1. Fluorometry
Ashis Kumar Podder

2. Luminescence It is of 3 types. Fluorescence Phosphorescence
Chemiluminescence

3. 1. Fluorescence
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 fluorescent substances.

4. 2. Phosphorescence
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.

5. 3. Chemiluminescence
Emission of light as a result of a chemical reaction at environmental temperatures.

6. Fluorometry
An analytical technique for identifying and characterizing minute amounts of a substance by excitation of the substance with a beam of ultraviolet/visible light and detection and measurement of the characteristic wavelength of fluorescent light emitted.

7. Photoluminescence and structure
Compounds with fused ring are found to be especially fluorescent, and the extent of fluorescence is found to be directly proportional to the number of rings in the molecule
The structural rigidity in a molecule favors fluorescence
Aliphatic and alicyclic carbonyl compounds or highly conjugated double bond structures also show fluorescence.

8. Theory of Fluorescence and Phosphorescence
a) Ground singlet state: A molecular electronic state in which all of the electrons are paired are called singlet state. Ground state is associated with many vibrational energy levels.

9. b) Excitation of the molecule: A molecule moves from its ground state to excited state by absorbing energy in the form of UV or visible radiation. Excited state is associated with many vibrational energy levels. Molecules are distributed in various vibrational energy levels. Two vibrational energy levels-
excited singlet state
excited triplet state

10. Excited singlet state
Excited state is usually excited singlet state
Spins paired
No net magnetic field
Average lifetime: 10-8 sec
Excited triplet state
Lying between excited singlet state and ground singlet state
Spins unpaired
Net magnetic field
Average lifetime: 10-4 – 10 sec
Ground singlet state

12. From the excited singlet state one of the following phenomenon occurs-
c) Resonance fluorescence: Molecules at each vibrational level of excited state loose energy by emitting photons and fall to original condition of ground state. So, energy and wavelength of emitted light will be equal to that of absorbed light. This process is called resonance fluorescence.

13. d) Radiation less processes
Vibration relaxation: loss of excess vibrational energy through collisions.
Internal conversion: 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.
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.
Intersystem crossing: a process where there is a crossover between electronic states of different multiplicity.

14. e) Fluorescence:
Transition from excited singlet state to ground singlet state.
Due to vibrational relaxation, the radiation emitted is of lower energy and longer wavelength.
The process is instantaneous and ceases immediately as the light source is removed.
Life time of the excited state is s.

15. f) Phosphorescence From excited triplet state to ground singlet state.
Characterized by an afterglow due to long life of triplet state.
At favorable conditions: low temperature and absence of oxygen
Life time of the excited triplet state is 10-4 to several seconds.

16. Factors affecting fluorescence
Nature of molecule
Nature of substituent
Concentration of fluorescing species
Adsorption
Oxygen pH
Temperature & viscosity
Inner filter effect

17. 1. Nature of molecule
All the molecules cannot show the phenomenon of fluorescence.
Only the molecules absorb uv/visible radiation can show this phenomenon.
Greater the absorbency of the molecule the more intense its fluorescence.

18. 2. Nature of substituent
Electron donating group enhances fluorescence. e.g.: NH2, OH etc.
Electron withdrawing groups decrease or destroy fluorescence. e.g.: COOH, NO2, N=N etc.
High atomic no. atom introduced into  electron system decreases fluorescence.

19. 3. Concentration of fluorescing species
The intensity of fluorescence increases as the concentration of fluorescing species increases. But this relationship is more complex than that between absorbance and the concentration of absorbing species.

20. 4. Adsorption
Extreme sensitiveness of the method requires very dilute solution.
Adsorption of the fluorescent substances on the container wall creates serious problems.

21. 5. Oxygen The presence of oxygen may interfere in 2 ways.
by direct oxidation of the fluorescent substances to non fluorescent
by quenching of fluorescence (para-magnetism)

22. 6. pH
Alteration of the pH of the solution will have significant effect on fluorescence.
Fluorescent spectrum is different for ionized and un-ionized species.

23. 7. Temperature & viscosity
Increase in temperature/decrease in viscosity will decrease fluorescence.
The increase in temperature ↓
Increase in thermal motion of molecules
Favors the intermolecular collision
Helps in dissipation of excited energy
1°C rise in temperature results in a decrease of about 1% in intensity of fluorescence.

24. 8. Inner filter effect
If non fluorescent solutes absorb either excitation or emission radiation, there is a reduction in measured intensity of fluorescence. This effect is inner filter effect. Example: in Na2CO3 solution, K2Cr2O7 exhibit this effect.

25. Quenching
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

26. 1. Self quenching
Deviations at higher concentrations can be attributed to self-quenching or self-absorption.

