Wednesday, March 11, 2020

Principle and Applications of Fluorimetry

What is Fluorometry?

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


Principle of Fluorometry:

The phosphorescence and fluorescence are processes of the photon emission, which arise from electronically excited states throughout molecular relaxation. These photonic mechanisms cause the polyatomic fluorescent molecules (fluorophores) between vibrational and electronic states. Fluorophores play an essential role in the fluorescence spectroscopy. 
Fluorophores are the components that produce fluorescence in molecules. The samples that have been excited electronically 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 rapid from the ground state to the excited state on the order of 10 to 15 seconds. 
The molecule is quickly relaxed after excitation, to the lowest vibrational point of the excited electronic state. The quick process of vibrational relaxation takes place on the time scale of femtoseconds to picoseconds. The emission and excitation spectra of fluorescence, respectively, reflect the vibrational level structures in the ground and the excited electronic states.

The different electronic states in fluorimetry are as follows.
Singlet excited state: It is a state in which the electrons from the opposite spin are unpaired out.
Triplet state: It is a state in which unpaired electrons of the identical spin are present
Doublet state: It is a state which contains unpaired electrons.
Singlet ground state: It is a state in which all electrons are paired within a molecule.

Applications of Fluorescence Spectroscopy:

  • The application of fluorometry is significant as a potent and valuable tool for studying the physical and chemical behavior of macro-molecules.
  • Fluorescence spectroscopy used in environmental analysis.
  • Fluorescence spectroscopy is used where the sample is scared and complex to process.
  • Fluorescence spectroscopy used in food analysis.
  • It is used to determine several types of analytes in serum.
  • Each form of fluorescence activity is to assist to apply fluorescent probes in polymer systems.
  • Fluorescence spectroscopy used in dairy processing.

The Advantages of Fluorescence Spectroscopy are as Follows.

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

The Disadvantages of Fluorescence Spectroscopy are as Follows.

  • The major disadvantage of fluorescence spectroscopy is that it can only analyze fluorescent molecules.
  • It has limitations associated with loss of photostability and recognition capability
  • Fluorophores have a short lifespan.
  • It is also susceptible to the auto-fluorescence of the solution.
  • It can susceptible to interference due to changes in sample pH and oxygen levels.

Difference between spectrofluorometer and spectrophotometer:

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

Commonly asked questions on fluorometry are as follows.

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

What is the basic principle of fluorescence spectroscopy?
A molecule is absorbed and excited by incidental electromagnetic radiation. In its excited state, it is unstable, and by emitting radiation, it returns to the ground state.

What are the types of fluorometer?
The filter fluorometer and the spectrofluorometer are the two basic types of the fluorometer.

What is the major advantage of fluorescence spectroscopy?
The high sensitivity and its very low detection limit are the major advantages of fluorescence spectroscopy.



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