Spectrofluorimetry

Spectrofluorimetry

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  ITM UNIVERSITY GWALIOR Assignmenton - Spectrofluorimetry Presented to - Mr. Hero Khan Pathan Sir Presented by - Mineeta Mahra M. Pharma (1st sem.)
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  •Introduction •Principle •Theory •Instrumentation •Factor affecting •Application •Reference Table ofcontents
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  Introduction Absorption of UV-VISradiation causes transition of electrons from ground state to excited state. - As excited state is not stable so, excess energy is released by Collisional deactivation photoluminescence This study or measurement of this emitted radiation is the principle of Fluorimetry. Phosphorescence is also related phenomena, which is study of emitted radiation when electron undergoes transition from triplet state to singlet ground state. • Singlet ground state : state in which electrons in a molecule are paired. [||] • Singlet excited state: state in which electrons are unpaid but of opposite spins. [||] • Triplet state: state in which unpaired electrons of Same spin are present. [||] • Excitation process: absorption of energy or light followed by conversion from ground state to excite state. • Relaxation process: process by which atom or molecule losses energy & returns to ground state.
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  It is twotype Luminescence is the emission of light by a substance. It occurs when an electron returns to the electronic ground state from an excited state and loses its excess energy as a phot on. 1. Fluorescence Spectroscopy 2. Phosphorescence spectroscopy 1. fluorescence Spectroscopy  When a beam of light is incident on certain substances they emit visible light or radiations. This is known as fluorescence.  Fluorescence starts immediately after the absorption of light and stops as soon as the incident light is cut off.  The substances showing this phenomenon are known as fluorescent substances. 2. Phosphorescence spectroscopy  When light radiation is incident on certain substances they emit light continuously even after the incident light is cut off.  This type of delayed fluorescence is called phosphorescence.  Substances showing phosphorescence are phosphorescent substances.
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  Principle  It isan analytical device depends on the fluorescence phenomenon which is a short- lived type of photoluminescence created by electromagnetic excitation.  That is, fluorescence is generated when a molecule transmits from its ground state So to one of several vibrational energy levels in the first excited electronic state, S1, or the second electronic excited state, S2, both of which are singlet states.  Relaxation to the ground state from these excited states occurs by emission of energy through heat and/or photons.
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  Theory  A molecularelectronic state in which all of the electrons are paired are called singlet state.  In a singlet state molecules are diamagnetic.  Most of the molecules in their ground state are paired.  When such a molecule absorbs uv/visible radiation, one or more of the paired electron raised to an excited singlet state /excited triplet state. From the excited singlet state one of the following phenomenon occurs i. Fluorescence ii. Phosphorescence iii. Radiation less process • Vibrational relaxation • Internal conversion • External conversion • Intersystem crossing
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  Instrumentation Components of instrumentationare following -  Light  Excitations monochromator  Sample holder  Emission monochromator  Detector  Read out device
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  Light source: Xenonarc lamp or mercury lamp A high pressure xenon arc lamp is used for spectrophotometer, which provides intense, stable and continuous beam in UV and visible region. Filter fluorimeters generally use low-pressure mercury vapour lamp which emits distinct line spectra in the UV and visible region. Two sets of monochromators are used: primary monochromator and secondary monochromator.  Primary monochromator: It includes slits and dispersive device to isolate the wavelength for excitation of sample.  Secondary monochromator: Isolates the wavelength of emitted fluorescence.  Light  Excitations monochromator
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   Sample holder Cuvettes:Usually quartz cuvettes are used. Cylindrical or rectangular cells fabricated of silica or glass used. Path length is usually 10mm or 1cm. DAII the surfaces of the sample holder are polished in fluorimetry.  Detector A phototube or photo multiplier are used.  Read out device A galvanometer or potentiometer is used readout our device.
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  Factor affecting  Concentration Incident light  Quantum yield  Absorption  PH  Oxygen  Temperature & viscosity  Scatter
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   Concentration  Thefluorescence intensity of a substance is proportional to concentration only when the absorbance in a 1 cm cell is less than 0.02. If the concentration of the fluorescent substance is so great that all incident radiation is absorbed, the equation will be: F = 1°9 That is the fluorescence is independent of concentration, and proportional to the intensity of incident radiation only, a property that may be utilized to determine the approximate emission characteristics of a light source.  Incident light  An increase in the intensity of light incident on the sample produces a proportional increase in the fluorescence intensity. The intensity of incident light depends on the intensity of light emitted from the lamp.
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   Quantum yield Thisis the ratio - Ø = No. Of Photon emitted/ no. of Photon absorbed Since some absorbed energy is lost by radiation less pathways, the quantum efficiency is less than 1. Highly fluorescent substances take o value near 1, which shows that most of the absorbed energy is re-emitted as fluorescence. For e.g.; fluorescein in 0.1 M NAOH and quinine in 0.05 M H,SO, have, o values of 0.85 and 0.54 respectively at 23°C. Non-fluorescent substances have o = 0.  Absorption The extreme sensitivity of the method requires very dilute solution, 10-100 times, weaker than those employed in absorption spectrophotometry. Adsorption of the fluorescent substance on the container walls may therefore presents serious problems and strong stock solutions must be kept and diluted as required. Quinine is a typical example of a substance which is adsorbed onto cell walls.
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   PH It isto be expected that alteration of the pH of a solution will have a significant effect on fluorescence if the absorption spectrum of the solute is changed.  Oxygen The presence of oxygen may interfere in two ways: By direct oxidation of the fluorescent substance to non- fluorescent products. By quenching of fluorescence.  Temperature & viscosity Variation in temperature and viscosity will cause variations in the frequency of collision between molecules. Thus, an increase in the temperature or the decrease in the viscosity is likely to decrease the fluorescence by deactivation of the excited molecules by collision.  Scatter The excitation and emission monochromators are at the same wavelengths, scattered light of the same wavelength as the incident light will be detected by the photomultiplier arising from colloidal particles in the sample (Tyndall scatter) and from the molecules (Rayleigh scatter).
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  Application  It isused for the chemical modification suchoxydation ,reduction hydrolysis, couping and self condensation.  The determination and comparison of both excitation and fluorescence spectra of a compound of may help to identify it.  The assay of vitamin in food sluffs NADH in like hormone, cortisone ,drug pharmaceutical ,cholesterol and Porphyrins.  Enzyme assay and kinetic analysis.  Study of protein structure.
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  Reference 1. Douglas a,Skoog , Holler, PRINCIPLE OF INSTRUMENTALANALYSIS, 5th edition , Saunders college, west Washington square, Philidhepia . 2. Dr Ravishankar, A TEXTBOOK OF PHARAMACEUTICAL ANALYSIS, 3rd edition, Rx pub., 57, west Tiruneveli-627006 car street. 3. Principle and techniques of BIOCHEMISTRY and MOLECULAR BIOLOGY-keith Wilson and john walker. * TIETZ-fundamentals of clinical chemistry. * Wikipedia . * Google .