Molecular fluorescence and phosphorescence spectroscopy TYBSC

1. Satish Pradhan
Dnyanasadhana College, Thane
Department of Chemistry
T.Y.B.Sc. Analytical Chemistry
Molecular Fluorescence
And
Phosphorescence Spectroscopy
1

2. Content
3.2.1 Introduction and Principle
3.2.2 Relationship of Fluorescence intensity with
concentration
3.2.3 Factors affecting Fluorescence and
Phosphorescence
3.2.4 Instrumentation and applications
3.2.5 Comparison of Fluorimetry and
Phosphorimetry
3.2.6 Comparison with Absorption methods
2

3. Introduction
• The term ‘fluorescence’
was coined by G. G.
Stokes in 1852 on the
name of the mineral
fluorspar (CaF2) that
emits visible light on
illumination with the UV
light.
3

4. Emission:
 When substance heated to about 725 K temperature ,it emits
radiation in the form of ultraviolet or visible energy is called
as Emission.
Photoluminescence:
 A body emits previously absorbed radiations even below this
temperature is called as Photoluminescence or cold light.
When such Photoluminescent substance is excited, it reemits
radiations of the same wavelength or longer wavelength.
Fluorescence.
• If the emission takes place in a time of approximately10-8 sec. or
less after absorption process is termed as Fluorescence.
4
Basic Terms

5. Lo
ng
er
wa
vel
en
gth
Molecule
U. V. Light
Re-emission
of
Radiation
in less than
10-8 sec.
5
Fluorescence : If the
emission takes place in a
time of approximately 10-8
sec. or less after
absorption process is
termed as Fluorescence.
Excited State

6. Molecule
U.V.Light
Re-emission
of
Radiation
in more than
10-8 sec.
6
Phosphorescence:
If the time elapsed
between the
absorption and
re-emission of
radiation becomes
more than 10-8 sec.
is termed as
Phosphorescence
Excited State

7. 7
Motions in a
molecule?
Rotational Translational Vibrational

8. 8
What is a
vibration in a
molecule?
Stretching of
bonds
Bending of
bonds
Internal
rotation around
single bonds.
Any change in
shape of the
molecule-

9. What is Vibration
For a C-C bond with a bond
length of 154 pm, the
variation is about 10 pm.
For C-C-C bond angle a
change of 4o is typical.
This moves a carbon atom
about 10 pm.
4o 10 pm
10 pm
154 pm
Stretching vibration
Bending vibration
9

10. For a C-C bond with a bond
length of 154 pm, the variation
is about 10 pm.
Bond length 154 pm,
10 pm.
10
Stretching vibration

11. C
C
C
4o
10 pm
11
For C-C-C bond angle a change of 4o is typical. This moves a carbon atom about
10 pm.
Bending vibration

12. How does the mass influence the vibration?
H2
I2
MM =2 g/mole
MM =254 g/mole
The greater the mass - the lower the wavenumber
12

13. 13
To begin with, the molecule is in the electronic
ground state.
In this state, the molecular orbital's are occupied
by two electrons.
You would recall from the knowledge about
Pauli’s principle, the spins of the two electrons
in the same orbital must be antiparallel.
This implies that the total spin, S, of the molecule
in the ground state is zero
G0
[½ +& -½)].
Theory:
Energy Level of Electron in a molecule

14. Total spin, S, of the molecule in the
ground state is zero [½ + ( ½)].
• This energy state is called “singlet state” and
is labeled as S0.
Singlet
state
S0.
14

15. Excitation of Electron in a
molecule
 When the molecule is excited by U.V. light
The electron spins in the excited state is called
a singlet (antiparallel) state.
 It is represented by S1.(S0—S1)
15

16. Excited singlet S1
Singlet
(antiparallel)
S1
16
S1
S0

17. Excitation of Electron in a molecule
 Similarly excited electrons will be in
parallel situation this state is called as
triplet (parallel) state.
 It is represented by T1.
17

18. Triplet
(parallel state)
T1
Excited Triplet T1 18
Excited state T1
Ground state S0

19. •Deactivation
Excited molecule can undergo a
number of relaxation processes during
the time it spends in the excited state
,this may be called as vibrational
relaxation. (S1 higher to S1 lower)
 (S1 higher to S1 lower)
19

20. Every
state of
energy
consist of
number of
vibrational
levels .
Ground state Singlet S0=
V1,V2,V3,V4
-
Excited state Singlet S1=V1,V2,V3,V4
-
---
Excited state Triplet T1=V1,V2,V3,V4
---
20

