Fluorimetry/ Fluoroscences

Instrumental metthods of chemical analysis

1. Presented By- ROHIT M.Pharmacy (Pharmaceutics)

2.       I N T R OD UC TI O N DEFINITION THEORY FACTORS AFFECTING FLOURESCENCE APPLICATIONS IN PHARMACY INSTRUMENTATION

3. 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. It is of 3 types. Fluorescence spectroscopy. Phosphorescence spectroscopy. Chemiluminescence spectroscopy

4. 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 flourescent substances .

5.  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 .

6.  A molecular electronic state in which all of the electrons are paired are called singlet state.  In a singlet state molecules are diamagnetic.  Most of the molecules in their ground state are paired.  When such a mole cule absorbs uv/visible radiation, one or more of the paired electron raised to an excited singlet state /excited triplet state.

7. triplet state s p i n s u np a i r e d G r o u n d singlet states excited singlet state spin paired no net mag.field net mag.field

8.  Fluorescence  Phosphorescence  Radiation less processes  Vibration relaxation  Internal conversion  External conversion  Intersystem crossing

9. LIGHT EMITING AT ONCE SOURCE STARTS & STOPS WHEM SOURCE STOPS

10. J AB L O N S K I ENE R G Y DIAGRAM

11. FLUORESCENCE & THEIR 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.

12. CO N TD …  Simple heterocyclic do not exhibit fluorescence.

13.  Fusion of heterocyclic nucleus to benzene ring increases fluorescence .

14. Fluorescence is favored in molecules with structural rigidity. organic chelating agents complexed with metal ion increases fluorescence .( A chelating agent is a substance whose molecules can form several bonds to a single metal ion).

15.  Nature of molecule  Nature of substituent  Effect of concentration  Adsorption  Light  Oxygen  pH  Temp . &viscosity  Intensity of incident light  Path length

16. 1. Temperature: it is inversely proportional t o luminescence. Increase in temp. causes increase in collision of molecules and finally increase in F & P. D ec re a s e i n t e m p. Ca u s es d e cr e a s e i n co llisi o n of molecules and finally decrease in F & P. 2 . V i s c os it y : It i s d i re ct l y p r o p o r t i o n a l to luminescence. In c r e a s e i n vis c os i ty c au se s d e cr e a s e i n m o l e c u l e collision and finally increase in F & P. D ec re a s e i n vis c os it y c au se s i nc r e a s e i n m o l e c ul e s collisions and finally decrease in F& P.

17. 3. Oxygen: It decrease the fluorescence in 2 ways; Ox i d ise s t h e f l u o r e s c e nt s u b s t a n c e t o non fluorescent substance. D ec re a s e f l u o r esce n c e b e c a u s e o f p a ra m a g ne t i c property as it has triplet ground state. 4. pH: its effects depends on chemical structure of molecules. E.g- Aniline in neutral & alkaline medium gives visible flourescence & in acidic medium gives fluorescence in uv region only. Phenols in acidic condition do not give f but in alkaline medium they give fluorescence.

18. Rigidity in structure: F l u o r i ne h a s r i g i d st r u c tu r e and i t s h o w s m o re fluorescence. B i p h e nyl h as flex i bl e st ruc t ure and s h o w s less fluorescence. Nature of group: E l e c t r o n d on a t i ng g r o u p s l i ke a m i no, h y d r o x y l etc causes increase in fluorescence. E l e c t r o n w i t h d r a w i ng g r o u p s li k e N O 2 , C O OH etc causes decrease in fluorescence.

19.  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 C h e m ic a l q u e n c h i n g Static quenching Collision quenching

20. Fluorescence Concentration of fluorescing species De vi a t i o n s a t h i g h e r c on ce n t r a t i o n s ca n b e attributed to self- quenching or self- absorption . Fluorescence Concentration of fluorescing species Calibration curve (Low con) calibration curve (High con)

21.  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.  H eavy m e t a l s - l eads t o q u e nc h i n g , b e c a us e of collisions of triplet ground state.

22. o f r i b o fl av i n by This occurs due to complex formation. e.g- caffeine reduces flourescence complex formation. COLLISIONAL QUENCHING  It reduces fluorescence by collision. where no. of collisions increased hence quenching takes place.

