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Spectrofluorimetry
This document discusses spectrofluorimetry and the dye dilution technique. It begins with an introduction to spectrofluorimetry, including its principle of exciting compounds to emit different colored radiation. Some organic and inorganic compounds exhibit fluorescence which can be used qualitatively and quantitatively. It then discusses the phenomenon of fluorescence, factors affecting fluorescence rates, and components of a fluorescence instrument. The document concludes with an overview of the dye dilution technique which uses fluorimetric reagents to make non-fluorescent substances fluorescent and quantifiable via spectrofluorimetry.
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Spectrofluorimetry
- 1. HANNAN ABDUL KAHLIQ ZMPI16-04 PUNJAB UNIVERSITY ZOOLOGY DEPARTMENT SUBJECT: ADVANCED ANALYTICAL TECHNIQUES
- 2. Spectrofluorimetry “Determination of Fluid volumesby Dye Dilution Technique”
- 3. Spectrofluorimetry •Introduction – Principle – Purpose •Phenomenonof fluorescence •Instrument •Factors affecting the rate of fluorescence
- 4. Introduction • Principle: – Someorganic or inorganic compounds, when hit by exciting radiation, emit different colored radiations, called fluorescence after de-excitation and can be detected. Emitted wavelength is always longer than incident one. • Purpose: • Qualitative: Tells us what fluorescent species is present using the wavelength of emitted radiation. • Quantitative: Tells us how much of that species is present.
- 5. •Introduction – Principle – Purpose •Phenomenonof fluorescence •Instrument •Factors affecting the rate of fluorescence
- 6. • Concept ofPhotoluminiscence: Emission of light after excitation. • Phosphorescence: Energy loss without light i.e. heat • Fluorescence: Energy loss in the form of light • Excited states of Electrons: – Singlet: Excited state of paired electrons, anti parallel spin. – Triplet: Excited state of unpaired electrons, parallel spin. Phenomenon of Fluorescence
- 7. These excited states playa vital role in fluorescence or phosphorescence
- 8.
- 9. •Phenomenon of fluorescence •Instrument •Factorsaffecting the rate of fluorescence
- 10. • Light source(Xe lamp striking UV light) • Monochromator (Desired wavelength) • Slit • Sample cuvette • Grating (selecting) • Photomultiplier • Detector • Computer PARTS
- 11. Incident light Absorption =Excitation
- 12. Stoke’s Law • “Thewavelength of emitted light is always longer than the wavelength”
- 13. •Instrument •Factors affecting therate of fluorescence
- 14. • Structure • Scatter •Quenchers • pH • Concentration • Intensity of incident light • Temperature and viscosity • Air (oxygen) Factors
- 15. • Aromatic compoundsare ideal • Compounds having π- π* transition fluoresce • Linear or highly conjugated molecules also do but less than aromatic compounds • More the number of rings, more will be the fluorescence • Fused ringed structure also fluoresce • Fusion of benzene rings to a heterocyclic nucleus increases the absorption • Addition of halogens decreases the chances of fluorescence because it increases the chance of phosphorescence Structure
- 16. • Effect ofstructural rigidity: – More rigid the structure, more will be the fluorescence. – Lack of rigidity will enhance the internal conversion and hence “phosphorescence”.
- 17. Scatter • Rayleigh Scattering: –Re-Emission of small fraction of excitation light in all directions form solute molecules. • Raman Diffusion: • Waves emitted by the solvent molecules as a result of transfer of vibrational energy from solute molecules. Incident Light Rayleigh Scattering Raman Diffutsion
- 18. Quenchers • “Reduction ofthe fluorescence intensity by the presence of substances in the sample other than the fluorescent analyte.” – Dynamic/Collisional quenching: direct Due to collision – Static Quenching: Absorbance of fluorescence
- 19. pH • Transfer ofcharge and hence the acidity of a molecule affect the occurrence of fluorescence. • More ionic a substance, more will be the fluorescence. • For example: aniline has several resonance (ionic) forms but anilinium has only one.
- 20. Concentration • Quantum yield: •For constant conditions, fluorescence is directly proportional to concentration. (F) = (Io- IT) Ф • Value for highly fluorescenct substances may be equal to one which is the Maximum value. • If the concentration is too great, all light will be absorbed and the above expression will be: F = IoФ Where: Io = Incident light IT = Transmitted light Ф = Quantum yield of fluorescence
- 21. Intensity of incidentlight • Higher the intensity of light higher will be the fluorescence. • If the intensity is too high too effects can happen: – Spectral overlap – Photo decomposition: The molecule of solution or changes its composition due to photochemical effect thereby affecting our results.
- 22. Temperature & Viscosity •Increase in temperature and decrease in viscosity will affect the frequency of collision between the molecules and hence the fluorescence will be affected. Air (Oxygen) • Direct oxidation of molecules can happen • Oxygen is a very effective quencher
- 23. Spectrofluorimetry “Determination of Fluid volumesby Dye Dilution Technique”
- 24. • “Dye dilutionor fluorescence derivatization is a technique in which some dyes (fluorimteric reagents) are added to such substance which cannot fluoresce and hence making to do so.” • This technique is used to Qunatify some of the substances through fluorimetric techniques. • fluorimteric reagents: – Organic aromatic reagents which are added to non- fluorescence compounds for spectrofluorimetry.
- 25. Examples • Determination ofprimary amines, amino acids, peptides etc. through reaction with fluorescamine reagent: Fluorescamine Amino Acid Fluorescent compound
- 26. Determination of primaryand secondary aliphatic amines through: a) Reaction with 4-chloro-7-nitrobenzo-2-oxa-l,3-diazole ( NBD- CI ) give yellow fluorescence: b) Reaction with l-dimethylaminonaphthalene-5-sulphonyl chloride (Dansyl, chloride):
- 27. • Thiamine HCIin pharmaceutical preparations such as tablets and elixirs and in food stuffs such as flour is relatively easily determined by oxidation to highly fluorescent thiochrome. The product is soluble in 2-methyl-propan-1-ol and hence is easily extracted from the reaction mixture for measurements.
- 28.