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    Assignment Analysis Final2

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    Mahadi Hasan Khan
    1) Fluorometry is an analytic method that uses a fluorometer to detect and measure fluorescence intensity from samples excited by specific wavelengths of light. 2) When electrons in a sample absorb light, they move to a higher energy state and emit fluorescent light of a longer wavelength upon returning to the ground state. 3) Factors like concentration, incident light intensity, pH, and temperature can affect fluorescence intensity, while the presence of quenchers or oxygen can decrease it.

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    Assignment Analysis Final2

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    Mahadi Hasan Khan
    77 views8 pages
    1) Fluorometry is an analytic method that uses a fluorometer to detect and measure fluorescence intensity from samples excited by specific wavelengths of light. 2) When electrons in a sample absorb light, they move to a higher energy state and emit fluorescent light of a longer wavelength upon returning to the ground state. 3) Factors like concentration, incident light intensity, pH, and temperature can affect fluorescence intensity, while the presence of quenchers or oxygen can decrease it.

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    1) Fluorometry is an analytic method that uses a fluorometer to detect and measure fluorescence intensity from samples excited by specific wavelengths of light. 2) When electrons in a sample absorb light, they move to a higher energy state and emit fluorescent light of a longer wavelength upon returning to the ground state. 3) Factors like concentration, incident light intensity, pH, and temperature can affect fluorescence intensity, while the presence of quenchers or oxygen can decrease it.

    Copyright:

    © All Rights Reserved
    Download as DOCX, PDF, TXT or read online from Scribd
    Download as docx, pdf, or txt
    77 views8 pages

    Assignment Analysis Final2

    Uploaded by

    Mahadi Hasan Khan
    1) Fluorometry is an analytic method that uses a fluorometer to detect and measure fluorescence intensity from samples excited by specific wavelengths of light. 2) When electrons in a sample absorb light, they move to a higher energy state and emit fluorescent light of a longer wavelength upon returning to the ground state. 3) Factors like concentration, incident light intensity, pH, and temperature can affect fluorescence intensity, while the presence of quenchers or oxygen can decrease it.

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    © All Rights Reserved
    Download as DOCX, PDF, TXT or read online from Scribd
    Download as docx, pdf, or txt
    of 8

    Assignment-1

    Course: Pharmaceutical Analysis (PHA305) Sec-1


    Course Instructor: Syeda Neureen Ahmed
    Submitted by: Ayesha Rahman (1920560),
    Usayatun Mehera (1921143),
    Nusrat Jahan Mim (1920495)
    Submitted Date:
    Topic: Fluorometry and Polarimetry
    Fluorometry:
    Fluorometry is an analytic method for detecting and measuring fluorescence intensity at a
    particular wavelength with the aid of the fluorometer. The fluorometer is the instrument with
    which the intensity of the fluoroscene from different objects can be measured. When the
    electrons in the sample are excited by the specific wavelength of the light, the electrons move
    from lower energy state to higher energy state by emitting fluoresce light of a different
    wavelength. Different fluorescent object emit different kinds of light at different wavelengths.

    Principle

    Molecules consist of bonding electrons such as Sigma and Pi electrons. These electrons have the
    least energy. They are more stable so they stay at the ground state. Molecules remain in their
    ground vibration level at room temperature. When the molecules get energy in the form of light
    with specific frequency, it would absorb light with 10-15 seconds. Due to this absorption, the
    molecules move from ground state to the first excited singlet electronic state and the excitation
    molecules jump to in any one of the vibrational levels in the first excited electronic state. With
    the increase of energy in the form of the light increase the excited level of the electron from
    ground state to one, two, three and so on. These excited levels are very unstable for the excited
    electrons. They tend to go back to their original energy state with the emission of light when the
    source of energy is being removed.
    Fluoroscene and Chemical structure

    Florescence is the substance that emits the absorbed light as the electron moves from a higher
    energy state to a lower energy level and the emitted light has a longer wavelength than the
    absorption. Fluorescence is generally observed in compound with rigid structure, organic
    chelates agent complexed with metal ion and substances which contain aromatic functional
    group with lower energy. Heterocyclic compounds do not show fluorescence properties such as
    pyridine, pyrrole furan but if these heterocyclic compounds combine with benzene rings it would
    increase fluorescence such as indole, quinoline, and isoquinoline. This is because a substitute on
    the benzene increases the wavelength to the maximum and there are corresponding changes to
    the florescent. The increase of the atomic number of the compound decreases the florescent.
    Unsubstituted aromatic hydrocarbon increases fluorescence as quantum efficiency increases with
    the number of rings and degree of condensation but if aromatic rings are substituted by the
    carboxylic acid, it does not show fluorescence property.

