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    Fluroscence microscopy

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    acerlaptop1007
    Fluroscence microscopy

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    Fluroscence microscopy

    Uploaded by

    acerlaptop1007
    4 views11 pages
    Fluroscence microscopy

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    © © All Rights Reserved

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    Fluroscence microscopy

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    4 views11 pages

    Fluroscence microscopy

    Uploaded by

    acerlaptop1007
    Fluroscence microscopy

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    of 11

    FLUORESCENCE SPECTROSCOPY

    FLUORESCENCE SPECTROSCOPY
    1. Fluorescence spectroscopy (fluorometry or spectrofluorometry), is a type of
    electromagnetic spectroscopy which analyzes fluorescence from a sample.
    2. It involves using a beam of light, usually ultraviolet light, that excites the electrons in
    molecules of certain compounds and causes them to emit light of a lower energy,
    typically, but not necessarily, visible light. This shift to longer wavelength is called the
    Stokes shift.
    3. Devices that measure fluorescence are called fluorometers or fluorimeters.
    The first observation of fluorescence from a quinine solution in sunlight was reported by Sir
    John Frederick William Herschel in 1845.

    Quinine
    The quinine in tonic water is excited by the ultraviolet light from the sun. Upon return to the
    ground state the quinine emits blue light with a wavelength near 450 nm.
    Fluorescence excitation and emission spectra for a solution
    of quinine.
    RELAXATION PROCESSES
    Once the molecule is excited to S1 or S2 several processes can occur that cause the molecule to
    lose its excess energy.
    Various mechanism of relaxation:
    1. Fluorescence
    2. Phosphorescence
    3. Vibrational relaxation
    4. Internal conversion
    5. External conversion
    6. Inter system crossing
    1. Fluorescence – the emission is basically immediate and therefore generally only
    visible
    2. Phosphorescence – store the absorbed light energy for some time and release light
    later, resulting in an afterglow
    3. Vibrational relaxation – excited electron gives some of its vibrational energy to
    another electron in the same or a different molecule, in the form of kinetic energy
    4. Internal conversion – radiation less transition between energy states of the same
    spin state
    5. External conversion – deactivation of an excited electronic state by interaction
    and energy transfer between the excited molecule and solvent or other solutes
    6. Intersystem crossing – the relaxation of the molecule from a singlet excited state
    to a lower energy, triplet excitation state
    Light source
    Xenon lamps Mercury-vapour lamp

    Filters and/or monochromators


    Grating monochromator

    Detectors
    Photomultiplier tube
    Factors interfering with
    fluorescence intensity
    1. Concentration
    2. Transition type in fluorescence
    3. Structure
    4. Temperature and solvent
    5. Impurities present in the solution
    APPLICATIONS
    1. Determination of fluorescent drugs in low-dose formulations in the presence of non-
    fluorescent excipients.
    2. In carrying out the limit tests where the impurity is fluorescent.
    3. Useful for studying the binding of drugs to component in complex formulations.
    4. Widely used in bioanalysis for measuring small amounts of drug and for studying drug-
    protein binding.

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