21. • Vibrational relaxation
• When the molecule in the excited
state (S1) relaxes down to the
lowest vibrational level it may emit
a photon and come down to the
electronic ground state (S0). This
process is called fluorescence and
takes about 10-9 s.
21

22. Intersystem Crossing
22
In this , the molecule in the vibrational states of a
singlet excited state may cross over to a vibrational
level of a triplet state if the two have same energy.
This process is called intersystem crossing.
(S1 to T1)

23. PHOSPHORESCENCE
In this relaxation process the excited molecule that
had crossed over to the triplet excited state by
intersystem crossing and has relaxed to the vibrational
ground state in the triplet excited state.
In such a case the molecule emits a photon and comes
down to a vibrational mode of the electronic ground
state, S0. This phenomenon is called
phosphorescence.
As the transition from a triplet state to a singlet
state is difficult because both electrons have same
spin (parallel) it take time to emit light.
23

24. Fluorescence is
instant
phenomenon
Thus, the
fluorescence
emission can take
place within 10-9 −
10-6 seconds,
Transition from
S1—S0
Phosphorescence
is delayed
fluorescence
phosphorescence
requires at least 10-4
seconds and may
take as long as 10-2
seconds.
Transition from
T1—S0
24

25. Joblonski
Diagram
•Molecular energy
level diagram
showing the
ground state and
the excited states
25

26. 26

27. Excited Singlet state S1
Ground state
singlet SO
Excited
Triplet
state S1
27
Phosphorescence
Fluorescence
Intersystem
crossing
Vibrational relaxation
E
N
E
R
G
Y

28. Instrumentation
Instruments for the
measurement of fluorescence
are known as fluorimeters or
Spectrofluorimeters.
The essential parts of a simple
fluorimeter are as follows;
28

29. Components of
fluorimeter
Radiation Source
A primary filter
A sample container,
A secondary filter:
Detector
Read out device
29

30. 30
U.V.Light
Visible
light
1. Radiation Source:
A light from a mercury-vapour lamp (or
other source of ultraviolet light) can work as
source of radiation in this technique.
Other lamps such as xenon lamp can also
work as source of radiation.

31. U.V.Light &
visible light
31
U.V.Light
Primary filter
2. A primary filter:
A primary filter transmits the part of beam
which can cause excitation of atoms to induce
fluorescence.
It select only U.V. Light but absorbs Visible
light

32. 32
 3. Sample Cell /Sample holder
Cells are usually made of silica or glass,
In practice fluorescence cells are normally
transparent on all four faces, so that except for
work of the highest precision it does not matter
much which way round the cell is placed into the
sample holder.

33. fluorescent radiation
& U.V. light
33
fluorescent
radiation
Secondary filter
Sample cell
4. A Secondary filter:
It allows only fluorescent radiation and
absorbs Visible light.

34. 4.Detector
Photocell can be used as
detector in this technique.
As sample emits fluorescent
radiation in all direction, to
measure the intensity,
Detector is placed at right
angle to that of incident
radiation.
At the other angles
scattering from solution cell
walls can cause error.
5.Readout Device
The output from the
detector is suitably
amplified and displayed on
a read out device like a
meter or digital display.
The sensitivity of the
amplifier can be changed so
as to be able to analyse
samples of varying
concentrations.
34

35. U.V. Light &
visible light
35
Photocell
/PMT
detector
Primary filter Secondary filter
Sample Cell
0. 0 0 0
Instrument: Single Beam Fluorimeter

36. 36
Primary filter
Secondary Filter
Single Beam Fluorimeter

37. 37
0.002
Primary filter
Mercury vapour lamp
Blank cuvette
Sample cuvette
Secondary filter
PMT detector Read Out Device
0. 0 0 0
Double Beam Fluorimeter

38. 38
Primary filter
Mercury vapour lamp
Blank cuvette
Sample cuvette
Secondary filter
PMT detector
Read Out Device
0. 0 0 0
Double Beam Fluorimeter

39. FACTORS AFFECTING
FLUORESCENCE AND
PHOSPHORESCENCE
Temperature
pH
Dissolved oxygen
Solvent
39
The fluorescence spectrum and intensity of a molecule
often depend strongly on the molecule’s environment.

