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Fluorimetry
Introduction/Theory: -Instrument that measure the intensity of fluorescence are called fluorimeter. -Spectrophotometer that measures the fluorescent intensity at variable wavelengths of excitation & emission, & are able to produce fluorescence spectra, are called spectrofluorimeter. -Fluorescence spectroscopy is related to the electronic & vibrational transition. -In ground energy state of molecules, electronic spin of electrons are paired/anti parallel. -After absorption of UV/Vis. light , the excited molecular species are raised to one of the various vibrational states in the electronic state. -These excited molecules are extremely short lived. -Deactivation occurs due to : i)Internal collision(internal conversion) ii)Cleavage of chemical bonds, iii)Re-emission as(luminescence) -When the molecular electrons are shielded from normal deactivation, luminescence or fluorescence occur. -Normal deactivation means deactivation by collisions. -In case fluorescence phenomena , after excitation, first of all, vibrational energy is lost or relaxation occurs by collisions(with molecules & solvent).

-Immediately after vibrational relaxation, fluorescence begins. -Fluorescence involves a transition from the lowest vibrational level of an excited state to one of the vibrational states in ground singlet state. -Its rapid & takes place within 10-6 -10-9s. -Some molecules undergo intersystem crossover to triplet excited state. -On returning from triplet excited to ground singlet state, phosphorescenc occurs(forbidden). -Most molecules lose less energy by emission than they gained from absorption. -So that fluorescence spectrum appears at longer wavelength than the excitation spectrum. -Fluorescence(excitation) & fluorescence (emission) spectra are theoretically mirror images of each other. Transition types in fluorescence: -Fluorescence results from the absorption of UV radiation of more than 250nm. - * transitions is seldom observed for fluorescence. - * & * - n transitions are common in fluorescence. - Only * leads to significant fluorescence.

Instrumentation: -Similar to that of UV/Vis spectrophotometer. -Light sources are mercury vapour lamp/high pressure xenon arc lamp. -Generally double beam optics are used. -This is to compensate for fluctuations in radiant power.

-The upper sample beam first passes through an excitation wavelength selector. -Filter is used for photofluorimeter, Monochromator for spectrofluorimeter. -The filter transmits radiation that excites fluorescence but excludes or limits radiation of the fluorescence emission . -Fluorescence is emitted from the sample in all directions. -It is observed right angles to the excitation beam. -Right angled observation, minimizes scattering & intense source radiation. -The emitted radiation passes through the emission selector(filter/monochromator). -It isolates the fluorescence emission. -The isolated radiation is converted to electrical signal by photo-transducer. -The lower reference beam passes through an attenuator. -It reduces power of the radiation to approx. that of the fluorescence radiation(100 times reduction or more). -The reference beam then received by a second transducer. -The beam is then converted to an electrical signal. -Electronics & a computer data system process the signals. -Ratio of the fluorescence emission intensity to the excitation source intensity is computed. -Digital fluorescence/spectrum are produced. Quantum yield/Quantum efficiency: -To fluoresce, molecules are to absorb radiation. -Not all molecules which absorb UV/Vis radiation fluoresce. -Those which fluoresce, their fluorescence can be quantify. -This is done by quantum yield() as: ()=No. photons emitted =Quantity of light emitted No.of photons absorbed Quantity of light absorbed or, =No. of molecules luminesce Total No. of excited molecules - is the inherent property of a molecule. -It is largely determined by structure. - Its value is 1. Quantitative aspects of fluorimetry: Total fluorescent(F) is given = F/Ia or, F= Ia, where , Ia is intensity of light absorption,& is quantum efficiency, Since, we know, I0 = Ia + It , where is intensity of incident light , It is intensity of transmitted light.

So that, F=(I0 It ) , According to Beer-Lambert law I0 / It = 10abc or, F = I0(1- e-2.3abc) Application: 1)Qualitative , 2)Quantitative 1)Qualitative: -Some information about molecular structure may be derived from excitation & emission spectra. -But qualitative application is very rare. 2)Quantitative analysis of drug substances: -Drugs & drug metabolites in blood, urine, & other biological products which are in very low Concn. -Quantitative studies of rates & mechanisms of drug absorption , metabolism and excretion. Fluorimetric analysis of drug substances. i) Assay of drugs that possess intrinsic fluorescence. Quinine,Chlorzoxazone, p-aminosalicylate, ergometrine, griesofulvin etc. ii) Derivatisation non-fluorescing drug to fluorescing. Amino acid to dansyl amino acid, Lactam formation of Chlordizepoxide , Oxidation product of reserpine etc. iii)Development of a fluorescent molecule by extensive molecular change. Oxidation of thiamine to thiochrome by ferricyanide.