, PhD)
Assistant Professor
Sanjivani College of Pharmaceutical Education
and Research (Autonomous),
Kopargaon, Ahmednagar-423603 (M.S.), INDIA
Mobile: +91-9689942854

4. • TWO ORBITAL
• 1. SINGLET :
• A. SINGLET GROUND STATE(SGS):
• B. SINGLET EXCITED STATE(SES)
• 2. TRIPLET:
• A. TRIPLET GROUND STATE(TGS)
• B. TRIPLET EXCITED STATE(TES)

5. SES
SGS
TES
TGS
Paired electrons
Unpaired electron
Dimagnetic
effect
Paramagnetic effect

9. FLUORESCENCE
 It is a phenomenon of emission of
radiation when the molecules are
exited by radiation at certain
wavelength.

10.  FLUORIMETRY:- It is measurement of fluorescence
intensity at a particular wavelength with the help of a
filter and detector used in fluorimeter or a
spectrofluorimeter

11.  PRINCIPLE:- Molecule contains electrons, electrons
and non bonding (n) electron.
 The electrons may be present in bonding molecular
orbital. It is called as highest occupied molecular orbital
(HOMO).It has lest energy and more stable.
 When the molecules absorbs radiant energy from a
light source, the bonding electrons may be promoted to
anti bonding molecular orbital (LUMO).
 LUMO is least Occupied Molecular orbital.It has more
energy and hence less stable.
 The process of promotion of electrons from HOMO to
LUMO with absorption of energy is called as excitation.

15.  Process involved in Absorption and emission process:
 When light of appropriate wavelength is absorbed by a
molecule the electrons are promoted from singlet
ground state to singlet excited state. once the molecule
is in this excited state relaxation can occur via several
process.
 For ex by emission of radiation . The process can be
the following
 1) Collisional deactivation process
 2)Fluorescence
 3)Phosphorescence.

16. FIGURE 1

18.  Collisional de activation :- In which entire energy
lost due to collision de activation and no radiation
emitted.
 Fluorescence:-excited singlet state is highly
unstable. Relaxation of electrons from excited
singlet to singlet ground state with emission of light.
 Phosphorescence:-At favorable condition like low
temperature and absence of oxygen there is
transition from excited singlet state to triplet state
which is called as inner system crossing. The
emission of radiation when electrons undergo
transition from triplet state to singlet ground state is
called as phosphorescence.

19.  Deactivation Processes
 A molecule that is excited can return to the ground state
by several combinations of mechanical steps that will be
described below and shown in Figure.
 The deactivation process of fluorescence and
phosphorescence involve an emission of a photon
radiation.
 The wiggly arrows in Figure are deactivation processes
without the use of radiation.
 The favored deactivation process is the route that is
most rapid and spends less time in the excited state.
 If the rate constant for fluorescence is more favorable in
the radiation less path, the fluorescence will be less
intense or absent.

20.  1. Intersystem crossing:
 Intersystem crossing is a process where there is a
crossover between electronic states of different
multiplicity as demonstrated in the singlet state to a
triplet state (S1 to T1).
 The probability of intersystem crossing is
enhanced if the vibration levels of the two states
overlap.
 Intersystem crossing is most commonly observed
with molecules that contain heavy atom such as
iodine or bromine.
 The spin and orbital interaction increase and the
spin become more favorable.
 Paramagnetic species also enhances intersystem
crossing, which consequently decreases
fluorescence.

21.  2. Internal conversion: Internal conversion is an
intermolecular process of molecule that passes to a lower
electronic state without the emission of radiation.
 It is a crossover of two states with the same multiplicity
meaning singlet-to-singlet or triplet-to-triplet states.
 The internal conversion is more efficient when two electronic
energy levels are close enough that two vibrational energy
levels can overlap as shown in between S1 and S2.
 Internal conversion can also occur between S0 and S1 from a
loss of energy by fluorescence from a higher excited state, but
it is less probable. The mechanism of internal conversion from
S1 to S0 is poorly understood.
 For some molecules, the vibrational levels of the ground state
overlaps with the first excited electronic state, which leads to
fast deactivation.
 These usually occur with aliphatic compounds (compound that
do not contain ring structure), which would account for the
compound is seldom fluorescing.
 Deactivation by energy transfer of these molecules occurs so
rapidly that the molecule does not have time to fluoresce.

22.  3. Vibrational Relaxation: A molecule maybe to
promoted to several vibrational levels during the
electronic excitation process.
 Collision of molecules with the excited species and
solvent leads to rapid energy transfer and a slight
increase in temperature of the solvent.
 Vibrational relaxation is so rapid that the lifetime of a
vibrational excited molecule (<10-12) is less than the
lifetime of the electronically excited state.
 For this reason, fluorescence from a solution always
involves the transition of the lowest vibrational level of
the excited state.
 Since the space of the emission lines are so close
together, the transition of the vibrational relaxation can
terminate in any vibrational level of the ground state.

23.  4. External Conversion: Deactivation of the
excited electronic state may also involve the
interaction and energy transfer between the excited
state and the solvent or solute in a process
called external conversion.
 Low temperature and high viscosity leads to
enhanced fluorescence because they reduce the
number of collision between molecules, thus
slowing down the deactivation process.

24.  5. Phosphorescence:
 Deactivation of the electronic excited state is also involved in
phosphorescence.
 After the molecule transitions through intersystem crossing to the
triplet state, further deactivation occurs through internal or external
fluorescence or phosphorescence.
 A triplet-to-singlet transition is more probable than a singlet-to-
singlet internal crossing.
 In phosphorescence, the excited state lifetime is inversely
proportional to the probability that the molecule will transition back
to the ground state.
 Since the lifetime of the molecule in the triplet state is large (10-
4 to 10 second or more), transition is less probable which suggest
that it will persist for some time even after irradiation has stopped.
 Since the external and internal conversion compete so effectively
with phosphorescence, the molecule has to be observed at lower
temperature in highly viscous media to protect the triplet state.