40. • As temperature increases fluorescence decreases.
Due to change in temperature viscosity of the medium
changes.(less Viscosity)
Change in viscosity increases the number of collisions of
the molecules of the fluorophore with solvent molecules.
No. of collisions increases the probability for deactivation
by internal conversion and vibrational relaxation.
To overcome this , it is recommended to use thermo stated
cell holders.
40
Effect of Temperature

41. Effect of pH
Relatively small changes in pH can cause
considerable changes in the fluorescence intensity
and spectral characteristics of fluorescence.
The molecules containing acidic or basic functional
groups undergoes ionisation due to the changes
in pH of the medium.
It 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 41

42. Dissolved Oxygen
Oxygen and many transition metals with unpaired
electrons are paramagnetic which 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.(phosphorescence)
Presence of dissolved oxygen influences phosphorescence
too and causes a large decrease in the phosphorescence
intensity. This is actually the oxygen emission and not the
phosphorescence.
Therefore, it is advisable to make phosphorescence
measurement in the absence of dissolved oxygen.
42

43. Solvent
The changes in the “polarity” or hydrogen
bonding ability of the solvent affect the
fluorescent behaviour of the analyte.
Solvent viscosity and solvents with heavy atoms
also affect fluorescence and phosphorescence.
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.
43

44. Applications of
Fluorimetry
Inorganic
analysis
Organic
analysis
Fluorescent
Indicators
Fluorescent
Indicators
44

45. Element or compound Fluorescent emission
maximum (nm)
Example of application
Uranium Uranium sample is fused
with NaF to give Uranium
fluoride &NaF.
Uranium up to 5x10-9 gm
in 1 gm sample of Uranium
salt Nuclear Research
Ruthenium Ruthenium ion forms a
complex with 5-methyl-
1,10-phenonthroline which
forms fluorescent colour at
pH 6
Ruthenium ion in presence
of platinum
Boron as benzoin complex 450 nm Water samples and soils
,steel
Aluminium as alizarin
(garnet red complex)
580 nm Water samples and soils
CALCIUM
fluorescent chelate forms
between calcium ions and
calcein [ fluorescein
(methy1iminodiacetic acid)]
in alkaline solution
calcium in biological
materials3'
45
Inorganic analysis

46. Application in
Acid Base Titration
Name of Indicator Approx. pH range Colour change
Eosin 3-4 Colourless to
green
Fluorescein 4-6 Colourless to
green
Acridine 5.2-6.6 Green -Violet
Alpha
napthaquinone
4.4-6.3 Blue to
colourless
46
Fluorescent Indicators

47. Element or compound Fluorescent emission
maximum (nm)
Example of application
Vitamin A 500 nm Foodstuffs, vitamin tablets
Vitamin B1 (Thiamine) and B2
(riboflavin)
Oxidation product
thiochrome is
fluorescent
Food samples like meat,
cereals, vitamin tablets
Amphetamine
Codeine, Morphine
282-300 nm
345 nm
Drug preparations and body
fluids
Polyaromatic hydrocarbons 320-550 nm Environmental sam
Study of protein structure Typtophan and FAD are
produce fluorescent
colour
Study of protein
degradation,
Clinical study
47
Organic analysis

48. Enzyme Assay and Kinetic study
Enzyme Wave
length
Application
4-
Methylumbeelliferone
+ Enzyme 
Fluorescent colour
450 nm To carry
Enzyme
Assay and
Kinetic study
in
Biomlecules
48

49. Sample Cell:
• The sample cell is a narrow quartz tube of an
internal diameter of 1 to 3 mm.
Phosphoroscope
• The sample tubes are placed in liquid Nitrogen
held in a quartz Dewar flask which is then placed
in the sample holder called Phosphoroscope
Rotating- can
Phosphoroscope:
• Rotating- can Phosphoroscope: It is Hollow
cylinder having two slits and rotated by variable
speed motor.
49
Phosphorimetry : Instrumentation:

50. • Common solvent for
Phosphormetric Studies
• Solvent: EPA : mixture of ethyl
ether, isopentane and ethanol
• The ratio of solvent is 5:5:2
50

51. 51
Dewar Vessel / flask

52. 52
Phosphorimeter
Read Out Device

53. Fluorimetry
Experimental set up is
easy
Studies done at room
temperature
Less sensitive
Complex sample can not
analyzed successfully
Phosphorimetry
Experimental set up is
complicated
Studies done at low
temperature -196Oc
More sensitive
Complex sample can be
analyzed successfully
53
Comparison

54. 54
Applications of Phosphorimetry
Element /Compound LOD /LOL Application
Aspirin 0.02-1.00 mg/cm3 Aspirin in blood serum
Procaine, cocaine,
phenonbarbital ,
Chloromazine
In blood serum
Cocaine, atropine (NADA ) Analysis of Urine
sample of Sports player
Alkoloids such as
nicotine,
nornicotine
And anabasine
In combination
with PC and TLC
Tobacco sample
Environmental studies

55. Phosphorimeter
55
PMT
Detector
Monochromator
Xenon lamp
Read out
Device
Rotating shutter
Sample Cell

56. •All the
best
56