Fluorescence spectroscopy

Komal 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).

stoke’s shift= λab-λemt 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 are

Solvent 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

ApplicationsUsed 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.