Fluorometry

A fluorometer or fluorimeter is a device used to measure parameters of fluorescence: its intensity and wavelength distribution of emission spectrum after excitation by a certain spectrum of light. These parameters are used to identify the presence and the number of specific molecules in a medium.

1. Presentation On : Fluorometry

2. Group No.: 08 Name: Reg. No.: 1. Kamrul Hasan Sojib 15103088 2. Smaranika Rahman 15103092 3. Sadikur Rahman 15103093 4. Zobaida Mostarin Nishi 15103094 5. Saqline Mostaq 14203061

3. Contents: Introduction. Theory of fluorometry. Fluorescence and chemical structure. Instrumentation. Factors effecting fluorescence intensity. Application. Advantages. Disadvantages.

4. History: The term fluorescence comes from the mineral fluorspar (calcium fluoride) when Sir George G. Stokes observed in 1852 that fluorspar would give off visible light (fluoresce) when exposed to electromagnetic radiation in the ultraviolet wavelength.

5. Luminescence: Luminescence is the emission of light by a substance. It occurs when an electron returns to the electronic ground state from an excited state and loses its excess energy as a photon. Luminescence

6. Classification of luminescence: Chemiluminescence. Photoluminescence. Bioluminescence. Electroluminescence. Thermoluminescence. Cathodoluminescence etc.

7. Fluorescence: When the luminescence stops within 10^-8 to 10^-4 sec after the source of excitation is removed is called fluorescence. Phosphorescence: When the luminescence continues for a slightly longer period of time ( 10^-4 to 10 sec) after the source of excitation is removed is called phosphorescence.

8. Classification of fluorescence: Based on the wavelength of emitted radiation when compared to absorbed radiation. Stokes fluorescence: wavelength of emitted radiation is longer than absorbed radiation. Anti-stokes’s fluorescence: wavelength of emitted radiation is shorter than absorbed radiation. Resonance fluorescence: wavelength of emitted radiation is equal to that of absorbed radiation.

9. Theory of fluorometry : Absorption of ultraviolet and visible by molecules of an irritated sample generates populations of molecules in excited states. There have a presence of vibrational energy levels. This is usually singlet state and the electrons are paired and they spin about their own axes in opposite direction. Molecules from the excited state can loss energy by several mechanism. If the excited molecules emit radiation at the same wavelength the process is termed resonance Fluorescence (rare phenomena). If the molecules loss energy through vibrational energy where the vibrational energy is thought to be lost to solvent molecules. This process called Vibrational relaxation. If a molecule return to the ground state by photo emission is called Fluorescence. A particular process by which a molecule can easily drop to the ground state through small energy loss called Internal Conversion.

10. Continued:

11. Continued: In some compounds a process is take place is called Intersystem crossing. Triplet state is formed in this process. It is characterized by unpairing of two electrons and this is contrast to the singlet state. In this state two electron spin their own axes in same direction. From the triplet state a molecule can drop to the ground state by emission of radiation, its called Phosphorescence. A molecule in the triplet state can also undergo radiation less conversion to the ground state.

12. Fluorescence and chemical structure: Fluorescence is most commonly observed in compounds containing aromatic functional groups with low energy. Most unsubstituted aromatic hydrocarbons show fluorescence - quantum efficiency increases with the no of rings and degree of condensation. Simple heterocyclic do not exhibit fluorescence.

13. Fusion of heterocyclic nucleus to benzene ring increases Fluorescence.

14. Substitution on the benzene rings shift wavelengths of absorbance maxima and corresponding changes in fluorescence peak. Fluorescence decreases with increasing atomic number of the Halogen. Substitution of carboxylic group in aromatic rings inhibit fluorescence.

15. Instrumentation:

16. Instrument design: Single beam fluorometer. Double beam fluorometer. Spectrofluorometer(double beam).

17. Single beam fluorometer:

18. Double beam fluorometer:

19. Spectrofluorometer:

20. Working of fluorometer:

21. Factors Affecting Fluorescence Intensity : Nature of molecule. Scatter. Nature of substituent. Effect of concentration. Adsorption. Light. Oxygen. pH. Temperature & viscosity. Intensity of incident light. Path length. Photochemical decomposition.

22. Nature of Molecules: Only the molecules absorbs uv/visible radiation can show this phenomenon. Greater the absorbency of the molecule the more intense its fluorescence. Scatter: Scatter is mainly due to colloidal particles in solution. Scattering of incident light after passing through the sample leads to decrease in fluorescence intensity.

23. 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.

