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

2. Contents 3.2 Molecular Fluorescence and Phosphorescence Spectroscopy
3.2.1 Theory of Molecular Fluorescence and Phosphorescence
Spectroscopy.
3.2.2 Jablonski Diagram of Energy Levels.
3.2.3 Relationship between Fluorescence intensity and concentration,
Factors affecting Fluorescence and Phosphorescence.
3.2.4 Instrumentation and Applications of Fluorimetry and Phosphorimetry.
3.2.5 Comparison ofFluorimetry and Phosphorimetry.
3.2.6 Comparison of Fluorimetry with Absorption methods.

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.

4. Basic Terms
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.
If the emission takes place in a time of approximately10-8 sec. or less after absorption process is termed as Fluorescence.

5. Fluorescence :If the emission takes place in a time of approximately sec. or less after absorption process is termed as Fluorescence.
Re-emission of
Radiation
in less than 10-8 sec.
U. V. Light
Molecule

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

7. Motions in a molecule?
Rotational
Translational
Vibrational

8. What is a vibration in a molecule?
Any change in shape of the molecule- stretching of bonds, bending of bonds, or internal rotation around single bonds.

9. vibration in a molecule internal rotation around single bonds
stretching of bonds
bending of bonds
internal rotation around single bonds

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

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

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

13. The greater the mass - the lower the wavenumber
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

14. Theory: Energy Level of Electron in a molecule
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
[½ +& -½)].

15. 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.

16. 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)

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

18. 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.

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

20. Deactivation S0=V1,V2,V3,V4------(Ground state Singlet)
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)
Note: Ground state Singlet, excited singlet and Triplet state consist of number of vibrational levels .
S0=V1,V2,V3,V (Ground state Singlet)
S1=V1,V2,V3,V4-----(Excited state Singlet)
T1=V1,V2,V3,V4 ----(Excited state Triplet)

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.

22. Intersystem Crossing
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.

24. Fluorescence is instant phenomenon while phosphorescence is delayed fluorescence
Thus, the fluorescence emission can take place within 10-9 − 10-6 seconds, (the transition from S1—S0).
The transition from an excited triplet state to the ground state in case of phosphorescence requires at least seconds and may take as long as 10-2 seconds.

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

27. Intersystem crossing Fluorescence Phosphorescence
Excited Singlet state S1
Vibrational relaxation
Intersystem crossing
ENERGY
Excited
Triplet
state S1
Fluorescence
Phosphorescence
Ground state
singlet SO

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

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

30. 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.
U.V.Light
Visible light

31. 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
Primary filter
U.V.Light & visible light
U.V.Light

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

34. 5.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.

35. 6.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.

36. Instrument: Single Beam Fuorimeter
Sample Cell
U.V. Light & visible light
Primary filter
Secondary filter
Photocell /PMT detector
Read Out Device

37. Single Beam Fluorimeter
Primary filter
Secondary Filter

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

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

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

41. Temperature: 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.

42. 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

43. 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.

44. 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.

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

46. Example of application
Inorganic analysis
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'

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

48. Example of application
Organic analysis
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
LSD
>430 nm
Body fluids
Amphetamine
Codeine, Morphine nm
345 nm
Drug preparations and body
fluids
Polyaromatic hydrocarbons ( nm
Environmental sam
Study of protein structure
Typtophan and FAD are produce fluorescent colour
Study of protein degradation,
Clinical study

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

50. Phosphorimetry Instrumentation:
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: It is Hollow cylinder having two slits and rotated by variable speed motor.

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

52. Dewar Vessel / flask

53. Phosphorimeter
Read Out Device

54. Applications of Phosphorimetry
Element /Compound
LOD /LOL
Application
Aspirin
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. Comparison of Fluorimetry and Phosphorimetry
Experimental set up is easy
Studies done at room temperature
Less sensitive
Complex sample can not analyzed successfully
Experimental set up is complicated
Studies done at low temperature -196Oc
more sensitive
Complex sample can be analyzed successfully

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

57. All the best