27. 2. Chemical quenching
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 metal leads to quenching, because of collisions of triplet ground state.

28. 3. Static quenching
It is the process in which quencher binds with fluorescent molecule in ground state and inhibits the molecule to go to excited state. E.g.: caffeine reduces the fluorescence of riboflavin by complex formation.

29. 4. Collisional quenching
It is the process in which quencher binds with fluorescent molecule in excited state and transfer of energy to quenching molecule occurs. Thus excited energy is dissipated and no fluorescent occurs.

30. Instrumentation for fluorescence
Excitation light source
Filters or monochromators
Sample holder
Detector
Readout device

31. 1. Source of light
Mercury vapor lamp: Mercury (Hg) vapor in high pressure (8 atm.) gives intense lines on continuous background above 350nm.
Xenon arc lamp: gives more intense radiation.
Tungsten lamp: used if excitation has to be done in visible region.

32. 2. Filters and monochromators
Filters: a fluorometer usually measures fixed wavelengths by filters.
Primary filter: absorbs visible light & transmits UV light.
Secondary filter: absorbs UV radiations & transmits visible light.
Monochromator: a spectrofluorometer usually has adjustable excitation and emission wavelengths via a monochromator. Spectrofluorometers are more flexible.
Excitation monochromater: isolates only the radiation which is absorbed by the molecule.
Emission monochromater: isolates only the radiation emitted by the molecule

33. 3. Sample cells
Sample cells are cylindrical or polyhedral made up of color corrected fused glass & path length normally 10 mm to 1 cm.

34. 4. Detectors
Photovoltaic cell (creation of electric current in a material upon exposure to light)
Photo tubes (gas-filled or vacuum tube that is sensitive to light)
Photo multiplier tubes

35. Applications of Fluorometry
1) Fluorescent indicators
Mainly used in acid-base titration. e.g.:
Fluorescein: colorless-green.
Quinine sulphate: blue-violet.
Acridine: green-violet

36. Applications of Fluorometry
2) Pharmaceutical analysis
3) Determination of vitamin B1 & B2.
Compound
Reagent
Excitation wavelength
Fluorescence
Hydrocortisone
75% v/v H2SO4 in ethanol
460
520
Nicotinamide
Cyanogen chloride
250
430

37. Applications of Fluorometry
4) Liquid chromatography: Fluorescence is an important method of determining compounds as they appear at the end of chromatogram or capillary electrophoresis column.
5) Organic analysis: Qualitative and quantitative analysis of organic aromatic compounds present in cigarette smoke, air pollutants, automobile exhausts etc.

38. Applications of Fluorometry
6) Determination of inorganic substances
Determination of ruthenium ions in presence of other platinum metals.
Determination of aluminum (III) in alloys.
Determination of boron in steel by complex formed with benzoin.

39. Advantages of Fluorometry
Sensitivity: More sensitive when compared to other absorption techniques. Concentrations as low as μg/ml or ng/ml can be determined.
Specificity: As both excitation & emission wavelengths are characteristic it is more specific than absorption methods.

40. Immunoassay
An immunoassay is a biochemical test that measures the concentration of a substance in a biological liquid like serum or urine, using the reaction of an antibody or antibodies to its antigen.
In life science research, immunoassays are used in the study of biological systems by tracking different proteins, hormones, and antibodies. In industry, immunoassays are used to detect contaminants in food and water, and in quality control to monitor specific molecules used during product processing.
Types of Immunoassay
Radio Immunoassay (RIA)
Enzyme Immunoassay (EIA)
Fluorescence Polarization Immunoassay (FPIA)

41. Fluorescence Polarization Immunoassay (FPIA)
Fluorescence Poloarization Immunoassy (FPIA) is a type of homogeneous competitive fluoresence immunoassay.
With competitive binding, antigen from the specimen and antigen-fluorescein (AgF) labeled reagent compete for binding sites on the antibody.
FPIA is used to provide accurate and sensitive measurements of small toxicological analytes such as therapeutic drugs and drugs of abuse.

42. Fluorescein
Fluorescein is a fluorescence label that absorbs light at 490 nm and releases this energy at 520 nm.
FPIA results in an inverse response curve such that more antigen in the sample; less antigen-fluorescein (AgF) labeled reagent bound to antibody; lower emission of plane polarized light.

43. Fluorescein molecules in solution, excited with a plane-polarized light, emit light into a fixed plane if the molecules remain stationary during the fluorophore's excitation. When polarized light is absorbed by AgF, the molecule rotates quickly before the light is emitted as fluorescence. When the larger-sized Ab-AgF complex absorbs the polarized light, it rotates more slowly and the light is emitted in the same plane and the detector can measure it.