Fluorescence spectroscopy Komal Choudhary Lecturer School of Biotechnology DAVV Indore

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Text of Fluorescence spectroscopy Komal Choudhary Lecturer School of Biotechnology DAVV Indore

  • Fluorescence spectroscopyKomal ChoudharyLecturerSchool of BiotechnologyDAVV Indore

  • Fluorescence spectroscopyLuminescence It is the emission of light from any substance, and occurs from energetically excited states.

    It is divided into two categories

    (1) Fluorescence

    (2) phosphorescence

  • Non radioactive relaxation- excitation energy is dissipated as heat(vibration) to the solvent.

    It includes internal conversion(IC) intersystem conversion(ISC).

    Singlet state

    Triplet state

  • Jablonski energy diagram tell us about the relaxation mechanism for exited state molecule.Once a molecule has absorbed energy in the form of electromagnetic radiation there are numbers of routs by which it can return to ground state.If the photon emission occurs between same spin state(i.e. S1-S0), this is called fluorescence emission. (release of energy is faster)If the spin state of the initial and final energy level are different(i.e. T1-S0), this is called phosphorescence emission. (release of energy is slower)

  • Fluorescence spectroscopy or spectrofluorometry, is a type of electromagnetic spectroscopy which analyzes fluorescence from a sample.Fluorescence is a phenomenon where by molecule after absorbing radiation emit the radiation of longer wavelength (compound absorb UV radiation, emit visible light).stokes shift= ab-emt Best results are obtained from those compounds which showing large shifts.

  • Components of spectroflorimeterLight source-xenon or mercury lampsSlitsTwo monochromator(prism or differaction grating) (1) excitation monochromator (2) emission monochromatorSample holder(quartz cuvette)Detector-photocell (red-sensitive PMT)

  • Factors which affect fluorescence or quench the emission of fluorescence areSolvent polaritypHTemperatureViscosityPresence of oxygenConcentration of the sample moleculeIntensity of incident light

  • Fluorescent probes

    Ethidium bromideFlouresceinDensyl chloride 1-Anilinonapthalene 8-sulphonate(ANS)4-methylumbelliferoneAcridineGFP,RFP

    Extrinsic fluorescenceIntrinsic fluorescence

  • Applications

    Used in both qualitative and quantitative(major) estimation.Assays of vitamin B in food stuff ,NADH, hormones, drugs, pesticides, Carcinogens, chlorophyll, cholesterol, metal ions etc. enzyme assays and kinetic analysis.Protein structure analysis.Membrane structure analysis.Microspectrofluorimetry (used to detect malignant cell in biopsy tissue)FACS.