23. Types of flourescence ( w hen e x c i t ed b y Chemiluminescence (when excited by chemicals) Electrochemiluminescence electrochemical reaction) 3 . P h otoluminesce n c e ( w hen e xc i t ed b y electromagnetic radiation)  Based on the wavelength of emitted radiation 1 . St r o ke ’s f l o ure s cenc e ( w ave l e ngt h o f t h e em i t t ed radiation is longer than the absorbed radiation)

24. C on t d. Anti-stroke’s Flourescence ( wavelength of emitted radiation is shorter than absorbed radiation) Resonance Flourescence (wavelength of emitted radiation = absorbed radiation)  Based on Phenomena: Sensitised flourescence (when elements like Th, Zn, Cd or an alkali metal are added to mercury vapour these elements are sensitised & thus gives fluorescence. Thermally assisted Flourescence (the excitation is partly by electromagnetic radiation and partly by thermal energy)

25. I N ST R U ME N T A T I ON

26.  SOURCE OF LIGHT  FILTERS AND MONOCHROMATORS  SAMPLE CELLS  DETECTORS

27.  MERCURY ARC LAMP.  XENON ARC LAMP.  TUNGSTEN LAMP.  TUNABLE DYE LASERS .

28. M E RCUR Y A R C LA M P  Produce intense line spectrum above 350nm.  High pressure lamps give lines at 366,405, 436, 546,577,691,734nm.  Low pressure lamps give additional radiation at 254nm.

29.  I n t e n s e r a d i at i on b y p assa g e of c ur r en t t h r o u g h an atmosphere of xenon.  Spectrum is continuous over the range between over 250- 600nm,peak intensity about 470nm .

30.  Intensity of the lamp is low.  If excitation is done in the visible region this lamp is used.  It does not offer UV radiation.

31.  Pulsed nitrogen laser as the primary source.  Radiation in the range between 360 and 650 nm is produced.

32. FILTERS Primary filter-absorbs visible light & transmits uv light. Secondary filter-absorbs uv radiations & transmits visible light. MONOCHROMATORS Exitation monochromaters-isolates only the radiation which is absorbed by the molecule. Emission monochromaters-isolates only the radiation emitted by the molecule.

33.  The majority of fluorescence assays are carried out in solution.  Cylindrical or rectangular cells fabricated of silica or glass used.  Path length is usually 10mm or 1cm.  All the surfaces of the sample holder are polished in fluorimetry.

34.  PHOTOVOLTAIC CELL  PHOTO TUBE  PHOTOMULTIPLIER TUBES – Best and accurate.

35.  SINGLE BEAM FLUORIMETER  DOUBLE BEAM FLUORIMETER  SPECTROFLUORIMETER(DOUBLE BEAM)

36.  Tungsten lamp as source of light.  The primary filter absorbs visible radiation and transmits uv radiation.  Emitted radiation measured at 90 o by secondary filter.  Secondary filter absorbs uv radiation and transmits visible radiation.

37.  Similar to single beam instrument.  Two incident beams from light source pass through primary filters separately and fall on either sample or reference solution.  The emitted radiation from sample or reference pass separately through secondary filter.

38. Power su p p ly Source primary filter Sample cell Slit secondary filter Detector Data processor

39. The primary filter in double beam fluorimeter is replaced by excitation monochromaters. The secondary filter is replaced by emission monochromaters. The incident beam is split into sample and reference beam using a beam splitter. The detector is photomultiplier tube.

40. Power s u pp ly Source Excitation monochromator Emission monochromator Detector Sam p le cell Data processor

41. 1] 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.

42. 2]Nuclear research  Field determination of uranium salts. 3]Fluorescent indicators Mainly used in acid-base titration. e.g.: eosin- colorless-green. Fluorescein:colourless-green. Quinine sulphate: blue-violet. Acridine: green-violet

43. 5] organic analysis  Qualitative and quantitative analysis of organic aromatic compounds present in cigarette smoke, air pollutants, automobile exhausts etc . 6 ] Liquid chromatography  Fluorescence is an imp method of determining compounds as they appear at the end of chromatogram or capillary electrophoresis column. 7 ] determination of vitamin B1 &B2.