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    05 Fluorometer

    Uploaded by

    Hassan Gill
    The document discusses fluorescence and chemiluminescence techniques for analyzing samples. It describes how fluorophores in samples absorb energy from a light source and emit fluorescent light, which is measured by a fluorometer. The intensity of fluorescence emitted is directly proportional to the concentration of the analyte in the sample. It also explains how chemiluminescence works without an excitation source by generating excited intermediates through oxidation reactions. Enhanced chemiluminescence uses enzyme systems to increase the efficiency and duration of light emission compared to conventional techniques. Both fluorescence and chemiluminescence can achieve very high sensitivity but are sensitive to environmental changes.

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    © All Rights Reserved
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    05 Fluorometer

    Uploaded by

    Hassan Gill
    94 views26 pages
    The document discusses fluorescence and chemiluminescence techniques for analyzing samples. It describes how fluorophores in samples absorb energy from a light source and emit fluorescent light, which is measured by a fluorometer. The intensity of fluorescence emitted is directly proportional to the concentration of the analyte in the sample. It also explains how chemiluminescence works without an excitation source by generating excited intermediates through oxidation reactions. Enhanced chemiluminescence uses enzyme systems to increase the efficiency and duration of light emission compared to conventional techniques. Both fluorescence and chemiluminescence can achieve very high sensitivity but are sensitive to environmental changes.

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    © © All Rights Reserved

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    The document discusses fluorescence and chemiluminescence techniques for analyzing samples. It describes how fluorophores in samples absorb energy from a light source and emit fluorescent light, which is measured by a fluorometer. The intensity of fluorescence emitted is directly proportional to the concentration of the analyte in the sample. It also explains how chemiluminescence works without an excitation source by generating excited intermediates through oxidation reactions. Enhanced chemiluminescence uses enzyme systems to increase the efficiency and duration of light emission compared to conventional techniques. Both fluorescence and chemiluminescence can achieve very high sensitivity but are sensitive to environmental changes.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    94 views26 pages

    05 Fluorometer

    Uploaded by

    Hassan Gill
    The document discusses fluorescence and chemiluminescence techniques for analyzing samples. It describes how fluorophores in samples absorb energy from a light source and emit fluorescent light, which is measured by a fluorometer. The intensity of fluorescence emitted is directly proportional to the concentration of the analyte in the sample. It also explains how chemiluminescence works without an excitation source by generating excited intermediates through oxidation reactions. Enhanced chemiluminescence uses enzyme systems to increase the efficiency and duration of light emission compared to conventional techniques. Both fluorescence and chemiluminescence can achieve very high sensitivity but are sensitive to environmental changes.

    Copyright:

    © All Rights Reserved
    Download as PDF, TXT or read online from Scribd
    Download as pdf or txt
    of 26

    FLUOROMETER

    FLUOROMETER
    Principle

    “When a sample containing fluorophor is excited by a radiant


    energy from a light source. It emits light of a longer wavelength
    which is called fluorescence.”
    Fluorescence is directly proportional to the concentration of
    analyte in the sample.
    Absorption results in excitation of molecule
    which results in emission of energy (fluorescence)

    Electron

    Absorption
    Incident Energy
    Sample Molecule
    cell
    High
    Energy
    Fluorescent Orbit
    Energy
    Low Energy Orbit
    Stokes Shift
    Emitted energy is less than absorbed energy

    1 Collision

    2 Heat loss

    3 Transfer to other molecules

    4 Emission of radiant energy


    Both excitation and fluorescence energies
    are characteristic for a given molecular type
    Primary Filter

    Sample cell

    Light Source
    Attenuator

    Secondary Filter

    Fluorescence
    Display

    PM Tube
    Concentration of substance is directly
    proportional to Fluorescence
    1. Very Low Fluorescence
    2. Low Fluorescence
    3. High Fluorescence
    4. Very High Fluorescence

    Concentration of
    substance
    Gas discharge lamps (Radiant Energy Source)

    Mercury & xenon arc are most frequently used

    Incandescent tungsten lamps seldom used

    Mercury vapor lamps are used in filter fluorometers

    Most spectrofluorometers use high pressure xenon lamp


    Monochromators

    1 Grating

    2 Prism

    3 Filter
    Light detector: Photomultiplier Tube

    Double-beam instruments are used to compensate


    for instability due to electric power fluctuation
    Fluorescence concentration measurements
    are related to 4 factors
    Molar absorptivity of
    compound

    Intensity of incident
    radiation Fluorescence
    Measurements
    Quantum efficiency of
    emitted energy

    Length of the light


    path
    Concentration of substance is directly
    proportional to Fluorescence

    Fluorescence Intensity

    Concentration of naphthalene (mg/ml)


    2 Types of Fluorescence

    Fluorescence Fluorescence
    Polarization Depolarization
    (Large Molecule) (Small Molecule)

    When sample Small molecule emits


    (fluorophore) is excited depolarized light b/c it
    it emits polarized light rotates out of
    in the same plane as polarization plane during
    incident light excitation lifetime
    Fluorescence Depolarization: A competition of sample
    analyte with fluorophor labelled analyte for limited Ab

    Sample Analyte
    Conc.

    Macromolecular
    Antibody-analyte-fluorophore
    Complex formed &
    Depolarization of radiant light
    2 Advantages over spectrophotometry

    1000 times more sensitive as emitted radiation is


    measured directly & over a zero background

    More specific as absorption and fluorescence have


    different wavelengths
    4 Factors affecting intensity of fluorescence

    Change in pH affects availability of electrons

    Temperature changes the loss of energy by collision


    rather than fluorescence
    Contaminating chemicals or a change of solvents may
    change the structure

    UV light used for excitation can cause photochemical changes


    Biggest disadvantage: Very sensitive to
    environmental changes

    Any decrease in fluorescence resulting from any


    of these possibilities is known as quenching
    CHEMILUMINISCENCE
    Chemical energy generated is used

    Intermedi- Energy
    Measure- generated
    Emission ates decay
    ment by produces
    of photons to ground
    PM tube excited
    state intermediates
    Chemiluminiscence is different from fluorescence

    1 No excitation radiation is required

    2 No monochromators are needed

    3 Oxidation reactions of luminol, acridinium esters & dioxetanes


    Enhanced Chemiluminiscence

    Chemi- Enzyme
    luminiscence Enhancer
    system

    Enhanced
    Chemiluminiscence
    Increased
    efficiency
    Time course for light intensity in enhanced type is much
    longer than that for conventional reactions

    Conventional
    (30 sec)

    Enhanced
    (60 min)
    Rapid increase in intensity of emitted light
    followed by gradual decay
    Advantages

    1 Sub-picomolar detection limits

    2 Speed of measurement (For Flash type 10 sec)

    3 Ease of use (mostly one step procedures)

    4 Simple instrumentation
    Main disadvantage

    Impurities can cause a background signal that


    degrades sensitivity and specificity

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