Download to read offline
More Related Content
Similar to Spectrofluorimetry
Spectrofluorimetry
- 1. SPECTROFLUORIMETRY PREPARED BY~MAHARSHI PANDYA M.PHARM(PHARMACEUTICS) SEM 1 DEPARMENT OF PHARMACEUTICAL SCIENCES, SAURASHTRA UNIVERCITY 1
- 2. introduction Absorption ofuv/visible radiation causes transition of electrons from ground state (low energy) to exited state is not stable, excess energy is lost. Molecule contains electron, electron and non bonding (n) electron. 1) The electrons may be present in bonding molecular orbital. It is called as highest occupied molecular orbital (HOMO). It has lest energy and more stable. 2) When the molecules absorbs radiant energy from a light source, the bonding electrons may be promoted to antibonding molecular orbital (LUMO. It has more energy and hence less stable. 2
- 3. principle The processof promotion of electrons from H0MO to LUMO with absorption of energy is called as excitation. Singlet state :- a state in which all the electron in a molecule are paired ↓↑ Doublet state :- a state in which un paired electrons is present ↓ or ↑ Triplet state :- a state in which unpaired electrons of same spin present ↑↑ Singlet excited state :- a state in which electrons are unpaired but of opposite spin like ↑ ↓ (unpaired and opposite spin) 3
- 4. When lightof 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 to relaxation can occur via several process. For ex by emission of radiation. The process can be the following 1) Collisional deactivation 2) Fluorescence 3) Phosphorescence 4
- 5. 1) Collisional deactivation :- in which entire energy lost due to collision de activation and no radiation emitted. 2) Fluorescence :- excited singlet state is highly unstable. Relaxation of electrons from exited singlet ground state with emission of light. 3) Phosphorescence :- At favorable condition like low temperature and absence of oxygen there is transition from excited singlet state to triplet state which is called as inner system crossing. The emission of radiation when electrons undergo transition from triplet state to singlet ground state is called as phosphorescence. 5
- 6.
- 7. Advantages Moresensitive when compared to other absorption techniques. Concentration as low as цg/ml or ng/ml can be determined. Precision upto 1% can be achieved easily. As both excitation and emission wave lengths are characteristic it is more specific than absorption methods. Disadvantage The susceptibility to environmental condition and virtual impossibility of predicting whether a compound will fluorescence. The other major problem is quenching, whereby the energy transferred to the other molecules. 7
- 8. Factors effecting fluorescenceintensity 1) Concentration 2) Quantum yield of fluorescence 3) Intensity of incident light 4) Adsorption 5) Oxygen 6) PH 7) Temperature and viscosity 8) Photodecomposition 9) Quenchers 10) Scatter 8
- 9. Concentration :-Fluorescence intensity is proportional to concentration of substance only when the absorbance is less than 0.02 . Quantum yield of fluorescence Ø :- Number of photons emitted / Number of photons absorbed. It is always less than 1 since some energy is lost by radiation less pathways ( Collisional, Intersystem crossing, Vibrational relaxation) Intensity of incident light :- Increase in the intensity light on the sample fluorescence intensity also increases. Adsorption :- Adsorption of sample solution in the container may leads to a serious problem. Oxygen :- Oxidation of fluorescent species to a non fluorescent species, quenches fluorescent substance 9
- 10. Ph :-Alteration of ph of a solution will have significant effect on fluorescence. For ex Aniline in alkali medium gives visible fluorescence and in acid gives invisible region. Temperature and viscosity:- temperature increases can increase the collisional de activation and reduce fluorescent intensity. If viscosity of solution is more the frequency of collisions are reduced and increase in fluorescent intensity. Photochemical decomposition:- Absorption of intense radiation leads to photochemical decomposition of a fluorescent substance to less fluorescent or non fluorescent substance. 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 10
- 11. Quenchers:- Quenchingis the reduction of fluorescence intensity by the presence of substance in the sample other than the fluorescent analyte. o Quenching is following types:- Inner fluorescent effect :- absorption of incident (uv) light or emitted (fluorescent) light by primary and secondary filters leads to decrease in fluorescence intensity. Self quenching :- At low concentration linearity is observed, at high concentration of the same substance increase in fluorescent intensity is observed. This phenomena is called self quenching 11
- 12. Collisional quenching:- Collisions between the fluorescent substance and halide ions leads to reduction in fluorescence intensity. Static quenching :- This occurs because of complex formation between the fluorescent molecule and other molecules. Ex : caffeine reduces fluorescence of riboflavin. 12
- 13.
