Fluorescence spectroscopy

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Fluorescence spectroscopy
Komal Choudhary Lecturer School of
Biotechnology DAVV Indore
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Fluorescence spectroscopy

• Luminescence
• It is the emission of light from any
  substance, and occurs from energetically excited
  states.
• It is divided into two categories
• (1) Fluorescence
• (2) phosphorescence

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• Non radioactive relaxation- excitation energy is
  dissipated as heat(vibration) to the solvent.
• It includes internal conversion(IC) intersystem
  conversion(ISC).
• Singlet state
• Triplet state

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• Jablonski energy diagram tell us about the
  relaxation mechanism for exited state molecule.
• Once a molecule has absorbed energy in the form
  of electromagnetic radiation there are numbers of
  routs by which it can return to ground state.
• If the photon emission occurs between same spin
  state(i.e. S1-S0), this is called fluorescence
  emission. (release of energy is faster)
• If the spin state of the initial and final energy
  level are different(i.e. T1-S0), this is called
  phosphorescence emission. (release of energy is
  slower)

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• Fluorescence spectroscopy or spectrofluorometry,
  is a type of electromagnetic spectroscopy which
  analyzes fluorescence from a sample.
• Fluorescence is a phenomenon where by molecule
  after absorbing radiation emit the radiation of
  longer wavelength (compound absorb UV radiation,
  emit visible light).
• stokes shift ?ab-?emt
• Best results are obtained from
• those compounds which
• showing large shifts.

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Components of spectroflorimeter

• Light source-xenon or mercury lamps
• Slits
• Two monochromator(prism or differaction grating)
• (1) excitation monochromator
• (2) emission monochromator
• Sample holder(quartz cuvette)
• Detector-photocell (red-sensitive PMT)

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• Fluorescence is the property of compounds having
• p-p transition, p electron cloud.
• Fluorescence is measured at right angle to sample
  holder to avoid the measurement of transmitted
  light.
• Cuvettes are clear from all the sides unlike
  those used in spectrophotometer.
• Fluorescence depends upon concentration of the
  sample.(particular range)
• Quantum yieldno of photon emitted/ no. of photon
  absorbed (value lies always 0-1)
• Quantity of any compounds in nanogram can be
  measured.(sensitivity)

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Factors which affect fluorescence or quench the
emission of fluorescence are

• Solvent polarity
• pH
• Temperature
• Viscosity
• Presence of oxygen
• Concentration of the sample molecule
• Intensity of incident light

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Fluorescent probes

• Ethidium bromide
• Flourescein
• Densyl chloride
• 1-Anilinonapthalene 8-sulphonate(ANS)
• 4-methylumbelliferone
• Acridine
• GFP,RFP
• Extrinsic fluorescence
• Intrinsic fluorescence

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Applications

• Used in both qualitative and quantitative(major)
  estimation.
• Assays of vitamin B in food stuff ,NADH,
  hormones, drugs, pesticides, Carcinogens,
  chlorophyll, cholesterol, metal ions etc.
• enzyme assays and kinetic analysis.
• Protein structure analysis.
• Membrane structure analysis.
• Microspectrofluorimetry (used to detect malignant
  cell in biopsy tissue)
• FACS.