    There are 2 types of fluorescence :


    Intrinsic Fluorescence: a substance (molecules, ions, etc.) that emits light strongly in its
    original form
    Extrinsic Fluoroscene: a substance that emits light strongly through reaction with a
    fluorometric reagent.

     Depending on the molecule involved, the electron can move to different energy states:
    Ground State-in this state, two electrons are in pairs and opposite spins.

    Singlet Excited state-in this state, electrons are in a higher energy orbital with opposite spin
    similar to the ground state

    Triplet excited state-in this state, electrons are more excited and may have the same spin. This
    is known as an intersystem crossing.

    Instrumentations
    Sources of light such as 
    Xenon arc lamp- intense radiation is formed with the help of the current through creating an
    atmosphere of xenon and the range of the spectrum is between 250-600nm with a peak at 470nm

    Mercury arc lamp

    It produces an intense light spectrum above 350nm and high-pressure lamps show lines at
    366,405,536,546,577 and low-pressure lamps show lines at 254nm.

    Tungsten Lamp

    It is used if excitation is required at visible light and it does not give UV light and the intensity of
    the lamp is low.

    Tunable dye laser-


    It produces radiation between 360-650 nm with the help of pulsed nitrogen as a primary source.
    Filters and monochromators-

    The filter consists of two types – 


    Primary filter- it absorbs visible light and emits UV light
    Secondary filter-it absorbs UV radiations and emit visible light

    Monochromators-
    Excitation monochromator- it gives appropriate radiation for excitation molecules.
    Emission monochromators- only isolate the fluorescence radiation.

    Sample cell: these are used to hold the sample which is in cylindrical or rectangular shape made
    out of silica and glass. In Fluorometry, all the surfaces of the sample cell are polished.

    Detectors:
    Photomultiplier tube-
    This is the best detector as its sensitivity is high as it detects weak light intensity.

    Photovoltaic cell

     It has a copper plate coated with a thin layer of cuprous oxide. For good contact, silver is laid
    onto this plate. When light falls on them oxide moves from the oxide layer to the copper and thus
    oxide becomes positive and copper becomes negative.

    The three most common fluorometers are:


    Single beam fluorometer-
    It is very simple to build, inexpensive, and easy to operate. It has tungsten as a light source and
    an optical system as the primary filter. The secondary filter measured emitted radiation at 90
    degrees. Secondary filters absorb UV light and emit visible light.

    Double beam fluorometer-

    Two incident beams passes through the primary filter independently and fall on the sample or
    standard solutions. The emitted ray from there passes to the secondary filter.

    Spectrofluriometer-

    Here, the primary filter is being substituted with the excitation monochromator and the
    secondary filter is being replaced with an emission monochromator. With the help of the beam
    splitter, the incident beam is split into a sample and reference beam.
    Factor affecting the intensity of fluorescence
     Concentration-intensity of the fluorescence is directly proportional to the concentration
    when the absorbance is less than o.o2
     Quantum yield of fluorescence-it should be always less than 1
     The intensity of incident light-it is directly proportional to the intensity of fluorescence
     Adsorption-it causes serious damage if the sample solution adsorbs to the container.
     Oxygen-oxidation of fluorescence produces a non-fluorescence substance which
    decreases fluorescence intensity
     Ph- changing of ph will have a significant effect on the intensity of fluorescence
     Temperature and viscosity-increase in temperature cause more collusion which reduces
    fluorescence intensity. Increased viscosity decreases the fluorescence intensity.
     Photo decomposition-photochemical degradation reduces fluorescence intensity
     Quencher-it reduces fluorescence intensity by the presence of a substance in the sample 
     Scatter-Scattering of incident light through the solution causes a decrease in fluorescence
    intensity.

    References
    https://www.edinst.com/us/blog/what-are-absorption-excitation-and-emission-spectra/
    https://slideplayer.com/slide/10446081/
    https://www.slideshare.net/sreevidyavemuri/fluorimetry-45064390

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