Fluorimetery.

the presentation gives knowledge about principle or fluorometry, factors that affect fluorescence including quenching instruments used in fluorometry, and the applications of fluorometry. added references in the end for more knowledge.

1. Fluorimetry Yogeeta M.Pharma ( Ist year) Pharmacolgy

2. CONTENT : INTRODUCTION PRINCIPLE FACTORS AFFECTING FLUORESCENCE QUENCHING INSTRUMENTATION APPLICATION

3. Introduction of Fluorimetry : Absorption of UV/visible radiation causes transition of electrons from one state to another. (i.e. ground state to excited state). At the excited state, electron is not stable, so it emits the energy in the form radiation to returns to ground state. The study of this transition is called as fluorimetry .

4. Terminology related to state of electrons Singlet ground state : A state in which all the electrons in a molecule are paired. Doublet state : A state in which an unpaired electron is present . E.g. free radical or Triplet state : A state in which unpaired electrons of same spin are present (unpaired and same spin) Singlet excited state : A state in which electrons are unpaired but of opposite spin

5. Principle of fluorimetry : Fluorescence is the phenomena of emission of radiation when there is transition from singlet ground state to singlet excited state . wavelength of absorbed radiation is called Excitation wavelength wavelength of emitted radiation is called Emission wavelength . Phosphorescence is also a related phenomenon, which is the study of emitted radiation when electrons undergo transition from triplet state to singlet ground state

6. Jablonski diagram Electronic transition Effect produced π  →  π * Only absorption but no fluorescence π  →  π * (and singlet transition) Fluorescence π  →  π * (and triplet transition) Phosphorescence

7. Factors affecting Fluorescence : Conjugation ( i.e. unsaturated molecule with π   electrons) So conjugation molecule absorb UV/visible radiation which predominantly leads to more fluorescence 2. Nature of substituent group (electron donating groups like (NH2, OH) enhance fluorescence; whereas electrons withdrawing groups like (NO2,COOH) reduce fluorescence intensity) 3. Rigidity of structure (Rigid structure gives more fluorescence intensity, whereas Flexible structure gives less fluorescence intensity)

8. 4. Effect of temperature increase in temp leads to more collision as a results decrease fluorescence intensity; & decrease in temp. leads to less collisions and results in increased fluorescence intensity 5. Viscosity increase in viscosity leads to decreased collision of molecule, which leads to enhancement of fluorescence intensity . Decrease in viscosity causes increased collision of molecule , which results in decrease fluorescence intensity. 6. Effect of pH eg : aniline in neutral or alkaline medium gives visible fluorescence but in acidic condition gives fluorescence in UV region only.

9. 7. Oxygen : It can decrease the fluorescence in two ways : it oxidises fluorescent substance to non fluorescent substance it quenches (decrease)fluorescence, because of the paramagnetic properties of molecule energy, as it has triplet ground state

10. Quenching : Fluorescence quenching is a physicochemical process that lowers the intensity of emitted light from fluorescent molecules. A variety of factors affect fluorescence- due to concentration, pH, pressure of chemical substance, temperature, viscosity etc. Types of Quenching : Self quenching Chemical quenching Static quenching Collision quenching

11. 1. Static quenching : This occurs because of complex formation in molecule(in ground state that is before excited state) Gain property like nonfluorescent for . eg _-caffeine reduces the fluorescence intensity of riboflavin by complex formation. 2.Collision quenching : It results of several factors like presence of halides, heavy metals, increased temp. and decrease in viscosity, where no. of collisions are increased. And quenching take place.

12. 3. Chemical Quenching : It is due to various factors like change in pH, presence of oxygen, halides and electrons, withdrawing groups, heavy metals etc. - change in pH: aniline in neutral or alkaline medium gives visible fluorescence at 290 nm but in acidic condition gives fluorescence. - Oxygen : oxidation leads to fluorescent substance to non fluorescent substance 3. Concentration Quenching or self quenching: It is a kind of self quenching. It occurs when the concentration of the fluorescence molecule increases in a sample solution the fluorescence intensity is reduced in highly concentrated solution

13. Fluorimetry:

14. Instrumentation: 1. Source of light : In Fluorimetry there are mainly three types of lamps are used A. Mercury vapour lamp :- -At high pressure Mercury lamp give 350 nm to 734 nm region radiation. -At low pressure Mercury vapour give addition line at 254 nm is used as source in filter type of Fluorimetry . B. Xenon arc lamp :-  It give a more intensity radiation when compared to Mercury lamp. C. Tungsten lamp :-   when molecule are excited in visible region tungsten lamp are used. 2. Filter and monochromator : In Fluorimetry there are mainly two filter are present -Primary filter : – absorbed visible radiation and transmit uv radiation. -Secondary filter : – absorbed uv radiation and transmit visible radiation.

15. Continue……. Mainly two monochromator are used in flourimetry . Excitation monochromator : – provide a suitable radiation for excited of molecule. Emission monochromator : – Isolates only the radiation emitted by the flourescent molecule. 3 . Sample cell :  sample cell are cylindrical or quadrangular shape. The cell are made up of colour corrected fused glass and pathlength is normally 10 mm or 1 cm.

16. Continue…. 4. Detector :- Mainly photovoltaic cell, Photo multiplier tubes , Photo tube are used as detector. Photo multiplier tubes are the best and accurate. 5 . Instruments A. Single beam fluorimeter B. Double beam fluorimeter C. Spectrofluorometer

17. 1. Single beam fluorometer Tungsten lamp as source of light. The primary filter absorbs visible radiation and transmits UV radiation. Emitted radiation measured at 90 degree by secondary filter. Secondary filter absorbs UV radiation and transmits visible radiation. Advantages simple in construction Easy to use econimical

18. Disadvantage: It is not possible to use reference solution and sample solution at a time. Rapid scanning to obtain excitation and emission spectrum of the compound is not possible

19. 2. Double beam fluorometer : Two incidence beam of light source pass through primary filter separately and fall on either sample or reference solution. The emitted radiation from sample or reference pass separately through secondary filter. Advantages: Sample and reference solution can be analysed simultaneously. Disadvantage : Rapid scanning is not possible due to use of filter.

20. Applications: Determination of vitamin B1 and B2 Mainly used in acid base titration: fluoresce in: colourless - green quinine sulphate: blue violet acridine :green –violet Pharmaceutical analysis: compound reagent Excitation wavelength fluorescence hydrocortisone 75%v/v H2So4 460 520 nicotinamide Cyanogen chloride 250 430

21. Fluorimetry is employed to carry out both qualitative and quantitative analyses for various aromatic compounds present in cigarette smoke, air-pollutant, concentrates, and automobiles exhaust. Measurement of naturally fluorescent compound: like tyro sine, tryptophan, bilirubin, and vitamins Measurement of chemically induced fluorescence: like chloroquine, heroin, hydrocortisone, isoniazid.

22. Reference: Dr. Ravi Sankar, ‘text book of pharmaceutical Analysis’, Rx publications, page no. 3.1 to 3.18 Douglas A Skoog, Principle of instrumental analysis. Gurdeep R Chatwal, Instrumental methods of chemical analysis

23. Thank You…..