3.2 molecular fluorescence and phosphorescence spectroscopy

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

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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. 4 Basic Terms

5. Longer wavelength 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

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

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

13. 13 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

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22. Intersystem Crossing 22

23. PHOSPHORESCENCE 23

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

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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.

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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. 39 The fluorescence spectrum and intensity of a molecule often depend strongly on the molecule’s environment.

40. 40 Effect of Temperature

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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 (r iboflavin) 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-Methyl umbeelliferone + Enzyme  Fluorescent colour 450 nm To carry Enzyme Assay and Kinetic study in Biomlecules 48

49. 49 Phosphorimetry : Instrumentation:

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51. 51 Dewar Vessel / flask

52. 52 Phosphorimeter Read Out Device

53. 53 Comparison

54. 54 Applications of Phospho rimetry 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