Fluorescence spectroscopy

1. Fluorescence spectroscopy Komal Choudhary Lecturer School of Biotechnology DAVV Indore

2. 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

4. 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

5. 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)

6. 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= λab-λemt • Best results are obtained from those compounds which showing large shifts.

7. 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)

9. Fluorescence is the property of compounds having π-πtransition, π electron cloud. • Fluorescence is measured at right angle to sample holder to avoid the measurement of transmitted light. • Cuvettes are clear from all the sides unlike those used in spectrophotometer. • Fluorescence depends upon concentration of the sample.(particular range) • Quantum yield=no of photon emitted/ no. of photon absorbed (value lies always 0-1) • Quantity of any compounds in nanogram can be measured.(sensitivity)

10. 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

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

12. 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.