25. Rates of Absorption and Emission comparison.```
Process ` `
`````` ``````````````````````````````
`
10-15 (instantaneous)
Internal Conversion ```` `
`` Sn
* → Sn 10-12 to 10-10
Intersystem Crossing S1 → T1 10-11 to 10-6
Fluorescence S1 → S0 10-9 to 10-6
Phosphorescence T1 → S0 10-3 to 10
Non-Radiative Decay S1 → S0
T1 → S0
10-7 to 10-5
10-3 to 10

26. 1. Concentration
2. Quantum yield of fluorescence
3. Intensity of incident light
4. Adsorption
5. Oxygen
6. Ph
7. Temperature& viscosity
8. Photodecomposition
9. Quenchers
10. Scatter

27. Fluorescence intensity is praportnal to
concentration of substance only when the
absorbance is less than 0.02
A=log IoIt or A= abc
Io=intensity of incident light
a= absorptivity of constant
b= Pathlength
c= concentration
Concentration:-

28. ()=NUMBER OF PHOTONS EMITTEDNUMBER OF
PHOTONS ABSORBEDS
It Is Always Less Than 1.0 Since Some
Energy Is Lost By Radiation less Pathways
(Collisional, Intersystem Crossing, Vibrational
Relaxation)

29. Increase In The Intensity Of Incident Light On The
Sample Fluorescence Intensity Also Increases.

Adsorption Of Sample Solution In The Container
May Leads To A Serious Problem.
 OXYGEN:-
Oxidation of fluorescent species to a non
fluorescent species, quenches fluorescent substance.
 Ph:-
Alteration of ph of a solution will have significant
effect on fluorescence
For ex Aniline in alkali medium gives visible
fluorescence but in acidic condition gives fluorescence
in visible region.

30.  Temperature and viscosity:-
 Temperature Increases Can Increase the
collisional de activation, and reduce fluorescent
intensity.
 If viscosity of solution is more the frequency of
collisions are reduced and increase in fluorescent
intensity.
 Photochemical decomposition:-
 Absorption of intense radiation leads to photochemical
decomposition of a fluorescent substance to less
fluorescent or non fluorescent substance.

31.  Quenchers:-
 Quenching is the reduction of fluorescence intensity
by the presence of substance in the sample other
than the fluorescent analyte.
 Quenching is following types:-

32.  INNER FLUORESCENT EFFECT:- Absorption Of
Incident (uv) Light Or Emitted (fluorescent) Light By
Primary And Secondary Filters Leads To Decrease
In Fluorescence intensity.
 SELF QUENCHING:-At Low Concentration
Linearity Is Observed, At High Concentration Of
The Same Substance Increase In Fluorescent
Intensity Is Observed. This phenomena is called
self quenching.

33.  COLLISONAL QUENCHING:- Collisions between
the fluorescent substance and halide ions leads to
reduction in fluorescence intensity.
 STATIC QUENCHING:- This occurs because of
complex formation between the fluorescent
molecule and other molecules.
 Ex: caffeine reduces fluorescence of riboflavin.

34. Scatter is mainly due to colloidal
particles in solution. Scattering of incident light
after passing through the sample leads to
decrease in fluorescence intensity.

35. INSTRUMENTATION

36. FIGURE 2

37. INSTRUMENTATION
SOURCE OF LIGHT
FILTERSAND MONOCHROMATORS
SAMPLE CELLS
DETECTORS

38.  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.
FIGURE 3

39.  xenon arc lamp: It give more intense radiation
than mercury vapour lamp. it is used in
spectrofluorimeter.
 tungsten lamp:- If excitation has to be done in
visible region this can be used. It is used in low cost
instruments.
FIGURE 4
FIGURE 5

40.  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.
FIGURE 6

41.  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.
FIGURE 7

42.  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
FIGURE 8

43.  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.

44.  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

45. BARRIER LAYER CELL
FIGURE 9

46.  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.

47.  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.
.

48. FIGURE 10

49. INSTRUMENTS
The most common types are:-
 Single beam (filter) fluorimeter
 Double beam (filter )fluorimeter
 Spectrofluorimeter(double beam)

50.  Instruments:-
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.

51.  Single beam instruments are simple in construction
cheaper and easy to operate.

FIGURE 11

52. 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.

53. SPLITTER
FIGURE 12

54.  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.

55. FIGURE 13

56. APPLICATIONS
1)Determination of inorganic substances.
Al3+,Li+,ZN2+
2)Determination of thiamine Hcl.
3)Detemination of phenytoin.
4)Determination of indoles, phenols, &
phenothiazines
5) Determination of napthols, proteins, plant pigments
and steroids.
6) Fluorimetry ,nowadays can be used in detection of
impurities in nanogram level better than
absorbance spectrophotometer with special
emphasis in determining components of sample at
the end of chromatographic or capillary column.

57.  Determination of ruthenium ions in presence of other
platinum metals.
 Determination of boron in steel, aluminum in alloys,
manganese in steel.
 Determination of boron in steel by complex formed with
benzoin.
 Estimation of cadmium with 2-(2 hydroxyphenyl)
benzoxazole in presence of tartarate .
 Respiratory tract infections.

58. S.No Name of the
compound
Experimental
conditions/ PH
Emission
wavelength
1 Adrenaline 1 335
2 Cynacobalamine 7 305
3 Riboflavin 6 520
4 Morphine 7 350
5 Hydrocortisone Acidic 520
6 Pentobarbitone 13 440
7 Amylobarbitone 14 410
Table 1