- 14. Source oflight Mercury vapour lamp :- Hg vapour in high pressure ( 8 atm ) gives intense line on continuous background above 350nm. Xenon arc lamp :- Gives more intense radiation. Tungsten lamp :- Used if excitation has to be done in via region. 14
- 15. Filters andmonochromators:- Filters :- These are nothing but optical filters works on the principle of absorption of unwanted light and transmitting the required wavelength of light. Primary filter:- Absorbs visible radiation and transmit UV radiation. Secondary filter:- Absorbs UV radiation and transmit visible radiation. 15
- 16. Monochromators :-They convert polychromatic light into monochromatic light. They can isolate a specific range of wavelength or a particular wavelength of radiation from a source. Excitation monochromators :- provides suitable radiation for excitation of molecule. Emission monochromators :- isolate only the radiation emitted by the fluorescent molecules. Sample cells :- These are ment for holding liquid samples. These are made up of quartz and can have various shapes ex : cylindrical or rectangular. 16
- 17. Detectors :Photometric detectors are used they are : Barrier layer cell/ photo voltaic cells Photomultiplier cells 17
- 18. Types of instruments Single beam filter fluorimeter It contains tungsten lamp as a source of light and has an optical system consists of primary filter. The emitted radiation is measured at 90’ by using a secondary filter and detector. Primary filter absorbs visible radiation and transmit uv radiation which excites the molecule present in sample cell. In stead of 90 if we use 180 geometry as in colorimetry secondary filter has to be highly efficient other wise both the unabsorbed uv radiation and fluorescent will produce detector response and give false result. 18
- 19.
- 20. In doublebeam fluorimeter It is similar to single beam except that the two incident beams from a single light source pass through primary filters separately and fall on the another reference solution. Then the emitted radiations from the sample or reference sample pass separately through secondary filter and produce response combinly on a detector. 20
- 21.
- 22. In spectrofluorimeter In this primary filter in double beam fluorimeter is replaced by excitation monochromator and the secondary filter is replaced by emission monochromator. Incident beam is split into sample and reference beam by using beam splitter. 22
- 23.
- 24. Application of spectrofluorimetry Determination organic substances Plant pigments, steroids, proteins, naphthols etc. can be determined at low concentrations. Generally used to carry out qualitative as well as quantitative analysis for a great aromatic compounds present in cigarette smoking, air pollutant concentration and automatic exhausts. o Determination of inorganic substance o Extensively used in the field of nuclear research for the determination of uranium salts. o Determination of vitamin B1 (thiamine) in food sample like meat cereals etc. 24
- 25. Determination ofvitamin B2( riboflavin). This method is generally used to measure the amount of impurities present in the sample. Most important application are found in the analyses of food products, pharmaceuticals, clinical samples and natural products. Fluorescent indicators : Intensity and color of the fluorescence of many substance depend upon the pH of solutions. These are called as fluorescent indicators and are generally used in acid base titrations. Eg. Eosin – pH 3.0-4.0 – colorless to green Fluorescein – pH 4.0-6.0 – colorless to green 25
- 26. conclusion Fluorimetric methodsare not useful in qualitative analysis and much used in quantitative analysis. Fluorescence is the most sensitive analytical techniques. Detection studies will increase the development of fluorescence field. 26
- 27. references SKOOG, principleof instrumental analysis B.K. Sharma instrumental methods of chemical analysis Wikipedia Google source 27
- 28.