Fluorescence spectroscopy Komal Choudhary Lecturer School of Biotechnology DAVV Indore.

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Presentation transcript:

Fluorescence spectroscopy Komal Choudhary Lecturer School of Biotechnology DAVV Indore

Fluorescence spectroscopy Luminescence 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). stoke’s shift= λ ab- λ emt Best results are obtained from those compounds which showing large shifts.

Components of spectroflorimeter Light source-xenon or mercury lamps Slits Two monochromator(prism or differaction grating) (1) excitation monochromator (2) emission monochromator Sample holder(quartz cuvette) Detector-photocell (red-sensitive PMT)

Factors which affect fluorescence or quench the emission of fluorescence are Solvent polarity pH Temperature Viscosity Presence of oxygen Concentration of the sample molecule Intensity of incident light

Fluorescent probes Ethidium bromide Flourescein Densyl chloride 1-Anilinonapthalene 8-sulphonate(ANS) 4-methylumbelliferone Acridine GFP,RFP Extrinsic fluorescence Intrinsic 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.