Principle and applications of fluorimetry

Fluorometry is a type of spectroscopy and is also called fluorescence spectroscopy. It is used to identify and determine analyte concentrations in a sample. The mechanisms involve exciting a beam of ultraviolet light on molecules of a specific analyte and allowing them to emit visible light. At one wavelength, it is the molecular absorption of light energy and its almost instantaneous re-emission at a different, longer wavelength. Several molecules are intrinsically fluorescent, and others must be modified for fluorescence.


  1. What is fluorimetry?
  2. Principle of fluorimetry
  3. Applications of fluorescence spectroscopy
  4. Advantages of fluorescence spectroscopy
  5. Disadvantages of Fluorescence Spectroscopy
  6. Difference Between Spectrofluorimeter and Spectrophotometer
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Principle of fluorimetry:

Phosphorescence and fluorescence are photon emission processes, resulting from electronically excited states during molecular relaxation. These photonic mechanisms trigger polyatomic fluorescent molecules (fluorophores) between vibrational and electronic states. Fluorophores play an essential role in fluorescence spectroscopy. Fluorophores are the components that produce fluorescence in molecules. Samples that have been electronically excited after absorption of UV (200 nm to 400 nm), visible (400 nm to 800 nm) or NIR (700 nm to 1100 nm) radiation. The excitation method is very fast from ground state to excited state on the order of 10 to 15 seconds. The molecule relaxes rapidly after excitation, to the lowest vibrational point of the excited electronic state. The rapid vibrational relaxation process takes place on the femtosecond to picosecond time scale. The emission and excitation spectra of fluorescence, respectively, reflect vibrational level structures in the ground and excited electronic states.

The different electronic states in fluorimetry are as follows.

Excited state t-shirt: It is a state in which electrons of opposite spin are unpaired.

triplet status: It is a state in which there are unpaired electrons of identical spin.

Dual status: It is a state that contains unpaired electrons.

Fundamental state of the single: It is a state in which all the electrons are paired in a molecule.

Applications of fluorescence spectroscopy:

  • The application of fluorometry is important as a powerful and valuable tool to study the physical and chemical behavior of macromolecules.
  • Fluorescence spectroscopy used in environmental analysis.
  • Fluorescence spectroscopy is used when the sample is apprehensive and complex to process.
  • Fluorescence spectroscopy used in food analysis.
  • It is used to determine various types of analytes in serum.
  • Each form of fluorescence activity is intended to help you apply fluorescent probes to polymer systems.
  • Fluorescence spectroscopy used in the processing of dairy products.

The advantages of fluorescence spectroscopy are as follows.

  • Due to the unique optical properties of molecules, it has high precision.
  • Fluorescence spectroscopy can be used for the quantification of fluorescent species.
  • This can calculate the decay time, fluorescence intensity, and component concentration.
  • This method is less expensive than the other methods.
  • It has the capability of quick and rapid diagnosis.

The disadvantages of fluorescence spectroscopy are as follows.

  • The main disadvantage of fluorescence spectroscopy is that only fluorescent molecules can be analyzed.
  • It has limitations associated with loss of photostability and the ability to recognize
  • Fluorophores have a short lifespan.
  • It is also sensitive to autofluorescence from the solution.
  • May be susceptible to interference from changes in sample pH and oxygen levels.

Difference Between Spectrofluorimeter and Spectrophotometer:

The main difference between spectrofluorometer and spectrophotometer is that spectrofluorometer is used to determine the fluorescence of analytes whereas spectrophotometer is used to determine the intensity of electromagnetic radiation.

The most frequently asked questions about fluorimetry are as follows.

What is fluorescence spectroscopy?

Fluorescence spectroscopy is a tool for determining the fluorescence of its components, often as a means of measuring the nature of the substance that fluoresces.

What is the basic principle of fluorescence spectroscopy?

A molecule is absorbed and excited by incident electromagnetic radiation. In its excited state, it is unstable and, by emitting radiation, it returns to the ground state.

What are the types of fluorimeter?

Filter fluorometer and spectrofluorimeter are the two basic types of fluorometer.

What is the big advantage fluorescence spectroscopy?

High sensitivity and its very low detection limit are the main advantages of fluorescence spectroscopy.

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