naveen FLUORIMETRY and PHOSPHORIMETRY

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fluorometry

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FLUORIMETRY and PHOSPHORIMETRY:

FLUORIMETRY and PHOSPHORIMETRY By: NAVEEN KUMAR

Fluorescence:

Fluorescence When a beam of light is incident on certain substances they emit visible light or radiation .This phenomenon is called fluorescence. Substances showing this phenomenon are fluorescent substances. This phenomenon is Fluorescence instantaneous i.e start emitting radiation immediately after the absorption and stops when incident light is cut off. Fluorescent substances emit maximum radiation with in 10 -10 to 10 -8 sec of absorption. This is also called delayed fluorescence

Phosphorescence:

Phosphorescence When light is incident on certain substances ,they emit light continuously even after the light is cut off . This is called phosphorescence and such substances are called as phosphorescent substances. In the fluorescence process, the electron did not change its spin direction But under the appropriate conditions, a spin-flip can occur The light emission process must wait until electron undergoes a spin-flip to revert back to it original state Phosphorescent substances emit maximum radiation within 10 -4 to 20 sec or longer.

Theory :

Theory Singlet and triplet stage- A molecular electronic state in which all electronic spins are paired is called singlet state. Rate of absorption and emission- rate of absorption is very rapid and fluorescence emission occur at slower rate. Deactivation process- the favored route is one that minimize lifetime of excited state. if deactivation by fluorescence is rapid w.r.t radiation less procedure is rapid such emission observed or vice-versa depend on favored rate constant.

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TERMS INCLUDED - - vibration relaxation- (occurs in ~ 10 -12 s ) collision between molecule of excited species and those of solvent lead to rapid energy transfer with increase in temperature. - Stroke shift- shift towards lower frequency from adsorption band due to vibration relaxation. - internal conversion- by which a molecular passes to a lower electronic state without emission of radiation. - predissocation observed eg . aliphatic compound - external conversion- interaction and energy transfer between the excited molecule and the solvent or solute. - intersystem crossing- it is a process in which there is a crossover between electronic state of different multiplicity. - dynamic quenching ( collissional quenching)- nonradiative energy transfer from an excited species to other molecule is called quenching. And contact between excited species quenching agent is called dynamic quenching.

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What Factor Control Whether a Molecule Fluorescent? Quantum Yield A. Quantum Yield f = ________ kf ______________ kf + ki + kec + kic + kpd + kd kf = rate constant for fluorescence ki = rate constant for intersystem crossing kec = rate constant for external conversion kic = rate constant for internal conversion kpd = rate constant for pre-dissociation kd = rate constant for dissociation

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B. Structure: 1.) Aromatic 2.) Rigid structures exhibit more 3.) Heavy atoms will decrease fluorescence 4.) Fluorescence will increase when molecule is adhered to surface C. Temperature and Solvent Effects 1.) Lower temperature increases fluorescence 2.) Solvent contains heavy atoms will decrease fluorescence but increase phosphorescence Factors affecting fluorescence and phosphorescence

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Factors affecting fluorescence and phosphorescence Effect of concentration Adsorption Intensity Oxidation PH Photodecomposition

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Excitation and emission spectra- excitation is obtained by measuring luminescence intensity at fixed wavelength Because first step in fluorescence is absorption of radiation and excitation spectra is essential.

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INSTRUMENTATION SOURCE - Magnitude of output signal increase with increase in radiant power Lamps used- 1 Tungsten or Deuterium lamp 2 Mercury Vapor lamp 3 Xenon arc lamp( continuum) 4 Light emitting diode Laser used- 1 pulsed nitrogen laser

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INSTRUMENTATION Filter and Monochromater- Interference or adsorption filter have been used in flourometer for wavelength selection. Transducer- signal is quite low in intensity so sensitive transducer are required. eg. Photomultiplier tubes, charge coupled devices. Cells and cell compartments- rectangular cells of glass or silica.

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Instrument design of flourometer - double beam optics for compensating fluctuation in radiant power. upper sample beam first passes through excitation wavelength selector. transmit radiation that excites fluorescence but exclude others. fluorescence observed in right angle to other beams

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Instrument design of flourometer-

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- source stability - source spectral distibution - inner filter distibution - efficiencies of optical components Correction and compensation scheme

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SENSTIVITY : This technique can measure concentration as low as microgram/ml or even ng /ml. SPECIFICITY : More specific than absorption technique. PRECISION : Up to 1% can easily achieved in fluorimetery . Advantages

Limitations:

Limitations Change in pH affects fluorescent intensity. Dissolved oxygen may affect fluorescent intensity. Traces of halides, heavy metals, etc can affect fluorescent intensity.

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Applications of photoluminescence methods 1 Determination of uranium salt by flourimetry 2 Determination of inorganic species -by direct method involve formation of fluorescing chelates . eg . aromatic structure with two aromatic donor function group that permit chelate formation with metal ion -decrease in fluorescing emission because of quenching 8-hydroxy quinoline benzoin

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3 Organic analysis- (adenine, antranilic acid, uric acid, morphine, proteins etc..) 4 Determination of aspirin in blood serum with high sensitivity by phosphometric at liq. Nitrogen temperature. - low conc. of procaine, cocaine, chlorpromazine can be determined. 5 Determination of Vit B1(Thiamine). - determination of Vit B2(riboflavin)

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Comparison of fluorimetry and phosphorimetry with absorption methods. Sensitivity of luminescence methods is generally 10 to 10 3 times greater than sensitivity of absorption method. Luminescence method have greater specificity and selectivity than absorption methods.

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references Willarad H. Hobart ,Instrumental methods of analysis 17 edition ,CBS publishers and distributers , New Delhi. Skoog DA, West DM, Holler FJ and Crouch S R, Analytical Chemistry: An Introduction, 7th ed.

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