24. Effect of Concentration: Fluorescence is directly proportional to concentration. f ∝ Conc .

25. Adsorption:   Extreme sensitiveness of the method requires very dilute solution. Adsorption of the fluorescent substances on the container wall create serious problems. Hence strong solutions must be diluted. Photochemical decomposition: Absorption of intense radiation leads to photochemical decomposition of a fluorescent substance to less fluorescent or non fluorescent substance.

26. Light: Monochromatic light is essential for the excitation of fluorescence because the intensity will vary with wavelength. Oxygen: The presence of oxygen may interfere in 2 ways: By direct oxidation of the fluorescent substances to non-fluorescent. By quenching of fluorescence.

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

28. Temperature & viscosity: Increase in temperature/decrease in viscosity will decrease fluorescence.

29. Intensity of incident light: The intensity of light depends upon: 1. Light emitted from the lamp. 2. Excitation monochromaters. 3. Excitation slit width. Path length: Use of micro cuvette does not reduce the fluorescence. Use of microcell may reduce interferences and increases the measured fluorescence.  

30. Quenching: Decrease in FI due to various factors like; chemical rearrangement, energy transfer, chemical complex etc . TYPES: 1.Self or concentration quenching. 2.Chemical quenching. 3. Static quenching. 4. Collisional quenching.

31. 1.Self or concentration quenching:

32. 2.Chemical quenching: Here decrease in fluorescence intensity due to the factors like change in pH, presence of oxygen, halides &heavy metals. 3.Static quenching: This occurs due to complex formation. e.g. caffeine reduces the fluorescence of riboflavin by complex formation. 4.Collisional quenching: It reduces fluorescence by collision. Where no. of collisions increased hence quenching takes place.

33. Application: Environmental Significance: To detect environmental pollutants such as polycyclic aromatic hydrocarbons: pyrene, benzopyrene, organothiophosphorous pesticides, carbamate insecticides. Generally used to carry out qualitative as well as quantitative analysis for a great aromatic compounds present in cigarette smoking, air pollutant concentrates & automobile exhausts.

34. Geology: Many types of calcite and amber will fluoresce under shortwave UV. Rubies, emeralds, and the Diamond exhibit red fluorescence under short-wave UV light; diamonds also emit light under X ray radiation.

35. Analytical Chemistry: To detect compounds from HPLC flow. TLC plates can be visualized if the compounds or a coloring reagent is fluorescent. Plant pigments, steroids, proteins, naphthol etc. can be determined at low concentrations. Biochemistry: Used generally as a non-destructive way of tracking or analysis of biological molecules (proteins). Possible direct or indirect analysis aromatic amino acids (phenylalanine- tyrosine-tryptophan). Fingerprints can be visualized with fluorescent compounds such as ninhydrin.

36. Medicine: Blood and other substances are sometimes detected by fluorescent reagents, particularly where their location was not previously known. There has also been a report of its use in differentiating malignant, bashful skin tumors from benign. Pharmacy: Possible direct or indirect analysis drugs such as: vitamins (vitamin A ,vitamin B2 , vitamin B6, vitamin B12, vitamin E, folic acid),Catecholamines (dopamine, norepinephrine) Other drugs (quinine, salicylic acid, morphine, barbiturates, lysergic acid diethylamide (LSD)) To measure the amount of impurities present in the sampl e.

37. Fluorescent indicators: Intensity and color of the fluorescence of many substances depend upon the pH of solutions. These are called as fluorescent indicators and are generally used in acid base titrations. E.g.: Eosin: pH 3.0-4.0 – colorless to green. Fluorescein: pH 4.0-6.0 – colorless to green. Quinine sulphate: blue-violet. Acridine: green-violet.

38. 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. Estimation of cadmium with 2-(2 hydroxyphenyl) benzoxazole in presence of tartarate. Nuclear Research: Field determination of uranium salts.

39. Advantages: SENSITIVITY : It is more sensitive as concentration is low as µg/ml or ng/ml. PRECISION : Upto 1 % can be achieved. SPECIFICITY : More specific than absorption method where absorption maxima may be same for two compounds. RANGE of APPLICATION : Even non fluorescent compounds can also be converted to fluorescent compounds by chemical compounds.

40. Disadvantages: Not useful for identification. Not all compounds fluorescence. Contamination can quench the fluorescence and hence give false or no results.

41. Reference : https://www.shimadzu.com/an/hplc/support/lib/lctalk/chemiluminescence_detection.htm https://www.slideshare.net/sreevidyavemuri/fluorimetry-45064390?next_slideshow=1 https://www.slideshare.net/tatya922050/spectrofluorimetry-or-fluorimetry-wwwredicalscom https://www.youtube.com/watch?v=B22ZsUOFM98 https://www.youtube.com/watch?v=YhAy-exSwZo https://www.youtube.com/watch?v=YhAy-exSwZo

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