FLUORIMETRY principle and applications.pptx

Instrumental analysis unit-1 chapter 2: fluorimetry.
presented by. It is a instrument used for identifying the intensity of fluorescence emitted by substance. In fluorimetry the light emitted is known as fluorescence. It is based on the principle of fluorescence and phosphorescence.

1. FLUORIMETRY Presented by B. Likhitha B. pharmacy

2. CONTENT INTRODUCTION DEFINITION THEORY PRINCIPLE FACTORS AFFECTING FLUORESCENCE QUENCHING INSTRUMENTATION APPLICATIONS

3. INTRODUCTION A large number of substances are known which can absorb UV or visible light energy. But these substance loses its excess energy as heat and emit the remaining energy as electromagnetic radiation of a wavelength longer than that absorbed. This process of emitting radiation (fluorescence and phosphorescence) is collectively called as Luminescence.

4. definition Fluorimetry (or fluorescence spectrometry ) is an analytical technique used to measure the intensity of fluorescence emitted by a substance after it has absorbed light. When a sample is exposed to light of a specific wavelength, the molecules in the sample may absorb that energy and move to an excited state. As the molecules return to their ground state, they release energy in the form of light, which is typically of a longer wavelength than the absorbed light. This emitted light is called fluorescence.

5. theory According to the Pauli Exclusion Principle, only two electrons can occupy each orbital where they must have opposite spin states. SPIN STATES DESCRIPTION ORBITALS Singlet ground state All the π electrons in a molecule are paired (paired and opposite). ↑↓ Doublet state Unpaired π electrons are present. e.g. – free radicals. ↑ or ↑ Triplet state Unpaired electrons of same spin are present. (unpaired and same spin). Singlet excited state Electrons are unpaired but of opposite spin. (unpaired and opposite). SPIN STATES DESCRIPTION ORBITALS Singlet ground state All the π electrons in a molecule are paired (paired and opposite). ↑↓ Doublet state Unpaired π electrons are present. e.g. – free radicals. ↑ or ↑ Triplet state Unpaired electrons of same spin are present. (unpaired and same spin). Singlet excited state Electrons are unpaired but of opposite spin. (unpaired and opposite).

6. principle when light of appropriate wavelength is absorbed by a molecule the electrons are promoted from singlet ground state to singlet excited state. Once the molecule is in this excited state, relaxation can occur via several processes by emission of radiation. This process involves: Conversion / collisional deactivation Fluorescence phosphorescence

7. principle

8. principle Conversion /collisional deactivation: In which entire energy lost due to collision de activation and no radiation emitted. Fluorescence : Part of energy loss due to vibrational transition and remaining energy is emitted as UV/Visible radiation of longer wavelength than the incident light. Principle: It is a phenomenon of emission of radiation; when there is transition from singlet excited state to singlet ground state. singlet excited emission of radiation → singlet ground state

9. principle c) Phosphorescence : When a light radiation is incident on certain substances, they emit light continuously even after the incident light is cut off. It is phenomenon of emission of radiation when there is transition from triplet excited state to singlet ground state. triplet excited state emission of radiation → singlet ground state

10. Factors affecting fluorescence Nature of molecule : Only those molecule which are able to absorb UV or Visible radiation can show the fluorescence and phosphorescence. Greater the absorbance more intense the luminescence. Aromatic ring, molecule having conjugated double ( π ) - show more luminescence bond. Aliphatic and cyclic organic compound - do not show any luminescence. Compound which do not exhibit fluorescence – heterocyclics, such as pyridine, furan, thiophene and pyrrole.

11. Factors affecting fluorescence 2. Rigidity of structure : Rigid structure – more fluorescence intensity Flexible structure – less fluorescence intensity 3. Nature of substituent group : Electron donating groups – increase luminescence Electron donating e.g. –OH, - Electron withdrawing groups – decrease luminescence Electron withdrawing e.g. –COOH, -CHO, , SH-X (halides like F, Cl). Group having no effect on luminescence H,  

12. Factors affecting fluorescence 4. Effect of temperature : Less temperature – decrease collisions of molecules, increase luminescence. High temperature – increase collision of molecules, decrease luminescence. 5. viscosity : Increase in viscosity → decrease collisions of molecules → increase luminescence. Decrease in viscosity → increase collisions of molecules → decrease luminescence.

13. Factors affecting fluorescence 6. Effect of dissolved oxygen : presence of oxygen may decrease the fluorescent intensity in two ways: Oxidation of fluorescent species to a non-fluorescent species. Quenches fluorescent substance because paramagnetic properties of molecular energy. 7. Number of rings :

14. factors affecting fluorescence 8. Effect of pH : Effect of pH depends on the chemical structure of the molecule.

15. quenching Quenching is the reduction of fluorescence intensity by the presence of substance in the sample other than the fluorescence. This effect may be due to various factors like: Concentration quenching: concentration quenching is a kind of self-quenching. It occurs when the concentration of the fluorescing molecule increase in a sample solution. Chemical quenching: Due to various factors like change in pH, presence of oxygen, halides, and electron-withdrawing groups, heavy metals, etc.

16. Fluorescent indicators Name of indicator pH range color change Eosin 3 – 4 color less to green Fluorescein 4 – 6 color less to green Quinine sulphate 3 – 5 blue to violet Acridine 5.2 – 6.6 green to violet blue 2 - Naphthoquinone 4.4 – 6.3 blue to color less

17. INSTRUMENTATION Fluorimetry : measurement of fluorescence intensity (emitted radiation; when electron undergoes excited singlet state to ground state) at a particular wavelength with the help of a filter fluorimeter or a spectrofluorometer.

18. INSTRUMENTATION PARTS OF INSTRUMENT DESCRIPTION Source of light Mercury vapor lamp: It is used as source in filter type fluorimeter. Xenon arc lamp: It give more intense radiation than mercury vapour lamp. It is used in spectrofluorimeter . Tungsten lamp: use if excitation has to be done in visible region. Filters primary filter: Absorbs visible radiation and transmit UV radiation. Secondary filter: Absorbs UV radiation and transmit visible radiation. Monochromators Excitation monochromator: provides suitable radiation for excitation of molecule. Emission monochromator: Isolate only the radiation emitted by the fluorescent molecules.

19. INSTRUMENTATION PARTS OF INSTRUMENT DESCRIPTION Sample holder Made up of quartz or glass Detectors which transforms light energy into electrical signals that are observed on recorder. PMT (photomultiplier tube) is commonly used as detectors.

20. APPLICATIONS Determination of inorganic substance. Determination of phenytoin. Determination of thiamine. Determination of phenols, indoles. Detection of impurities at nanogram quantity.

21. Thank you